Detection of Low-Level Cardinium and Wolbachia Infections in Culicoides

PubMed Central

Mee, Peter T.; Weeks, Andrew R.; Walker, Peter J.; Hoffmann, Ary A.

2015-01-01

Bacterial endosymbionts have been identified as potentially useful biological control agents for a range of invertebrate vectors of disease. Previous studies of Culicoides (Diptera: Ceratopogonidae) species using conventional PCR assays have provided evidence of Wolbachia (1/33) and Cardinium (8/33) infections. Here, we screened 20 species of Culicoides for Wolbachia and Cardinium, utilizing a combination of conventional PCR and more sensitive quantitative PCR (qPCR) assays. Low levels of Cardinium DNA were detected in females of all but one of the Culicoides species screened, and low levels of Wolbachia were detected in females of 9 of the 20 Culicoides species. Sequence analysis based on partial 16S rRNA gene and gyrB sequences identified “Candidatus Cardinium hertigii” from group C, which has previously been identified in Culicoides from Japan, Israel, and the United Kingdom. Wolbachia strains detected in this study showed 98 to 99% sequence identity to Wolbachia previously detected from Culicoides based on the 16S rRNA gene, whereas a strain with a novel wsp sequence was identified in Culicoides narrabeenensis. Cardinium isolates grouped to geographical regions independent of the host Culicoides species, suggesting possible geographical barriers to Cardinium movement. Screening also identified Asaia bacteria in Culicoides. These findings point to a diversity of low-level endosymbiont infections in Culicoides, providing candidates for further characterization and highlighting the widespread occurrence of these endosymbionts in this insect group. PMID:26150447

[Isolation and identification of Cronobacter (Enterobacter sakazakii) strains from food].

PubMed

Dong, Xiaohui; Li, Chengsi; Wu, Qingping; Zhang, Jumei; Mo, Shuping; Guo, Weipeng; Yang, Xiaojuan; Xu, Xiaoke

2013-05-04

This study aimed to detect and quantify Cronobacter in 300 powdered milk samples and 50 non-powdered milk samples. Totally, 24 Cronobacter (formerly Enterobacter sakazakii) strains isolated from powdered milk and other foods were identified and confirmed. Cronobacter strains were detected quantitatively using most probable number (MPN) method and molecular detection method. We identified 24 Cronobacter strains using biochemical patterns, including indole production and dulcitol, malonate, melezitose, turanose, and myo-Inositol utilization. Of the 24 strains, their 16S rRNA genes were sequenced, and constructed phylogenetic tree by N-J (Neighbour-Joining) with the 16S rRNA gene sequences of 17 identified Cronobacter strains and 10 non-Cronobacter strains. Quantitative detection showed that Cronobacter strains were detected in 23 out of 350 samples yielding 6.6% detection rate. Twenty-four Cronobacter strains were isolated from 23 samples and the Cronobacter was more than 100 MPN/100g in 4 samples out of 23 samples. The 24 Cronobacter spp. isolates strains were identified and confirmed, including 19 Cronobacter sakazakii strains, 2 C. malonaticus strains, 2 C. dubliensis subsp. lactaridi strains, and 1 C. muytjensii strain. The combination of molecular detection method and most probable number (MPN) method could be suitable for the detection of Cronobacter in powdered milk, with low rate of contamination and high demand of quantitative detection. 24 isolated strains were confirmed and identified by biochemical patterns and molecular technology, and C. sakazakii could be the dominant species. The problem of Cronobacter in powdered milk should be a hidden danger to nurseling, and should catch the government and consumer's attention.

An Advanced Approach to Simultaneous Monitoring of Multiple Bacteria in Space

NASA Technical Reports Server (NTRS)

Eggers, M.

1998-01-01

The utility of a novel microarray-based microbial analyzer was demonstrated by the rapid detection, imaging, and identification of a mixture of microorganisms found in a waste water sample from the Lunar-Mars Life Support Test Project through the synergistic combination of: (1) judicious RNA probe selection via algorithms developed by University of Houston scientists; (2) tuned surface chemistries developed by Baylor College of Medicine scientists to facilitate hybridization of rRNA targets to DNA probes under very low salt conditions, thereby minimizing secondary structure; and (3) integration of the microarray printing and detection/imaging instrumentation by Genometrix to complete the quantitative analysis of microorganism mixtures.

Development of Quantitative PCR Assays Targeting the 16S rRNA Genes of Enterococcus spp. and Their Application to the Identification of Enterococcus Species in Environmental Samples

PubMed Central

Ryu, Hodon; Henson, Michael; Elk, Michael; Toledo-Hernandez, Carlos; Griffith, John; Blackwood, Denene; Noble, Rachel; Gourmelon, Michèle; Glassmeyer, Susan

2013-01-01

The detection of environmental enterococci has been determined primarily by using culture-based techniques that might exclude some enterococcal species as well as those that are nonculturable. To address this, the relative abundances of enterococci were examined by challenging fecal and water samples against a currently available genus-specific assay (Entero1). To determine the diversity of enterococcal species, 16S rRNA gene-based group-specific quantitative PCR (qPCR) assays were developed and evaluated against eight of the most common environmental enterococcal species. Partial 16S rRNA gene sequences of 439 presumptive environmental enterococcal strains were analyzed to study further the diversity of enterococci and to confirm the specificities of group-specific assays. The group-specific qPCR assays showed relatively high amplification rates with targeted species (>98%), although some assays cross-amplified with nontargeted species (1.3 to 6.5%). The results with the group-specific assays also showed that different enterococcal species co-occurred in most fecal samples. The most abundant enterococci in water and fecal samples were Enterococcus faecalis and Enterococcus faecium, although we identified more water isolates as Enterococcus casseliflavus than as any of the other species. The prevalence of the Entero1 marker was in agreement with the combined number of positive signals determined by the group-specific assays in most fecal samples, except in gull feces. On the other hand, the number of group-specific assay signals was lower in all water samples tested, suggesting that other enterococcal species are present in these samples. While the results highlight the value of genus- and group-specific assays for detecting the major enterococcal groups in environmental water samples, additional studies are needed to determine further the diversity, distributions, and relative abundances of all enterococcal species found in water. PMID:23087032

Competitor internal standards for quantitative detection of mycoplasma DNA.

PubMed

Sidhu, M K; Rashidbaigi, A; Testa, D; Liao, M J

1995-05-01

Homologous internal controls were used as competitor DNA in the polymerase chain reaction for the quantitative detection of mycoplasma DNA. PCR primer sets were designed on the basis of the most conserved nucleotide sequences of the 16S rRNA gene of mycoplasma species. Amplification of this gene was examined in five different mycoplasma species: Mycoplasma orale, M. hyorhinus, M. synoviae, M. gallisepticum and M. pneumoniae. To evaluate the primers, a number of different cell lines were assayed for the detection of mycoplasma infections. All positive cell lines showed a distinct product on agarose gels while uninfected cells showed no DNA amplification. Neither bacterial nor eukaryotic DNA produced any cross-reaction with the primers used, thus confirming their specificity. Internal control DNA to be used for quantitation was constructed by modifying the sizes of the wild-type amplified products and cloning them in plasmid vectors. These controls used the same primer binding sites as the wild-type and the amplified products were differentiated by a size difference. The detection limits for all the mycoplasma species by competitive quantitative PCR were estimated to range from 4 to 60 genome copies per assay as determined by ethidium bromide-stained agarose gels. These internal standards also serve as positive controls in PCR-based detection of mycoplasma DNA, and therefore accidental contamination of test samples with wild-type positive controls can be eliminated. The quantitative PCR method developed will be useful in monitoring the progression and significance of mycoplasma in the disease process.

PCR detection and quantitation of predominant anaerobic bacteria in human and animal fecal samples.

PubMed Central

Wang, R F; Cao, W W; Cerniglia, C E

1996-01-01

PCR procedures based on 16S rRNA gene sequences specific for 12 anaerobic bacteria that predominate in the human intestinal tract were developed and used for quantitative detection of these species in human (adult and baby) feces and animal (rat, mouse, cat, dog, monkey, and rabbit) feces. Fusobacterium prausnitzii, Peptostreptococcus productus, and Clostridium clostridiiforme had high PCR titers (the maximum dilutions for positive PCR results ranged from 10(-3) to 10(-8)) in all of the human and animal fecal samples tested. Bacteroides thetaiotaomicron, Bacteroides vulgatus, and Eubacterium limosum also showed higher PCR titers (10(-2) to 10(-6)) in adult human feces. The other bacteria tested, including Escherichia coli, Bifidobacterium adolescentis, Bifidobacterium longum, Lactobacillus acidophilus, Eubacterium biforme, and Bacteroides distasonis, were either at low PCR titers (less than 10(-2)) or not detected by PCR. The reported PCR procedure including the fecal sample preparation method is simplified and rapid and eliminates the DNA isolation steps. PMID:8919784

Use of real-time qPCR to quantify members of the unculturable heterotrophic bacterial community in a deep sea marine sponge, Vetulina sp.

PubMed

Cassler, M; Peterson, C L; Ledger, A; Pomponi, S A; Wright, A E; Winegar, R; McCarthy, P J; Lopez, J V

2008-04-01

In this report, real-time quantitative PCR (TaqMan qPCR) of the small subunit (SSU) 16S-like rRNA molecule, a universal phylogenetic marker, was used to quantify the relative abundance of individual bacterial members of a diverse, yet mostly unculturable, microbial community from a marine sponge. Molecular phylogenetic analyses of bacterial communities derived from Caribbean Lithistid sponges have shown a wide diversity of microbes that included at least six major subdivisions; however, very little overlap was observed between the culturable and unculturable microbial communities. Based on sequence data of three culture-independent Lithistid-derived representative bacteria, we designed probe/primer sets for TaqMan qPCR to quantitatively characterize selected microbial residents in a Lithistid sponge, Vetulina, metagenome. TaqMan assays included specificity testing, DNA limit of detection analysis, and quantification of specific microbial rRNA sequences such as Nitrospira-like microbes and Actinobacteria up to 172 million copies per microgram per Lithistid sponge metagenome. By contrast, qPCR amplification with probes designed for common previously cultured sponge-associated bacteria in the genera Rheinheimera and Marinomonas and a representative of the CFB group resulted in only minimal detection of the Rheiheimera in total DNA extracted from the sponge. These data verify that a large portion of the microbial community within Lithistid sponges may consist of currently unculturable microorganisms.

Evaluation of nearest-neighbor methods for detection of chimeric small-subunit rRNA sequences

NASA Technical Reports Server (NTRS)

Robison-Cox, J. F.; Bateson, M. M.; Ward, D. M.

1995-01-01

Detection of chimeric artifacts formed when PCR is used to retrieve naturally occurring small-subunit (SSU) rRNA sequences may rely on demonstrating that different sequence domains have different phylogenetic affiliations. We evaluated the CHECK_CHIMERA method of the Ribosomal Database Project and another method which we developed, both based on determining nearest neighbors of different sequence domains, for their ability to discern artificially generated SSU rRNA chimeras from authentic Ribosomal Database Project sequences. The reliability of both methods decreases when the parental sequences which contribute to chimera formation are more than 82 to 84% similar. Detection is also complicated by the occurrence of authentic SSU rRNA sequences that behave like chimeras. We developed a naive statistical test based on CHECK_CHIMERA output and used it to evaluate previously reported SSU rRNA chimeras. Application of this test also suggests that chimeras might be formed by retrieving SSU rRNAs as cDNA. The amount of uncertainty associated with nearest-neighbor analyses indicates that such tests alone are insufficient and that better methods are needed.

Determination of Bifidobacterium and Lactobacillus in breast milk of healthy women by digital PCR.

PubMed

Qian, L; Song, H; Cai, W

2016-09-01

Breast milk is one of the most important sources of postnatal microbes. Quantitative real-time polymerase chain reaction (qRT-PCR) is currently used for the quantitative analysis of bacterial 16S rRNA genes in breast milk. However, this method relies on the use of standard curves and is imprecise when quantitating target DNA of low abundance. In contrast, droplet digital PCR (DD-PCR) provides an absolute quantitation without the need for calibration curves. A comparison between DD-PCR and qRT-PCR was conducted for the quantitation of Bifidobacterium and Lactobacillus 16S RNA genes in human breast milk, and the impacts of selected maternal factors were studied on the composition of these two bacteria in breast milk. From this study, DD-PCR reported between 0-34,460 16S rRNA gene copies of Bifidobacterium genera and between 1,108-634,000 16S rRNA gene copies of Lactobacillus genera in 1 ml breast milk. The 16S rRNA gene copy number of Lactobacillus genera was much greater than that of Bifidobacterium genera in breast milk. DD-PCR showed a 10-fold lower limit of quantitation as compared to qRT-PCR. A higher correlation and agreement was observed between qRT-PCR and DD-PCR in Lactobacillus quantitation as compared to Bifidobacterium quantitation. Based on our DD-PCR quantitation, a low abundance of Bifidobacterium bacteria in breast milk was correlated to higher pre-pregnancy body mass index (BMI). However, no significant difference was observed for these two bacteria in breast milk between mothers who had vaginal deliveries and caesarean deliveries. This study suggests that DD-PCR is a better tool to quantitate the bacterial load of breast milk compared to the conventional qRT-PCR method. The number of breast milk Bifidobacterium bacteria is influenced by maternal pre-pregnancy BMI.

The Applicability of TaqMan-Based Quantitative Real-Time PCR Assays for Detecting and Enumerating Cryptosporidium spp. Oocysts in the Environment

PubMed Central

Staggs, Sarah E.; Beckman, Erin M.; Keely, Scott P.; Mackwan, Reena; Ware, Michael W.; Moyer, Alan P.; Ferretti, James A.; Sayed, Abu; Xiao, Lihua; Villegas, Eric N.

2013-01-01

Quantitative real-time polymerase chain reaction (qPCR) assays to detect Cryptosporidium oocysts in clinical samples are increasingly being used to diagnose human cryptosporidiosis, but a parallel approach for detecting and identifying Cryptosporidium oocyst contamination in surface water sources has yet to be established for current drinking water quality monitoring practices. It has been proposed that Cryptosporidium qPCR-based assays could be used as viable alternatives to current microscopic-based detection methods to quantify levels of oocysts in drinking water sources; however, data on specificity, analytical sensitivity, and the ability to accurately quantify low levels of oocysts are limited. The purpose of this study was to provide a comprehensive evaluation of TaqMan-based qPCR assays, which were developed for either clinical or environmental investigations, for detecting Cryptosporidium oocyst contamination in water. Ten different qPCR assays, six previously published and four developed in this study were analyzed for specificity and analytical sensitivity. Specificity varied between all ten assays, and in one particular assay, which targeted the Cryptosporidium 18S rRNA gene, successfully detected all Cryptosporidium spp. tested, but also cross-amplified T. gondii, fungi, algae, and dinoflagellates. When evaluating the analytical sensitivity of these qPCR assays, results showed that eight of the assays could reliably detect ten flow-sorted oocysts in reagent water or environmental matrix. This study revealed that while a qPCR-based detection assay can be useful for detecting and differentiating different Cryptosporidium species in environmental samples, it cannot accurately measure low levels of oocysts that are typically found in drinking water sources. PMID:23805235

A role for Dehalobacter spp. in the reductive dehalogenation of dichlorobenzenes and monochlorobenzene.

PubMed

Nelson, Jennifer L; Fung, Jennifer M; Cadillo-Quiroz, Hinsby; Cheng, Xu; Zinder, Stephen H

2011-08-15

Previously, we demonstrated the reductive dehalogenation of dichlorobenzene (DCB) isomers to monochlorobenzene (MCB), and MCB to benzene in sediment microcosms derived from a chlorobenzene-contaminated site. In this study, enrichment cultures were established for each DCB isomer and each produced MCB and trace amounts of benzene as end products. MCB dehalogenation activity could only be transferred in sediment microcosms. The 1,2-DCB-dehalogenating culture was studied the most intensively. Whereas Dehalococcoides spp. were not detected in any of the microcosms or cultures, Dehalobacter spp. were detected in 16S rRNA gene clone libraries from 1,2-DCB enrichment cultures, and by PCR using Dehalobacter-specific primers in 1,3-DCB and 1,4-DCB enrichments and MCB-dehalogenating microcosms. Quantitative PCR showed Dehalobacter 16S rRNA gene copies increased up to 3 orders of magnitude upon dehalogenation of DCBs or MCB, and that nearly all of bacterial 16S rRNA genes in a 1,2-DCB-dehalogenating culture belonged to Dehalobacter spp. Dehalobacter 16S rRNA genes from DCB enrichment cultures and MCB-dehalogenating microcosms showed considerable diversity, implying that 16S rRNA sequences do not predict dehalogenation-spectra of Dehalobacter spp. These studies support a role for Dehalobacter spp. in the reductive dehalogenation of DCBs and MCB, and this genus should be considered for its potential impact on chlorobenzene fate at contaminated sites.

DETECTION OF STACHYBOTRYS CHARTARUM USING rRNA, tri5, AND Β-TUBULIN PRIMERS AND DETERMINING THEIR RELATIVE COPY NUMBER BY REAL TIME PCR

EPA Science Inventory

This research utilizes the quantitative polymerase chain reaction (qPCR) to determine ribosomal copy number of fungal organisms found in unhealthy indoor environments. Knowing specific copy numbers will allow for greater accuracy in quantification when utilizing current pQCR tec...

Evaluating the reproducibility of quantifying modified nucleosides from ribonucleic acids by LC–UV–MS

PubMed Central

Russell, Susan P.; Limbach, Patrick A.

2013-01-01

Post-transcriptional chemical covalent modification of adenosine, guanosine, uridine and cytidine occurs frequently in all types of ribonucleic acids (RNAs). In ribosomal RNA (rRNA) and transfer RNA (tRNA) these modifications make important contributions to RNA structure and stability and to the accuracy and efficiency of protein translation. The functional dynamics, synergistic nature and regulatory roles of these posttranscriptional nucleoside modifications within the cell are not well characterized. These modifications are present at very low levels and isolation of individual nucleosides for analysis requires a complex multi-step approach. The focus of this study is to characterize the reproducibility of a liquid chromatography method used to isolate and quantitatively characterize modified nucleosides in tRNA and rRNA when nucleoside detection is performed using ultraviolet and mass spectrometric detection (UV and MS, respectively). Despite the analytical challenges of sample isolation and dynamic range, quantitative profiling of modified nucleosides obtained from bacterial tRNAs and rRNAs is feasible at relative standard deviations of 5% RSD or less. PMID:23500350

Dramatic Differences in Gut Bacterial Densities Correlate with Diet and Habitat in Rainforest Ants.

PubMed

Sanders, Jon G; Lukasik, Piotr; Frederickson, Megan E; Russell, Jacob A; Koga, Ryuichi; Knight, Rob; Pierce, Naomi E

2017-10-01

Abundance is a key parameter in microbial ecology, and important to estimates of potential metabolite flux, impacts of dispersal, and sensitivity of samples to technical biases such as laboratory contamination. However, modern amplicon-based sequencing techniques by themselves typically provide no information about the absolute abundance of microbes. Here, we use fluorescence microscopy and quantitative polymerase chain reaction as independent estimates of microbial abundance to test the hypothesis that microbial symbionts have enabled ants to dominate tropical rainforest canopies by facilitating herbivorous diets, and compare these methods to microbial diversity profiles from 16S rRNA amplicon sequencing. Through a systematic survey of ants from a lowland tropical forest, we show that the density of gut microbiota varies across several orders of magnitude among ant lineages, with median individuals from many genera only marginally above detection limits. Supporting the hypothesis that microbial symbiosis is important to dominance in the canopy, we find that the abundance of gut bacteria is positively correlated with stable isotope proxies of herbivory among canopy-dwelling ants, but not among ground-dwelling ants. Notably, these broad findings are much more evident in the quantitative data than in the 16S rRNA sequencing data. Our results provide quantitative context to the potential role of bacteria in facilitating the ants' dominance of the tropical rainforest canopy, and have broad implications for the interpretation of sequence-based surveys of microbial diversity. © The Author 2017. Published by Oxford University Press on behalf of the Society for Integrative and Comparative Biology. All rights reserved. For permissions please email: journals.permissions@oup.com.

Phylogenetic and gene expression analysis of cyanobacteria and diatoms in the twilight waters of the temperate northeast Pacific Ocean.

PubMed

Gao, Weimin; Shi, Xu; Wu, Jieying; Jin, Yuguang; Zhang, Weiwen; Meldrum, Deirdre R

2011-11-01

In this study, to explore the microbial community structure and its functionality in the deep-sea environments, we initially performed a 16S ribosomal RNA (rRNA)-based community structure analyses for microbial communities in the sea water collected from sites of 765-790 m in depth in the Pacific Ocean. Interestingly, in the clone library we detected the presence of both photoautotrophic bacteria such as cyanobacteria and photoheterotrophic bacteria, such as Chloroflexus sp. To further explore the existence and diversity of possible light-utilizing microorganisms, we then constructed and analyzed a 23S rRNA plastid gene cloning library. The results showed that the majority of this cloning library was occupied by oxygenic photoautotrophic organisms, such as diatoms Thalassiosira spp. and cyanobacterium Synechococcus sp. In addition, the diversity of these oxygenic photoautotrophic organisms was very limited. Moreover, both reverse-transcription PCR and quantitative reverse-transcription PCR approaches had been employed to detect expression of the genes involved in protein synthesis and photosynthesis of photoautotrophic organisms, and the positive results were obtained. The possible mechanisms underlying the existence of very limited diversity of photosynthetic organisms at this depth of ocean, as well as the positive detection of rRNA and mRNA of diatom and cyanobacteria, were discussed.

Single Cell Analysis Linking Ribosomal (r)DNA and rRNA Copy Numbers to Cell Size and Growth Rate Provides Insights into Molecular Protistan Ecology.

PubMed

Fu, Rao; Gong, Jun

2017-11-01

Ribosomal (r)RNA and rDNA have been golden molecular markers in microbial ecology. However, it remains poorly understood how ribotype copy number (CN)-based characteristics are linked with diversity, abundance, and activity of protist populations and communities observed at organismal levels. Here, we applied a single-cell approach to quantify ribotype CNs in two ciliate species reared at different temperatures. We found that in actively growing cells, the per-cell rDNA and rRNA CNs scaled with cell volume (CV) to 0.44 and 0.58 powers, respectively. The modeled rDNA and rRNA concentrations thus appear to be much higher in smaller than in larger cells. The observed rRNA:rDNA ratio scaled with CV 0.14 . The maximum growth rate could be well predicted by a combination of per-cell ribotype CN and temperature. Our empirical data and modeling on single-cell ribotype scaling are in agreement with both the metabolic theory of ecology and the growth rate hypothesis, providing a quantitative framework for linking cellular rDNA and rRNA CNs with body size, growth (activity), and biomass stoichiometry. This study also demonstrates that the expression rate of rRNA genes is constrained by cell size, and favors biomass rather than abundance-based interpretation of quantitative ribotype data in population and community ecology of protists. © 2017 The Authors. Journal of Eukaryotic Microbiology published by Wiley Periodicals, Inc. on behalf of International Society of Protistologists.

Archaea in the foregut of macropod marsupials: PCR and amplicon sequence-based observations.

PubMed

Klieve, A V; Ouwerkerk, D; Maguire, A J

2012-11-01

To investigate, using culture-independent techniques, the presence and diversity of methanogenic archaea in the foregut of kangaroos. DNA was extracted from forestomach contents of 42 kangaroos (three species), three sheep and three cattle. Four qualitative and quantitative PCR assays targeting the archaeal domain (16S rRNA gene) or the functional methanogenesis gene, mcrA, were used to determine the presence and population density of archaea in kangaroos and whether they were likely to be methanogens. All ruminal samples were positive for archaea, produced PCR product of expected size, contained high numbers of archaea and high numbers of cells with mcrA genes. Kangaroos were much more diverse and contradictory. Fourteen kangaroos had detectable archaea with numbers 10- to 1000-fold fewer than sheep and cattle. Many kangaroos that did not possess archaea were positive for the mcrA gene and had detectable numbers of cells with this gene and vice versa. DNA sequence analysis of kangaroos' archaeal 16S rRNA gene clones show that many methanogens were related to Methanosphaera stadmanae. Other sequences were related to non-methanogenic archaea (Thermoplasma sp.), and a number of kangaroos had mcrA gene sequences related to methane oxidising archaea (ANME). Discrepancies between qualitative and quantitative PCR assays for archaea and the mcrA gene suggest that the archaeal communities are very diverse and it is possible that novel species exist. Archaea (in general) were below detectable limits in many kangaroos, especially Red kangaroos; when present they are in lower numbers than in ruminants, and the archaea are not necessarily methanogenic. The determination of why this is the case in the kangaroo foregut could assist in reducing emissions from other ecosystems in the future. © 2012 The Authors Journal of Applied Microbiology © 2012 The Society for Applied Microbiology.

Sensitive detection of porcine DNA in processed animal proteins using a TaqMan real-time PCR assay.

PubMed

Pegels, N; González, I; Fernández, S; García, T; Martín, R

2012-01-01

A TaqMan real-time PCR method was developed for specific detection of porcine-prohibited material in industrial feeds. The assay combines the use of a porcine-specific primer pair, which amplifies a 79 bp fragment of the mitochondrial (mt) 12 S rRNA gene, and a locked nucleic acid (LNA) TaqMan probe complementary to a target sequence lying between the porcine-specific primers. The nuclear 18 S rRNA gene system, yielding a 77 bp amplicon, was employed as a positive amplification control to monitor the total content of amplifiable DNA in the samples. The specificity of the porcine primers-probe system was verified against different animal and plant species, including mammals, birds and fish. The applicability of the real-time PCR protocol to detect the presence of porcine mt DNA in feeds was determined through the analysis of 190 industrial feeds (19 known reference and 171 blind samples) subjected to stringent processing treatments. The performance of the method allows qualitative and highly sensitive detection of short fragments from porcine DNA in all the industrial feeds declared to contain porcine material. Although the method has quantitative potential, the real quantitative capability of the assay is limited by the existing variability in terms of composition and processing conditions of the feeds, which affect the amount and quality of amplifiable DNA.

Evaluation of new gyrB-based real-time PCR system for the detection of B. fragilis as an indicator of human-specific fecal contamination.

PubMed

Lee, Chang Soo; Lee, Jiyoung

2010-09-01

A rapid and specific gyrB-based real-time PCR system has been developed for detecting Bacteroides fragilis as a human-specific marker of fecal contamination. Its specificity and sensitivity was evaluated by comparison with other 16S rRNA gene-based primers using closely related Bacteroides and Prevotella. Many studies have used 16S rRNA gene-based method targeting Bacteroides because this genus is relatively abundant in human feces and is useful for microbial source tracking. However, 16S rRNA gene-based primers are evolutionarily too conserved among taxa to discriminate between human-specific species of Bacteroides and other closely related genera, such as Prevotella. Recently, one of the housekeeping genes, gyrB, has been used as an alternative target in multilocus sequence analysis (MLSA) to provide greater phylogenetic resolution. In this study, a new B. fragilis-specific primer set (Bf904F/Bf958R) was designed by alignments of 322 gyrB genes and was compared with the performance of the 16S rRNA gene-based primers in the presence of B. fragilis, Bacteroides ovatus and Prevotella melaninogenica. Amplicons were sequenced and a phylogenetic tree was constructed to confirm the specificity of the primers to B. fragilis. The gyrB-based primers successfully discriminated B. fragilis from B. ovatus and P. melaninogenica. Real-time PCR results showed that the gyrB primer set had a comparable sensitivity in the detection of B. fragilis when compared with the 16S rRNA primer set. The host-specificity of our gyrB-based primer set was validated with human, pig, cow, and dog fecal samples. The gyrB primer system had superior human-specificity. The gyrB-based system can rapidly detect human-specific fecal source and can be used for improved source tracking of human contamination. (c) 2010 Elsevier B.V. All rights reserved.

Sequence variation identified in the 18S rRNA gene of Theileria mutans and Theileria velifera from the African buffalo (Syncerus caffer).

PubMed

Chaisi, Mamohale E; Collins, Nicola E; Potgieter, Fred T; Oosthuizen, Marinda C

2013-01-16

The African buffalo (Syncerus caffer) is a natural reservoir host for both pathogenic and non-pathogenic Theileria species. These often occur naturally as mixed infections in buffalo. Although the benign and mildly pathogenic forms do not have any significant economic importance, their presence could complicate the interpretation of diagnostic test results aimed at the specific diagnosis of the pathogenic Theileria parva in cattle and buffalo in South Africa. The 18S rRNA gene has been used as the target in a quantitative real-time PCR (qPCR) assay for the detection of T. parva infections. However, the extent of sequence variation within this gene in the non-pathogenic Theileria spp. of the Africa buffalo is not well known. The aim of this study was, therefore, to characterise the full-length 18S rRNA genes of Theileria mutans, Theileria sp. (strain MSD) and T. velifera and to determine the possible influence of any sequence variation on the specific detection of T. parva using the 18S rRNA qPCR. The reverse line blot (RLB) hybridization assay was used to select samples which either tested positive for several different Theileria spp., or which hybridised only with the Babesia/Theileria genus-specific probe and not with any of the Babesia or Theileria species-specific probes. The full-length 18S rRNA genes from 14 samples, originating from 13 buffalo and one bovine from different localities in South Africa, were amplified, cloned and the resulting recombinants sequenced. Variations in the 18S rRNA gene sequences were identified in T. mutans, Theileria sp. (strain MSD) and T. velifera, with the greatest diversity observed amongst the T. mutans variants. This variation possibly explained why the RLB hybridization assay failed to detect T. mutans and T. velifera in some of the analysed samples. Copyright © 2012 Elsevier B.V. All rights reserved.

Analysis of ribosomal RNA stability in dead cells of wine yeast by quantitative PCR.

PubMed

Sunyer-Figueres, Merce; Wang, Chunxiao; Mas, Albert

2018-04-02

During wine production, some yeasts enter a Viable But Not Culturable (VBNC) state, which may influence the quality and stability of the final wine through remnant metabolic activity or by resuscitation. Culture-independent techniques are used for obtaining an accurate estimation of the number of live cells, and quantitative PCR could be the most accurate technique. As a marker of cell viability, rRNA was evaluated by analyzing its stability in dead cells. The species-specific stability of rRNA was tested in Saccharomyces cerevisiae, as well as in three species of non-Saccharomyces yeast (Hanseniaspora uvarum, Torulaspora delbrueckii and Starmerella bacillaris). High temperature and antimicrobial dimethyl dicarbonate (DMDC) treatments were efficient in lysing the yeast cells. rRNA gene and rRNA (as cDNA) were analyzed over 48 h after cell lysis by quantitative PCR. The results confirmed the stability of rRNA for 48 h after the cell lysis treatments. To sum up, rRNA may not be a good marker of cell viability in the wine yeasts that were tested. Copyright © 2018 Elsevier B.V. All rights reserved.

Rapid Quantitative Detection of Lactobacillus sakei in Meat and Fermented Sausages by Real-Time PCR

PubMed Central

Martín, Belén; Jofré, Anna; Garriga, Margarita; Pla, Maria; Aymerich, Teresa

2006-01-01

A quick and simple method for quantitative detection of Lactobacillus sakei in fermented sausages was successfully developed. It is based on Chelex-100-based DNA purification and real-time PCR enumeration using a TaqMan fluorescence probe. Primers and probes were designed in the L. sakei 16S-23S rRNA intergenic transcribed spacer region, and the assay was evaluated using L. sakei genomic DNA and an artificially inoculated sausage model. The detection limit of this technique was approximately 3 cells per reaction mixture using both purified DNA and the inoculated sausage model. The quantification limit was established at 30 cells per reaction mixture in both models. The assay was then applied to enumerate L. sakei in real samples, and the results were compared to the MRS agar count method followed by confirmation of the percentage of L. sakei colonies. The results obtained by real-time PCR were not statistically significantly different than those obtained by plate count on MRS agar (P > 0.05), showing a satisfactory agreement between both methods. Therefore, the real-time PCR assay developed can be considered a promising rapid alternative method for the quantification of L. sakei and evaluation of the implantation of starter strains of L. sakei in fermented sausages. PMID:16957227

Rapid quantitative detection of Lactobacillus sakei in meat and fermented sausages by real-time PCR.

PubMed

Martín, Belén; Jofré, Anna; Garriga, Margarita; Pla, Maria; Aymerich, Teresa

2006-09-01

A quick and simple method for quantitative detection of Lactobacillus sakei in fermented sausages was successfully developed. It is based on Chelex-100-based DNA purification and real-time PCR enumeration using a TaqMan fluorescence probe. Primers and probes were designed in the L. sakei 16S-23S rRNA intergenic transcribed spacer region, and the assay was evaluated using L. sakei genomic DNA and an artificially inoculated sausage model. The detection limit of this technique was approximately 3 cells per reaction mixture using both purified DNA and the inoculated sausage model. The quantification limit was established at 30 cells per reaction mixture in both models. The assay was then applied to enumerate L. sakei in real samples, and the results were compared to the MRS agar count method followed by confirmation of the percentage of L. sakei colonies. The results obtained by real-time PCR were not statistically significantly different than those obtained by plate count on MRS agar (P > 0.05), showing a satisfactory agreement between both methods. Therefore, the real-time PCR assay developed can be considered a promising rapid alternative method for the quantification of L. sakei and evaluation of the implantation of starter strains of L. sakei in fermented sausages.

Evaluation of genetic markers from the 16S rRNA gene V2 region for use in quantitative detection of selected Bacteroidales species and human fecal waste by real time PCR

EPA Science Inventory

Molecular methods for rapidly quantifying defined Bacteroidales species from the human gastrointestinal tract may have important clinical and environmental applications, ranging from diagnosis of infections to fecal source tracking in surface waters. In this study, sequences from...

Abundance and diversity of microbial inhabitants in European spacecraft-associated clean rooms.

PubMed

Stieglmeier, Michaela; Rettberg, Petra; Barczyk, Simon; Bohmeier, Maria; Pukall, Rüdiger; Wirth, Reinhard; Moissl-Eichinger, Christine

2012-06-01

The determination of the microbial load of a spacecraft en route to interesting extraterrestrial environments is mandatory and currently based on the culturable, heat-shock-surviving portion of microbial contaminants. Our study compared these classical bioburden measurements as required by NASA's and ESA's guidelines for the microbial examination of flight hardware, with molecular analysis methods (16S rRNA gene cloning and quantitative PCR) to further develop our understanding of the diversity and abundance of the microbial communities of spacecraft-associated clean rooms. Three samplings of the Herschel Space Observatory and its surrounding clean rooms were performed in two different European facilities. Molecular analyses detected a broad diversity of microbes typically found in the human microbiome with three bacterial genera (Staphylococcus, Propionibacterium, and Brevundimonas) common to all three locations. Bioburden measurements revealed a low, but heterogeneous, abundance of spore-forming and other heat-resistant microorganisms. Total cell numbers estimated by quantitative real-time PCR were typically 3 orders of magnitude greater than those determined by viable counts, which indicates a tendency for traditional methods to underestimate the extent of clean room bioburden. Furthermore, the molecular methods allowed the detection of a much broader diversity than traditional culture-based methods.

Characterization and Comparison of Galactomannan Enzyme Immunoassay and Quantitative Real-Time PCR Assay for Detection of Aspergillus fumigatus in Bronchoalveolar Lavage Fluid from Experimental Invasive Pulmonary Aspergillosis

PubMed Central

Francesconi, Andrea; Kasai, Miki; Petraitiene, Ruta; Petraitis, Vidmantas; Kelaher, Amy M.; Schaufele, Robert; Hope, William W.; Shea, Yvonne R.; Bacher, John; Walsh, Thomas J.

2006-01-01

Bronchoalveolar lavage (BAL) is widely used for evaluation of patients with suspected invasive pulmonary aspergillosis (IPA). However, the diagnostic yield of BAL for detection of IPA by culture and direct examination is limited. Earlier diagnosis may be facilitated by assays that can detect Aspergillus galactomannan antigen or DNA in BAL fluid. We therefore characterized and compared the diagnostic yields of a galactomannan enzyme immunoassay (GM EIA), quantitative real-time PCR (qPCR), and quantitative cultures in experiments using BAL fluid from neutropenic rabbits with experimentally induced IPA defined as microbiologically and histologically evident invasion. The qPCR assay targeted the rRNA gene complex of Aspergillus fumigatus. The GM EIA and qPCR assay were characterized by receiver operator curve analysis. With an optimal cutoff of 0.75, the GM EIA had a sensitivity and specificity of 100% in untreated controls. A decline in sensitivity (92%) was observed when antifungal therapy (AFT) was administered. The optimal cutoff for qPCR was a crossover of 36 cycles, with sensitivity and specificity of 80% and 100%, respectively. The sensitivity of qPCR also decreased with AFT to 50%. Quantitative culture of BAL had a sensitivity of 46% and a specificity of 100%. The sensitivity of quantitative culture decreased with AFT to 16%. The GM EIA and qPCR assay had greater sensitivity than culture in detection of A. fumigatus in BAL fluid in experimentally induced IPA (P ± 0.04). Use of the GM EIA and qPCR assay in conjunction with culture-based diagnostic methods applied to BAL fluid could facilitate accurate diagnosis and more-timely initiation of specific therapy. PMID:16825367

Distribution and diversity of Verrucomicrobia methanotrophs in geothermal and acidic environments.

PubMed

Sharp, Christine E; Smirnova, Angela V; Graham, Jaime M; Stott, Matthew B; Khadka, Roshan; Moore, Tim R; Grasby, Stephen E; Strack, Maria; Dunfield, Peter F

2014-06-01

Recently, methanotrophic members of the phylum Verrucomicrobia have been described, but little is known about their distribution in nature. We surveyed methanotrophic bacteria in geothermal springs and acidic wetlands via pyrosequencing of 16S rRNA gene amplicons. Putative methanotrophic Verrucomicrobia were found in samples covering a broad temperature range (22.5-81.6°C), but only in acidic conditions (pH 1.8-5.0) and only in geothermal environments, not in acidic bogs or fens. Phylogenetically, three 16S rRNA gene sequence clusters of putative methanotrophic Verrucomicrobia were observed. Those detected in high-temperature geothermal samples (44.1-81.6°C) grouped with known thermoacidiphilic 'Methylacidiphilum' isolates. A second group dominated in moderate-temperature geothermal samples (22.5-40.1°C) and a representative mesophilic methanotroph from this group was isolated (strain LP2A). Genome sequencing verified that strain LP2A possessed particulate methane monooxygenase, but its 16S rRNA gene sequence identity to 'Methylacidiphilum infernorum' strain V4 was only 90.6%. A third group clustered distantly with known methanotrophic Verrucomicrobia. Using pmoA-gene targeted quantitative polymerase chain reaction, two geothermal soil profiles showed a dominance of LP2A-like pmoA sequences in the cooler surface layers and 'Methylacidiphilum'-like pmoA sequences in deeper, hotter layers. Based on these results, there appears to be a thermophilic group and a mesophilic group of methanotrophic Verrucomicrobia. However, both were detected only in acidic geothermal environments. © 2014 Society for Applied Microbiology and John Wiley & Sons Ltd.

Quantification of Yeast and Bacterial Gene Transcripts in Retail Cheeses by Reverse Transcription-Quantitative PCR

PubMed Central

Straub, Cécile; Castellote, Jessie; Onesime, Djamila; Bonnarme, Pascal; Irlinger, Françoise

2013-01-01

The cheese microbiota contributes to a large extent to the development of the typical color, flavor, and texture of the final product. Its composition is not well defined in most cases and varies from one cheese to another. The aim of the present study was to establish procedures for gene transcript quantification in cheeses by reverse transcription-quantitative PCR. Total RNA was extracted from five smear-ripened cheeses purchased on the retail market, using a method that does not involve prior separation of microbial cells. 16S rRNA and malate:quinone oxidoreductase gene transcripts of Corynebacterium casei, Brevibacterium aurantiacum, and Arthrobacter arilaitensis and 26S rRNA and beta tubulin gene transcripts of Geotrichum candidum and Debaryomyces hansenii could be detected and quantified in most of the samples. Three types of normalization were applied: against total RNA, against the amount of cheese, and against a reference gene. For the first two types of normalization, differences of reverse transcription efficiencies from one sample to another were taken into account by analysis of exogenous control mRNA. No good correlation was found between the abundances of target mRNA or rRNA transcripts and the viable cell concentration of the corresponding species. However, in most cases, no mRNA transcripts were detected for species that did not belong to the dominant species. The applications of gene expression measurement in cheeses containing an undefined microbiota, as well as issues concerning the strategy of normalization and the assessment of amplification specificity, are discussed. PMID:23124230

Evaluation of Faecalibacterium 16S rDNA genetic markers for accurate identification of swine faecal waste by quantitative PCR.

PubMed

Duan, Chuanren; Cui, Yamin; Zhao, Yi; Zhai, Jun; Zhang, Baoyun; Zhang, Kun; Sun, Da; Chen, Hang

2016-10-01

A genetic marker within the 16S rRNA gene of Faecalibacterium was identified for use in a quantitative PCR (qPCR) assay to detect swine faecal contamination in water. A total of 146,038 bacterial sequences were obtained using 454 pyrosequencing. By comparative bioinformatics analysis of Faecalibacterium sequences with those of numerous swine and other animal species, swine-specific Faecalibacterium 16S rRNA gene sequences were identified and Polymerase Chain Okabe (PCR) primer sets designed and tested against faecal DNA samples from swine and non-swine sources. Two PCR primer sets, PFB-1 and PFB-2, showed the highest specificity to swine faecal waste and had no cross-reaction with other animal samples. PFB-1 and PFB-2 amplified 16S rRNA gene sequences from 50 samples of swine with positive ratios of 86 and 90%, respectively. We compared swine-specific Faecalibacterium qPCR assays for the purpose of quantifying the newly identified markers. The quantification limits (LOQs) of PFB-1 and PFB-2 markers in environmental water were 6.5 and 2.9 copies per 100 ml, respectively. Of the swine-associated assays tested, PFB-2 was more sensitive in detecting the swine faecal waste and quantifying the microbial load. Furthermore, the microbial abundance and diversity of the microbiomes of swine and other animal faeces were estimated using operational taxonomic units (OTUs). The species specificity was demonstrated for the microbial populations present in various animal faeces. Copyright © 2016 Elsevier Ltd. All rights reserved.

Nonenzymatic microorganism identification based on ribosomal RNA

NASA Astrophysics Data System (ADS)

Ives, Jeffrey T.; Pierini, Alicia M.; Stokes, Jeffrey A.; Wahlund, Thomas M.; Read, Betsy; Bechtel, James H.; Bronk, Burt V.

1999-11-01

Effective defense against biological warfare (BW) agents requires rapid, fieldable and accurate systems. For micro- organisms like bacteria and viruses, ribosomal RNA (rRNA) provides a valuable target with multiple advantages of species specificity and intrinsic target amplification. Vegetative and spore forms of bacteria contain approximately 104 copies of rRNA. Direct detection of rRNA copies can eliminate some of the interference and preparation difficulties involved in enzymatic amplification methods. In order to apply the advantages of rRNA to BW defense, we are developing a fieldable system based on 16S rRNA, physical disruption of the micro-organism, solid phase hybridization, and fluorescence detection. Our goals include species-specific identification, complete operation from raw sample to identification in 15 minutes or less, and compact, fieldable instrumentation. Initial work on this project has investigated the lysis and hybridization steps, the species-specificity of oligonucleotides probes, and the development of a novel electromagnetic method to physically disrupt the micro- organisms. Target bacteria have been Escherichia coli (E. coli) and Bacillus subtilis (B. subtilis). Continuing work includes further development of methods to rapidly disrupt the micro-organisms and release the rRNA, improved integration and processing, and extension to bacterial and mammalian viruses like MS2 and vesicular stomatitis virus.

Analysis of Pteridium ribosomal RNA sequences by rapid direct sequencing.

PubMed

Tan, M K

1991-08-01

A total of 864 bases from 5 regions interspersed in the 18S and 26S rRNA molecules from various clones of Pteridium covering the general geographical distribution of the genus was analysed using a rapid rRNA sequencing technique. No base difference has been detected amongst the three major lineages, two of which apparently separated before the breakup of the ancient supercontinent, Pangaea. These regions of the rRNA sequences have thus been conserved for at least 160 million years and are here compared with other eukaryotic, especially plant rRNAs.

Detection of Haemophilus influenzae in respiratory secretions from pneumonia patients by quantitative real-time polymerase chain reaction.

PubMed

Abdeldaim, Guma M K; Strålin, Kristoffer; Kirsebom, Leif A; Olcén, Per; Blomberg, Jonas; Herrmann, Björn

2009-08-01

A quantitative real-time polymerase chain reaction (PCR) based on the omp P6 gene was developed to detect Haemophilus influenzae. Its specificity was determined by analysis of 29 strains of 11 different Haemophilus spp. and was compared with PCR assays having other target genes: rnpB, 16S rRNA, and bexA. The method was evaluated on nasopharyngeal aspirates from 166 adult patients with community-acquired pneumonia. When 10(4) DNA copies/mL was used as cutoff limit for the method, P6 PCR had a sensitivity of 97.5% and a specificity of 96.0% compared with the culture. Of 20 culture-negative but P6 PCR-positive cases, 18 were confirmed by fucK PCR as H. influenzae. Five (5.9%) of 84 nasopharyngeal aspirates from adult controls tested PCR positive. We conclude that the P6 real-time PCR is both sensitive and specific for identification of H. influenzae in respiratory secretions. Quantification facilitates discrimination between disease-causing H. influenzae strains and commensal colonization.

Creation of a data base for sequences of ribosomal nucleic acids and detection of conserved restriction endonucleases sites through computerized processing.

PubMed Central

Patarca, R; Dorta, B; Ramirez, J L

1982-01-01

As part of a project pertaining the organization of ribosomal genes in Kinetoplastidae, we have created a data base for published sequences of ribosomal nucleic acids, with information in Spanish. As a first step in their processing, we have written a computer program which introduces the new feature of determining the length of the fragments produced after single or multiple digestion with any of the known restriction enzymes. With this information we have detected conserved SAU 3A sites: (i) at the 5' end of the 5.8S rRNA and at the 3' end of the small subunit rRNA, both included in similar larger sequences; (ii) in the 5.8S rRNA of vertebrates (a second one), which is not present in lower eukaryotes, showing a clear evolutive divergence; and, (iii) at the 5' terminal of the small subunit rRNA, included in a larger conserved sequence. The possible biological importance of these sequences is discussed. PMID:6278402

Panel of 23S rRNA Gene-Based Real-Time PCR Assays for Improved Universal and Group-Specific Detection of Phytoplasmas▿ †

PubMed Central

Hodgetts, Jennifer; Boonham, Neil; Mumford, Rick; Dickinson, Matthew

2009-01-01

Primers and probes based on the 23S rRNA gene have been utilized to design a range of real-time PCR assays for routine phytoplasma diagnostics. These assays have been authenticated as phytoplasma specific and shown to be at least as sensitive as nested PCR. A universal assay to detect all phytoplasmas has been developed, along with a multiplex assay to discriminate 16SrI group phytoplasmas from members of all of the other 16Sr groups. Assays for the 16SrII, 16SrIV, and 16SrXII groups have also been developed to confirm that the 23S rRNA gene can be used to design group-specific assays. PMID:19270148

A Complementary Isothermal Amplification Method to the U.S. EPA Quantitative Polymerase Chain Reaction Approach for the Detection of Enterococci in Environmental Waters

PubMed Central

2017-01-01

We report a novel molecular assay, based on helicase-dependent amplification (HDA), for the detection of enterococci as markers for fecal pollution in water. This isothermal assay targets the same Enterococcus 23S rRNA gene region as the existing quantitative polymerase chain reaction (qPCR) assays of U.S. Environmental Protection Agency Methods 1611 and 1609 but can be entirely performed on a simple heating block. The developed Enterococcus HDA assay successfully discriminated 15 enterococcal from 15 non-enterococcal reference strains and reliably detected 48 environmental isolates of enterococci. The limit of detection was 25 target copies per reaction, only 3 times higher than that of qPCR. The applicability of the assay was tested on 30 environmental water sample DNA extracts, simulating a gradient of fecal pollution. Despite the isothermal nature of the reaction, the HDA results were consistent with those of the qPCR reference. Given this performance, we conclude that the developed Enterococcus HDA assay has great potential as a qualitative molecular screening method for resource-limited settings when combined with compatible up- and downstream processes. This amplification strategy can pave the way for developing a new generation of rapid, low-cost, and field-deployable molecular diagnostic tools for water quality monitoring. PMID:28541661

Microbial ecology, bacterial pathogens, and antibiotic resistant genes in swine manure wastewater as influenced by three swine management systems.

PubMed

Brooks, John P; Adeli, Ardeshir; McLaughlin, Michael R

2014-06-15

The environmental influence of farm management in concentrated animal feeding operations (CAFO) can yield vast changes to the microbial biota and ecological structure of both the pig and waste manure lagoon wastewater. While some of these changes may not be negative, it is possible that CAFOs can enrich antibiotic resistant bacteria or pathogens based on farm type, thereby influencing the impact imparted by the land application of its respective wastewater. The purpose of this study was to measure the microbial constituents of swine-sow, -nursery, and -finisher farm manure lagoon wastewater and determine the changes induced by farm management. A total of 37 farms were visited in the Mid-South USA and analyzed for the genes 16S rRNA, spaQ (Salmonella spp.), Camp-16S (Campylobacter spp.), tetA, tetB, ermF, ermA, mecA, and intI using quantitative PCR. Additionally, 16S rRNA sequence libraries were created. Overall, it appeared that finisher farms were significantly different from nursery and sow farms in nearly all genes measured and in 16S rRNA clone libraries. Nearly all antibiotic resistance genes were detected in all farms. Interestingly, the mecA resistance gene (e.g. methicillin resistant Staphylococcus aureus) was below detection limits on most farms, and decreased as the pigs aged. Finisher farms generally had fewer antibiotic resistance genes, which corroborated previous phenotypic data; additionally, finisher farms produced a less diverse 16S rRNA sequence library. Comparisons of Camp-16S and spaQ GU (genomic unit) values to previous culture data demonstrated ratios from 10 to 10,000:1 depending on farm type, indicating viable but not cultivatable bacteria were dominant. The current study indicated that swine farm management schemes positively and negatively affect microbial and antibiotic resistant populations in CAFO wastewater which has future "downstream" implications from both an environmental and public health perspective. Published by Elsevier Ltd.

Ultra-Sensitive Detection of Plasmodium falciparum by Amplification of Multi-Copy Subtelomeric Targets

PubMed Central

Hofmann, Natalie; Mwingira, Felista; Shekalaghe, Seif; Robinson, Leanne J.; Mueller, Ivo; Felger, Ingrid

2015-01-01

Background Planning and evaluating malaria control strategies relies on accurate definition of parasite prevalence in the population. A large proportion of asymptomatic parasite infections can only be identified by surveillance with molecular methods, yet these infections also contribute to onward transmission to mosquitoes. The sensitivity of molecular detection by PCR is limited by the abundance of the target sequence in a DNA sample; thus, detection becomes imperfect at low densities. We aimed to increase PCR diagnostic sensitivity by targeting multi-copy genomic sequences for reliable detection of low-density infections, and investigated the impact of these PCR assays on community prevalence data. Methods and Findings Two quantitative PCR (qPCR) assays were developed for ultra-sensitive detection of Plasmodium falciparum, targeting the high-copy telomere-associated repetitive element 2 (TARE-2, ∼250 copies/genome) and the var gene acidic terminal sequence (varATS, 59 copies/genome). Our assays reached a limit of detection of 0.03 to 0.15 parasites/μl blood and were 10× more sensitive than standard 18S rRNA qPCR. In a population cross-sectional study in Tanzania, 295/498 samples tested positive using ultra-sensitive assays. Light microscopy missed 169 infections (57%). 18S rRNA qPCR failed to identify 48 infections (16%), of which 40% carried gametocytes detected by pfs25 quantitative reverse-transcription PCR. To judge the suitability of the TARE-2 and varATS assays for high-throughput screens, their performance was tested on sample pools. Both ultra-sensitive assays correctly detected all pools containing one low-density P. falciparum–positive sample, which went undetected by 18S rRNA qPCR, among nine negatives. TARE-2 and varATS qPCRs improve estimates of prevalence rates, yet other infections might still remain undetected when absent in the limited blood volume sampled. Conclusions Measured malaria prevalence in communities is largely determined by the sensitivity of the diagnostic tool used. Even when applying standard molecular diagnostics, prevalence in our study population was underestimated by 8% compared to the new assays. Our findings highlight the need for highly sensitive tools such as TARE-2 and varATS qPCR in community surveillance and for monitoring interventions to better describe malaria epidemiology and inform malaria elimination efforts. PMID:25734259

Development and application of two independent real-time PCR assays to detect clinically relevant Mucorales species.

PubMed

Springer, Jan; Goldenberger, Daniel; Schmidt, Friderike; Weisser, Maja; Wehrle-Wieland, Elisabeth; Einsele, Hermann; Frei, Reno; Löffler, Jürgen

2016-03-01

PCR-based detection of Mucorales species could improve diagnosis of suspected invasive fungal infection, leading to a better patient outcome. This study describes two independent probe-based real-time PCR tests for detection of clinically relevant Mucorales, targeting specific fragments of the 18S and the 28S rRNA genes. Both assays have a short turnaround time, allow fast, specific and very sensitive detection of clinically relevant Mucorales and have the potential to be used as quantitative tests. They were validated on various clinical samples (fresh and formalin-fixed paraffin-embedded specimens, mainly biopsies, n = 17). The assays should be used as add-on tools to complement standard techniques; a combined approach of both real-time PCR assays has 100 % sensitivity. Genus identification by subsequent sequencing is possible for amplicons of the 18S PCR assay. In conclusion, combination of the two independent Mucorales assays described in this study, 18S and 28S, detected all clinical samples associated with proven Mucorales infection (n = 10). Reliable and specific identification of Mucorales is a prerequisite for successful antifungal therapy as these fungi show intrinsic resistance to voriconazole and caspofungin.

Genetic speciation of environmental Legionella isolates in Thailand.

PubMed

Paveenkittiporn, Wantana; Dejsirilert, Surang; Kalambaheti, Thareerat

2012-10-01

Legionella-like organisms were isolated during 2003-2007 from various water resources by culturing on selective media of Wadowsky-Yee-Okuda agar. The 256 isolates were identified as belonging to the Legionella genus based on detection of 108 bp PCR product of the 5S rRNA gene, while the inclusion as Legionella pneumophila were confirmed by PCR detection of a specific mip gene region of 168 bp. The 50 isolates, identified as non-pneumophila, were then subjected to DNA tree analysis, based on mip gene of ~650 bp and rnpB genes product ranged from 304 to 354 bp. Phylogenetic tree was constructed to predict their species in relative to the available database. The isolates of which their speciation, based on those two genes were inconclusive, were then investigated for the almost full-length of 16S rRNA sequences. The isolates were assigned as 16 known Legionella species, and proposed seven novel species based on their unique 16S rRNA sequence. Copyright © 2012 Elsevier B.V. All rights reserved.

Bacterial and archaeal symbionts in the South China Sea sponge Phakellia fusca: community structure, relative abundance, and ammonia-oxidizing populations.

PubMed

Han, Minqi; Liu, Fang; Zhang, Fengli; Li, Zhiyong; Lin, Houwen

2012-12-01

Many biologically active natural products have been isolated from Phakellia fusca, an indigenous sponge in the South China Sea; however, the microbial symbionts of Phakellia fusca remain unknown. The present investigations on sponge microbial community are mainly based on qualitative analysis, while quantitative analysis, e.g., relative abundance, is rarely carried out, and little is known about the roles of microbial symbionts. In this study, the community structure and relative abundance of bacteria, actinobacteria, and archaea associated with Phakellia fusca were revealed by 16S rRNA gene library-based sequencing and quantitative real time PCR (qRT-PCR). The ammonia-oxidizing populations were investigated based on amoA gene and anammox-specific 16S rRNA gene libraries. As a result, it was found that bacterial symbionts of sponge Phakellia fusca consist of Proteobacteria including Gamma-, Alpha-, and Delta-proteobacteria, Cyanobacteria with Gamma-proteobacteria as the predominant components. In particular, the diversity of actinobacterial symbionts in Phakellia fusca is high, which is composed of Corynebacterineae, Acidimicrobidae, Frankineae, Micrococcineae, and Streptosporangineae. All the observed archaea in sponge Phakellia fusca belong to Crenarchaeota, and the detected ammonia-oxidizing populations are ammonia-oxidizing archaea, suggesting the nitrification function of sponge archaeal symbionts. According to qRT-PCR analysis, bacterial symbionts dominated the microbial community, while archaea represented the second predominant symbionts, followed by actinobacteria. The revealed diverse prokaryotic symbionts of Phakellia fusca are valuable for the understanding and in-depth utilization of Phakellia fusca microbial symbionts. This study extends our knowledge of the community, especially the relative abundance of microbial symbionts in sponges.

Occurrence and prevalence of antibiotic resistance in landfill leachate.

PubMed

Wang, Yangqing; Tang, Wei; Qiao, Jing; Song, Liyan

2015-08-01

Antibiotic resistance (AR) is extensively present in various environments, posing emerging threat to public and environmental health. Landfill receives unused and unwanted antibiotics through household waste and AR within waste (e.g., activated sludge and illegal clinical waste) and is supposed to serve as an important AR reservoir. In this study, we used culture-dependent methods and quantitative molecular techniques to detect and quantify antibiotic-resistant bacteria (ARB) and antibiotic resistance genes (ARGs) in 12 landfill leachate samples from six geographic different landfills, China. Five tested ARGs (tetO, tetW, bla(TEM), sulI, and sulII) and seven kinds of antibiotic-resistant heterotrophic ARB were extensively detected in all samples, demonstrating their occurrence in landfill. The detected high ratio (10(-2) to 10(-5)) of ARGs to 16S ribosomal RNA (rRNA) gene copies implied that ARGs are prevalent in landfill. Correlation analysis showed that ARGs (tetO, tetW, sulI, and sulII) significantly correlated to ambient bacterial 16S rRNA gene copies, suggesting that the abundance of bacteria in landfill leachate may play an important role in the horizontal spread of ARGs.

Viability-qPCR for detecting Legionella: Comparison of two assays based on different amplicon lengths.

PubMed

Ditommaso, Savina; Giacomuzzi, Monica; Ricciardi, Elisa; Zotti, Carla M

2015-08-01

Two different real-time quantitative PCR (PMA-qPCR) assays were applied for quantification of Legionella spp. by targeting a long amplicon (approx 400 bp) of 16S rRNA gene and a short amplicon (approx. 100 bp) of 5S rRNA gene. Purified DNA extracts from pure cultures of Legionella spp. and from environmental water samples were quantified. Application of the two assays to quantify Legionella in artificially contaminated water achieved that both assays were able to detect Legionella over a linear range of 10 to 10(5) cells ml(-1). A statistical analysis of the standard curves showed that both assays were linear with a good correlation coefficient (R(2) = 0.99) between the Ct and the copy number. Amplification with the reference assay was the most effective for detecting low copy numbers (1 bacterium per PCR mixture). Using selective quantification of viable Legionella by the PMA-qPCR method we obtained a greater inhibition of the amplification of the 400-bp 16S gene fragment (Δlog(10) = 3.74 ± 0.39 log(10) GU ml(-1)). A complete inhibition of the PCR signal was obtained when heat-killed cells in a concentration below 1 × 10(5) cells ml(-1) were pretreated with PMA. Analysing short amplicon sizes led to only 2.08 log reductions in the Legionella dead-cell signal. When we tested environmental water samples, the two qPCR assays were in good agreement according to the kappa index (0.741). Applying qPCR combined with PMA treatment, we also obtained a good agreement (kappa index 0.615). The comparison of quantitative results shows that both assays yielded the same quantification sensitivity (mean log = 4.59 vs mean log = 4.31). Copyright © 2015 Elsevier Ltd. All rights reserved.

Quantitative real-time reverse transcription polymerase chain reaction: normalization to rRNA or single housekeeping genes is inappropriate for human tissue biopsies.

PubMed

Tricarico, Carmela; Pinzani, Pamela; Bianchi, Simonetta; Paglierani, Milena; Distante, Vito; Pazzagli, Mario; Bustin, Stephen A; Orlando, Claudio

2002-10-15

Careful normalization is essential when using quantitative reverse transcription polymerase chain reaction assays to compare mRNA levels between biopsies from different individuals or cells undergoing different treatment. Generally this involves the use of internal controls, such as mRNA specified by a housekeeping gene, ribosomal RNA (rRNA), or accurately quantitated total RNA. The aim of this study was to compare these methods and determine which one can provide the most accurate and biologically relevant quantitative results. Our results show significant variation in the expression levels of 10 commonly used housekeeping genes and 18S rRNA, both between individuals and between biopsies taken from the same patient. Furthermore, in 23 breast cancers samples mRNA and protein levels of a regulated gene, vascular endothelial growth factor (VEGF), correlated only when normalized to total RNA, as did microvessel density. Finally, mRNA levels of VEGF and the most popular housekeeping gene, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), were significantly correlated in the colon. Our results suggest that the use of internal standards comprising single housekeeping genes or rRNA is inappropriate for studies involving tissue biopsies.

Molecular diversity of the rumen microbiome of Norwegian reindeer on natural summer pasture.

PubMed

Sundset, Monica A; Edwards, Joan E; Cheng, Yan Fen; Senosiain, Roberto S; Fraile, Maria N; Northwood, Korinne S; Praesteng, Kirsti E; Glad, Trine; Mathiesen, Svein D; Wright, André-Denis G

2009-02-01

The molecular diversity of the rumen microbiome was investigated in five semi-domesticated adult female Norwegian reindeer (Rangifer tarandus tarandus) grazing on natural summer pastures on the coast of northern Norway (71.00 degrees N, 25.30 degrees E). Mean population densities (numbers per gram wet weight) of methanogenic archaea, rumen bacteria and ciliate protozoa, estimated using quantitative real-time polymerase chain reaction (PCR), were 3.17x10(9), 5.17x10(11) and 4.02x10(7), respectively. Molecular diversity of rumen methanogens was revealed using a 16S rRNA gene library (54 clones) constructed using pooled PCR products from the whole rumen contents of the five individual reindeer. Based upon a similarity criterion of <97%, a total of 19 distinct operational taxonomic units (OTUs) were identified, nine of which are potential new species. The 16S rRNA sequences generated from the reindeer rumen exhibited a high degree of sequence similarity to methanogens affiliated with the families Methanobacteriaceae (14 OTUs) and Methanosarcinaceae (one OTU). Four of the OTUs detected belonged to a group of uncultivated archaea previously found in domestic ruminants and thought to be dominant in the rumen together with Methanobrevibacter spp. Denaturing gradient gel electrophoresis profiling of the rumen bacterial 16S rRNA gene and the protozoal 18S rRNA gene indicated a high degree of animal variation, although some bands were common to all individuals. Automated ribosomal intergenic spacer analysis (ARISA) profiling of the ruminal Neocallimastigales population indicated that the reindeer are likely to contain more than one type of anaerobic fungus. The ARISA profile from one animal was distinct from the other four. This is the first molecular investigation of the ruminal methanogenic archaea in reindeer, revealing higher numbers than expected based on methane emission data available. Also, many of the reindeer archaeal 16S rRNA gene sequences were similar to those reported in domesticated ruminants in Australia, Canada, China, New Zealand and Venezuela, supporting previous findings that there seems to be no host type or geographical effect on the methanogenic archaea community structure in ruminants.

New Insights into the RNA-Based Mechanism of Action of the Anticancer Drug 5′-Fluorouracil in Eukaryotic Cells

PubMed Central

Mojardín, Laura; Botet, Javier; Quintales, Luis; Moreno, Sergio; Salas, Margarita

2013-01-01

5-Fluorouracil (5FU) is a chemotherapeutic drug widely used in treating a range of advanced, solid tumours and, in particular, colorectal cancer. Here, we used high-density tiling DNA microarray technology to obtain the specific transcriptome-wide response induced by 5FU in the eukaryotic model Schizosaccharomyces pombe. This approach combined with real-time quantitative PCR analysis allowed us to detect splicing defects of a significant number of intron-containing mRNA, in addition to identify some rRNA and tRNA processing defects after 5FU treatment. Interestingly, our studies also revealed that 5FU specifically induced the expression of certain genes implicated in the processing of mRNA, tRNA and rRNA precursors, and in the post-transcriptional modification of uracil residues in RNA. The transcription of several tRNA genes was also significantly induced after drug exposure. These transcriptional changes might represent a cellular response mechanism to counteract 5FU damage since deletion strains for some of these up-regulated genes were hypersensitive to 5FU. Moreover, most of these RNA processing genes have human orthologs that participate in conserved pathways, suggesting that they could be novel targets to improve the efficacy of 5FU-based treatments. PMID:24223771

Next-Generation Sequencing Combined with Specific PCR Assays To Determine the Bacterial 16S rRNA Gene Profiles of Middle Ear Fluid Collected from Children with Acute Otitis Media

PubMed Central

Kramna, Lenka; Oikarinen, Sami; Sipilä, Markku; Rautiainen, Markus; Aittoniemi, Janne; Laranne, Jussi; Hyöty, Heikki; Cinek, Ondrej

2017-01-01

ABSTRACT The aim of the study was to analyze the bacteriome of acute otitis media with a novel modification of next-generation sequencing techniques. Outpatient children with acute otitis media were enrolled in the study, and middle ear fluids were collected during 90 episodes from 79 subjects aged 5 to 42 months (median age, 19 months). The bacteriome profiles of middle ear fluid samples were determined by a nested-PCR amplification of the 16S rRNA gene (V4 region), followed by mass sequencing. The profiling results were compared to the results of specific PCR assays targeting selected prevalent pathogens. Bacteriome profiling using nested amplification of low-volume samples was aided by a bioinformatic subtraction of signal contaminants from the recombinant polymerase, achieving a sensitivity slightly lower than that of specific PCR detection. Streptococcus pneumoniae was detected in 28 (31%) samples, Haemophilus influenzae in 24 (27%), Moraxella catarrhalis in 18 (20%), Staphylococcus spp. in 21 (23%), Turicella otitidis in 5 (5.6%), Alloiococcus otitidis in 3 (3.3%), and other bacteria in 14 (16%) using bacteriome profiling. S. pneumoniae was the dominant pathogen in 14 (16%) samples, H. influenzae in 15 (17%), M. catarrhalis in 5 (5.6%), T. otitidis in 2, and Staphylococcus auricularis in 2. Weaker signals of Prevotella melaninogenica, Veillonella dispar, and Veillonella montpellierensis were noted in several samples. Fourteen samples (16%) were not explainable by bacterial pathogens; novel causative agents were not detected. In conclusion, unbiased bacteriome profiling helped in depicting the true mutual quantitative ratios of ear bacteria, but at present, its complicated protocol impedes its routine clinical use. IMPORTANCE Although S. pneumoniae, H. influenzae, and M. catarrhalis have been long established as the most important pathogens in acute otitis media using culture and specific PCR assays, the knowledge of their mutual quantitative relations and possible roles of other bacteria is incomplete. The advent of unbiased bacteriome 16S rRNA gene profiling has allowed the detection of nearly all bacteria present in the sample, and it helps in depicting their mutual quantitative ratios. Due to the difficulties in performing mass sequencing in low-volume samples, only a few bacteriome-profiling studies of otitis media have been published, all limited to cases of chronic otitis media. Here, we present a study on samples obtained from young children with acute otitis media, successfully using a strategy of nested PCR coupled with mass sequencing, and demonstrate that the method can confer quantitative information hardly obtainable by other methods. PMID:28357413

Nested PCR and RFLP analysis based on the 16S rRNA gene

USDA-ARS?s Scientific Manuscript database

Current phytoplasma detection and identification method is primarily based on nested PCR followed by restriction fragment length polymorphism analysis and gel electrophoresis. This method can potentially detect and differentiate all phytoplasmas including those previously not described. The present ...

Evaluating the Detection of Hydrocarbon-Degrading Bacteria in 16S rRNA Gene Sequencing Surveys

PubMed Central

Berry, David; Gutierrez, Tony

2017-01-01

Hydrocarbonoclastic bacteria (HCB) play a key role in the biodegradation of oil hydrocarbons in marine and other environments. A small number of taxa have been identified as obligate HCB, notably the Gammaproteobacterial genera Alcanivorax, Cycloclasticus, Marinobacter, Neptumonas, Oleiphilus, Oleispira, and Thalassolituus, as well as the Alphaproteobacterial genus Thalassospira. Detection of HCB in amplicon-based sequencing surveys relies on high coverage by PCR primers and accurate taxonomic classification. In this study, we performed a phylogenetic analysis to identify 16S rRNA gene sequence regions that represent the breadth of sequence diversity within these taxa. Using validated sequences, we evaluated 449 universal 16S rRNA gene-targeted bacterial PCR primer pairs for their coverage of these taxa. The results of this analysis provide a practical framework for selection of suitable primer sets for optimal detection of HCB in sequencing surveys. PMID:28567035

Evaluating the Detection of Hydrocarbon-Degrading Bacteria in 16S rRNA Gene Sequencing Surveys.

PubMed

Berry, David; Gutierrez, Tony

2017-01-01

Hydrocarbonoclastic bacteria (HCB) play a key role in the biodegradation of oil hydrocarbons in marine and other environments. A small number of taxa have been identified as obligate HCB, notably the Gammaproteobacterial genera Alcanivorax, Cycloclasticus, Marinobacter, Neptumonas, Oleiphilus, Oleispira , and Thalassolituus , as well as the Alphaproteobacterial genus Thalassospira . Detection of HCB in amplicon-based sequencing surveys relies on high coverage by PCR primers and accurate taxonomic classification. In this study, we performed a phylogenetic analysis to identify 16S rRNA gene sequence regions that represent the breadth of sequence diversity within these taxa. Using validated sequences, we evaluated 449 universal 16S rRNA gene-targeted bacterial PCR primer pairs for their coverage of these taxa. The results of this analysis provide a practical framework for selection of suitable primer sets for optimal detection of HCB in sequencing surveys.

Microbial identification by immunohybridization assay of artificial RNA labels

NASA Technical Reports Server (NTRS)

Kourentzi, Katerina D.; Fox, George E.; Willson, Richard C.

2002-01-01

Ribosomal RNA (rRNA) and engineered stable artificial RNAs (aRNAs) are frequently used to monitor bacteria in complex ecosystems. In this work, we describe a solid-phase immunocapture hybridization assay that can be used with low molecular weight RNA targets. A biotinylated DNA probe is efficiently hybridized in solution with the target RNA, and the DNA-RNA hybrids are captured on streptavidin-coated plates and quantified using a DNA-RNA heteroduplex-specific antibody conjugated to alkaline phosphatase. The assay was shown to be specific for both 5S rRNA and low molecular weight (LMW) artificial RNAs and highly sensitive, allowing detection of as little as 5.2 ng (0.15 pmol) in the case of 5S rRNA. Target RNAs were readily detected even in the presence of excess nontarget RNA. Detection using DNA probes as small as 17 bases targeting a repetitive artificial RNA sequence in an engineered RNA was more efficient than the detection of a unique sequence.

Detection of Anaplasma marginale and A. phagocytophilum in Bovine Peripheral Blood Samples by Duplex Real-Time Reverse Transcriptase PCR Assay ▿

PubMed Central

Reinbold, James B.; Coetzee, Johann F.; Sirigireddy, Kamesh R.; Ganta, Roman R.

2010-01-01

Insufficient diagnostic sensitivity and specificity coupled with the potential for cross-reactivity among closely related Anaplasma species has made the accurate determination of infection status problematic. A method for the development of simplex and duplex real-time quantitative reverse transcriptase PCR (qRT-PCR) assays for the detection of A. marginale and A. phagocytophilum 16S rRNA in plasma-free bovine peripheral blood samples is described. The duplex assay was able to detect as few as 100 copies of 16S rRNA of both A. marginale and A. phagocytophilum in the same reaction. The ratio of 16S rRNA to 16S DNA copies for A. marginale was determined to be 117.9:1 (95% confidence interval [95% CI], 100.7:1, 135.2:1). Therefore, the detection limit is the minimum infective unit of one A. marginale bacterium. The duplex assay detected nonequivalent molar ratios as high as 100-fold. Additionally, the duplex assay and a competitive enzyme-linked immunosorbent assay (cELISA) were used to screen 237 samples collected from herds in which anaplasmosis was endemic. When the cELISA was evaluated by the results of the qRT-PCR, its sensitivity and specificity for the detection of A. marginale infection were found to be 65.2% (95% CI, 55.3%, 75.1%) and 97.3% (95% CI, 94.7%, 99.9%), respectively. A. phagocytophilum infection was not detected in the samples analyzed. One- and two-way receiver operator characteristic curves were constructed in order to recommend the optimum negative cutoff value for the cELISA. Percentages of inhibition of 20 and 15.3% were recommended for the one- and two-way curves, respectively. In conclusion, the duplex real-time qRT-PCR assay is a highly sensitive and specific diagnostic tool for the accurate and precise detection of A. marginale and A. phagocytophilum infections in cattle. PMID:20463162

Review: Diagnostic accuracy of PCR-based detection tests for Helicobacter Pylori in stool samples.

PubMed

Khadangi, Fatemeh; Yassi, Maryam; Kerachian, Mohammad Amin

2017-12-01

Although different methods have been established to detect Helicobacter pylori (H. pylori) infection, identifying infected patients is an ongoing challenge. The aim of this meta-analysis was to provide pooled diagnostic accuracy measures for stool PCR test in the diagnosis of H. pylori infection. In this study, a systematic review and meta-analysis were carried out on various sources, including MEDLINE, Web of Sciences, and the Cochrane Library from April 1, 1999, to May 1, 2016. This meta-analysis adheres to the guidelines provided by the Preferred Reporting Items for Systematic Reviews and Meta-Analyses report (PRISMA Statement). The clinical value of DNA stool PCR test was based on the pooled false positive, false negative, true positive, and true negative of different genes. Twenty-six of 328 studies identified met the eligibility criteria. Stool PCR test had a performance of 71% (95% CI: 68-73) sensitivity, 96% (95% CI: 94-97) specificity, and 65.6 (95% CI: 30.2-142.5) diagnostic odds ratio (DOR) in diagnosis of H. pylori. The DOR of genes which showed the highest performance of stool PCR tests was as follows: 23S rRNA 152.5 (95% CI: 55.5-418.9), 16S rRNA 67.9 (95%CI: 6.4-714.3), and glmM 68.1 (95%CI: 20.1-231.7). The sensitivity and specificity of stool PCR test are relatively in the same spectrum of other diagnostic methods for the detection of H. pylori infection. In descending order of significance, the most diagnostic candidate genes using PCR detection were 23S rRNA, 16S rRNA, and glmM. PCR for 23S rRNA gene which has the highest performance could be applicable to detect H. pylori infection. © 2017 John Wiley & Sons Ltd.

Application of Stochastic Labeling with Random-Sequence Barcodes for Simultaneous Quantification and Sequencing of Environmental 16S rRNA Genes.

PubMed

Hoshino, Tatsuhiko; Inagaki, Fumio

2017-01-01

Next-generation sequencing (NGS) is a powerful tool for analyzing environmental DNA and provides the comprehensive molecular view of microbial communities. For obtaining the copy number of particular sequences in the NGS library, however, additional quantitative analysis as quantitative PCR (qPCR) or digital PCR (dPCR) is required. Furthermore, number of sequences in a sequence library does not always reflect the original copy number of a target gene because of biases caused by PCR amplification, making it difficult to convert the proportion of particular sequences in the NGS library to the copy number using the mass of input DNA. To address this issue, we applied stochastic labeling approach with random-tag sequences and developed a NGS-based quantification protocol, which enables simultaneous sequencing and quantification of the targeted DNA. This quantitative sequencing (qSeq) is initiated from single-primer extension (SPE) using a primer with random tag adjacent to the 5' end of target-specific sequence. During SPE, each DNA molecule is stochastically labeled with the random tag. Subsequently, first-round PCR is conducted, specifically targeting the SPE product, followed by second-round PCR to index for NGS. The number of random tags is only determined during the SPE step and is therefore not affected by the two rounds of PCR that may introduce amplification biases. In the case of 16S rRNA genes, after NGS sequencing and taxonomic classification, the absolute number of target phylotypes 16S rRNA gene can be estimated by Poisson statistics by counting random tags incorporated at the end of sequence. To test the feasibility of this approach, the 16S rRNA gene of Sulfolobus tokodaii was subjected to qSeq, which resulted in accurate quantification of 5.0 × 103 to 5.0 × 104 copies of the 16S rRNA gene. Furthermore, qSeq was applied to mock microbial communities and environmental samples, and the results were comparable to those obtained using digital PCR and relative abundance based on a standard sequence library. We demonstrated that the qSeq protocol proposed here is advantageous for providing less-biased absolute copy numbers of each target DNA with NGS sequencing at one time. By this new experiment scheme in microbial ecology, microbial community compositions can be explored in more quantitative manner, thus expanding our knowledge of microbial ecosystems in natural environments.

Bacterial loads of Ureaplasma parvum contribute to the development of inflammatory responses in the male urethra.

PubMed

Deguchi, Takashi; Shimada, Yasushi; Horie, Kengo; Mizutani, Kohsuke; Seike, Kensaku; Tsuchiya, Tomohiro; Yokoi, Shigeaki; Yasuda, Mitsuru; Ito, Shin

2015-12-01

Ureaplasma parvum, which has been recognised as a coloniser in the male urethra, is detected in some men with non-gonococcal urethritis. In this study, we quantified the 16 S rRNA genes of U. parvum by a real-time polymerase chain reaction-based assay in first-voided urine from 15 symptomatic and 38 asymptomatic men who were positive only for U. parvum. We also determined the leukocyte counts by automated quantitative urine particle analysis in their first-voided urine. Positive correlations were observed between copies of the 16 S rRNA genes of U. parvum/ml and the leukocyte counts/µl in first-voided urine (p = 0.0019). The loads of ≥10(4) copies of the 16 S rRNA gene/ml, corresponding to ≥5 × 10(3) cells of U. parvum/ml, were significantly associated with the presence of ≥12.5 leukocytes/µl in first-voided urine that might document the presence of inflammatory responses in the urethra. However, a large portion of the subjects (83.0%) had bacterial loads of <5 × 10(3) cells of U. parvum/ml, and 79.5% of them showed <12.5 leukocytes/µl. The ambiguity of the pathogenic role of U. parvum in non-gonococcal urethritis could, in part, be due to its low bacterial loads, which might not give rise to inflammatory responses in the male urethra. © The Author(s) 2015.

Enzymic colorimetry-based DNA chip: a rapid and accurate assay for detecting mutations for clarithromycin resistance in the 23S rRNA gene of Helicobacter pylori.

PubMed

Xuan, Shi-Hai; Zhou, Yu-Gui; Shao, Bo; Cui, Ya-Lin; Li, Jian; Yin, Hong-Bo; Song, Xiao-Ping; Cong, Hui; Jing, Feng-Xiang; Jin, Qing-Hui; Wang, Hui-Min; Zhou, Jie

2009-11-01

Macrolide drugs, such as clarithromycin (CAM), are a key component of many combination therapies used to eradicate Helicobacter pylori. However, resistance to CAM is increasing in H. pylori and is becoming a serious problem in H. pylori eradication therapy. CAM resistance in H. pylori is mostly due to point mutations (A2142G/C, A2143G) in the peptidyltransferase-encoding region of the 23S rRNA gene. In this study an enzymic colorimetry-based DNA chip was developed to analyse single-nucleotide polymorphisms of the 23S rRNA gene to determine the prevalence of mutations in CAM-related resistance in H. pylori-positive patients. The results of the colorimetric DNA chip were confirmed by direct DNA sequencing. In 63 samples, the incidence of the A2143G mutation was 17.46 % (11/63). The results of the colorimetric DNA chip were concordant with DNA sequencing in 96.83 % of results (61/63). The colorimetric DNA chip could detect wild-type and mutant signals at every site, even at a DNA concentration of 1.53 x 10(2) copies microl(-1). Thus, the colorimetric DNA chip is a reliable assay for rapid and accurate detection of mutations in the 23S rRNA gene of H. pylori that lead to CAM-related resistance, directly from gastric tissues.

Multiplex quantification of 16S rDNA of predominant bacteria group within human fecal samples by polymerase chain reaction--ligase detection reaction (PCR-LDR).

PubMed

Li, Kai; Chen, Bei; Zhou, Yuxun; Huang, Rui; Liang, Yinming; Wang, Qinxi; Xiao, Zhenxian; Xiao, Junhua

2009-03-01

A new method, based on ligase detection reaction (LDR), was developed for quantitative detection of multiplex PCR amplicons of 16S rRNA genes present in complex mixtures (specifically feces). LDR has been widely used in single nucleotide polymorphism (SNP) assay but never applied for quantification of multiplex PCR products. This method employs one pair of DNA probes, one of which is labeled with fluorescence for signal capture, complementary to the target sequence. For multiple target sequence analysis, probes were modified with different lengths of polyT at the 5' end and 3' end. Using a DNA sequencer, these ligated probes were separated and identified by size and dye color. Then, relative abundance of target DNA were normalized and quantified based on the fluorescence intensities and exterior size standards. 16S rRNA gene of three preponderant bacteria groups in human feces: Clostridium coccoides, Bacteroides and related genera, and Clostridium leptum group, were amplified and cloned into plasmid DNA so as to make standard curves. After PCR-LDR analysis, a strong linear relationship was found between the florescence intensity and the diluted plasmid DNA concentrations. Furthermore, based on this method, 100 human fecal samples were quantified for the relative abundance of the three bacterial groups. Relative abundance of C. coccoides was significantly higher in elderly people in comparison with young adults, without gender differences. Relative abundance of Bacteroides and related genera and C. leptum group were significantly higher in young and middle aged than in the elderly. Regarding the whole set of sample, C. coccoides showed the highest relative abundance, followed by decreasing groups Bacteroides and related genera, and C. leptum. These results imply that PCR-LDR can be feasible and flexible applied to large scale epidemiological studies.

Spatio-Temporal Detection of the Thiomonas Population and the Thiomonas Arsenite Oxidase Involved in Natural Arsenite Attenuation Processes in the Carnoulès Acid Mine Drainage

PubMed Central

Hovasse, Agnès; Bruneel, Odile; Casiot, Corinne; Desoeuvre, Angélique; Farasin, Julien; Hery, Marina; Van Dorsselaer, Alain; Carapito, Christine; Arsène-Ploetze, Florence

2016-01-01

The acid mine drainage (AMD) impacted creek of the Carnoulès mine (Southern France) is characterized by acid waters with a high heavy metal content. The microbial community inhabiting this AMD was extensively studied using isolation, metagenomic and metaproteomic methods, and the results showed that a natural arsenic (and iron) attenuation process involving the arsenite oxidase activity of several Thiomonas strains occurs at this site. A sensitive quantitative Selected Reaction Monitoring (SRM)-based proteomic approach was developed for detecting and quantifying the two subunits of the arsenite oxidase and RpoA of two different Thiomonas groups. Using this approach combined with FISH and pyrosequencing-based 16S rRNA gene sequence analysis, it was established here for the first time that these Thiomonas strains are ubiquitously present in minor proportions in this AMD and that they express the key enzymes involved in natural remediation processes at various locations and time points. In addition to these findings, this study also confirms that targeted proteomics applied at the community level can be used to detect weakly abundant proteins in situ. PMID:26870729

Metagenomics uncovers gaps in amplicon-based detection of microbial diversity

DOE PAGES

Eloe-Fadrosh, Emiley A.; Ivanova, Natalia N.; Woyke, Tanja; ...

2016-02-01

Our view of microbial diversity has expanded greatly over the past 40 years, primarily through the wide application of PCR-based surveys of the small-subunit ribosomal RNA (SSU rRNA) gene. Yet significant gaps in knowledge remain due to well-recognized limitations of this method. Here in this paper, we systematically survey primer fidelity in SSU rRNA gene sequences recovered from over 6,000 assembled metagenomes sampled globally. Our findings show that approximately 10% of environmental microbial sequences might be missed from classical PCR-based SSU rRNA gene surveys, mostly members of the Candidate Phyla Radiation (CPR) and as yet uncharacterized Archaea. In conclusion, thesemore » results underscore the extent of uncharacterized microbial diversity and provide fruitful avenues for describing additional phylogenetic lineages.« less

Molecular method for detection of total coliforms in drinking water samples.

PubMed

Maheux, Andrée F; Boudreau, Dominique K; Bisson, Marc-Antoine; Dion-Dupont, Vanessa; Bouchard, Sébastien; Nkuranga, Martine; Bergeron, Michel G; Rodriguez, Manuel J

2014-07-01

This work demonstrates the ability of a bacterial concentration and recovery procedure combined with three different PCR assays targeting the lacZ, wecG, and 16S rRNA genes, respectively, to detect the presence of total coliforms in 100-ml samples of potable water (presence/absence test). PCR assays were first compared to the culture-based Colilert and MI agar methods to determine their ability to detect 147 coliform strains representing 76 species of Enterobacteriaceae encountered in fecal and environmental settings. Results showed that 86 (58.5%) and 109 (74.1%) strains yielded a positive signal with Colilert and MI agar methods, respectively, whereas the lacZ, wecG, and 16S rRNA PCR assays detected 133 (90.5%), 111 (75.5%), and 146 (99.3%) of the 147 total coliform strains tested. These assays were then assessed by testing 122 well water samples collected in the Québec City region of Canada. Results showed that 97 (79.5%) of the samples tested by culture-based methods and 95 (77.9%), 82 (67.2%), and 98 (80.3%) of samples tested using PCR-based methods contained total coliforms, respectively. Consequently, despite the high genetic variability of the total coliform group, this study demonstrated that it is possible to use molecular assays to detect total coliforms in potable water: the 16S rRNA molecular assay was shown to be as efficient as recommended culture-based methods. This assay might be used in combination with an Escherichia coli molecular assay to assess drinking water quality. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

Molecular Method for Detection of Total Coliforms in Drinking Water Samples

PubMed Central

Boudreau, Dominique K.; Bisson, Marc-Antoine; Dion-Dupont, Vanessa; Bouchard, Sébastien; Nkuranga, Martine; Bergeron, Michel G.; Rodriguez, Manuel J.

2014-01-01

This work demonstrates the ability of a bacterial concentration and recovery procedure combined with three different PCR assays targeting the lacZ, wecG, and 16S rRNA genes, respectively, to detect the presence of total coliforms in 100-ml samples of potable water (presence/absence test). PCR assays were first compared to the culture-based Colilert and MI agar methods to determine their ability to detect 147 coliform strains representing 76 species of Enterobacteriaceae encountered in fecal and environmental settings. Results showed that 86 (58.5%) and 109 (74.1%) strains yielded a positive signal with Colilert and MI agar methods, respectively, whereas the lacZ, wecG, and 16S rRNA PCR assays detected 133 (90.5%), 111 (75.5%), and 146 (99.3%) of the 147 total coliform strains tested. These assays were then assessed by testing 122 well water samples collected in the Québec City region of Canada. Results showed that 97 (79.5%) of the samples tested by culture-based methods and 95 (77.9%), 82 (67.2%), and 98 (80.3%) of samples tested using PCR-based methods contained total coliforms, respectively. Consequently, despite the high genetic variability of the total coliform group, this study demonstrated that it is possible to use molecular assays to detect total coliforms in potable water: the 16S rRNA molecular assay was shown to be as efficient as recommended culture-based methods. This assay might be used in combination with an Escherichia coli molecular assay to assess drinking water quality. PMID:24771030

Nodeomics: Pathogen Detection in Vertebrate Lymph Nodes Using Meta-Transcriptomics

USGS Publications Warehouse

Wittekindt, Nicola E.; Padhi, Abinash; Schuster, Stephan C.; Qi, Ji; Zhao, Fangqing; Tomsho, Lynn P.; Kasson, Lindsay R.; Packard, Michael; Cross, Paul C.; Poss, Mary

2010-01-01

The ongoing emergence of human infections originating from wildlife highlights the need for better knowledge of the microbial community in wildlife species where traditional diagnostic approaches are limited. Here we evaluate the microbial biota in healthy mule deer (Odocoileus hemionus) by analyses of lymph node meta-transcriptomes. cDNA libraries from five individuals and two pools of samples were prepared from retropharyngeal lymph node RNA enriched for polyadenylated RNA and sequenced using Roche-454 Life Sciences technology. Protein-coding and 16S ribosomal RNA (rRNA) sequences were taxonomically profiled using protein and rRNA specific databases. Representatives of all bacterial phyla were detected in the seven libraries based on protein-coding transcripts indicating that viable microbiota were present in lymph nodes. Residents of skin and rumen, and those ubiquitous in mule deer habitat dominated classifiable bacterial species. Based on detection of both rRNA and protein-coding transcripts, we identified two new proteobacterial species; a Helicobacter closely related to Helicobacter cetorum in the Helicobacter pylori/Helicobacter acinonychis complex and an Acinetobacter related to Acinetobacter schindleri. Among viruses, a novel gamma retrovirus and other members of the Poxviridae and Retroviridae were identified. We additionally evaluated bacterial diversity by amplicon sequencing the hypervariable V6 region of 16S rRNA and demonstrate that overall taxonomic diversity is higher with the meta-transcriptomic approach. These data provide the most complete picture to date of the microbial diversity within a wildlife host. Our research advances the use of meta-transcriptomics to study microbiota in wildlife tissues, which will facilitate detection of novel organisms with pathogenic potential to human and animals.

Alternative splicing of anciently exonized 5S rRNA regulates plant transcription factor TFIIIA

PubMed Central

Fu, Yan; Bannach, Oliver; Chen, Hao; Teune, Jan-Hendrik; Schmitz, Axel; Steger, Gerhard; Xiong, Liming; Barbazuk, W. Brad

2009-01-01

Identifying conserved alternative splicing (AS) events among evolutionarily distant species can prioritize AS events for functional characterization and help uncover relevant cis- and trans-regulatory factors. A genome-wide search for conserved cassette exon AS events in higher plants revealed the exonization of 5S ribosomal RNA (5S rRNA) within the gene of its own transcription regulator, TFIIIA (transcription factor for polymerase III A). The 5S rRNA-derived exon in TFIIIA gene exists in all representative land plant species but not in green algae and nonplant species, suggesting it is specific to land plants. TFIIIA is essential for RNA polymerase III-based transcription of 5S rRNA in eukaryotes. Integrating comparative genomics and molecular biology revealed that the conserved cassette exon derived from 5S rRNA is coupled with nonsense-mediated mRNA decay. Utilizing multiple independent Arabidopsis overexpressing TFIIIA transgenic lines under osmotic and salt stress, strong accordance between phenotypic and molecular evidence reveals the biological relevance of AS of the exonized 5S rRNA in quantitative autoregulation of TFIIIA homeostasis. Most significantly, this study provides the first evidence of ancient exaptation of 5S rRNA in plants, suggesting a novel gene regulation model mediated by the AS of an anciently exonized noncoding element. PMID:19211543

Genetic characterization of human-pathogenic Cyclospora cayetanensis parasites from three endemic regions at the 18S ribosomal RNA locus.

PubMed

Sulaiman, Irshad M; Ortega, Ynes; Simpson, Steven; Kerdahi, Khalil

2014-03-01

Cyclospora cayetanensis is an apicocomplexan parasite that infects the gastrointestinal tract and causes acute diarrheal disease in humans. In recent years, this human-pathogenic parasite has led to several foodborne outbreaks in the United States and Canada, mostly associated with imported produce. Understanding the biology and epidemiology of C. cayetanensis is difficult because little is known about its origin, possible zoonotic reservoirs, and genetic relationships with other coccidian parasites. Recently, we developed a 70kDa heat shock protein (HSP70) gene based nested PCR protocol for detection of C. cayetanensis parasite and sequenced the PCR products of 16 human isolates from Nepal, Mexico, and Peru. In this study, we have characterized the regions of 18S ribosomal RNA (rRNA) gene of 17 human C. cayetanensis isolates for molecular detection, and also to ascertain the genetic diversity of this parasite. The 18S rRNA primer sets were further tested by PCR amplification followed by nucleotide sequencing of the PCR amplified products of previously characterized C. cayetanensis isolates from three endemic regions at HSP70 locus. Although no genetic polymorphism was observed at the regions of HSP70 locus characterized in our previous study, the data analysis of this study revealed a minor genetic diversity at the 18S rRNA locus among the C. cayetanensis isolates. The 18S rRNA gene-based nested PCR protocol provides a useful genetic marker for the detection of C. cayetanensis parasite and confirms it as a genetically distinct species in genus Cyclospora. The results also supported lack of geographic segregation and existence of genetically homogeneous population for the C. cayetanensis parasites both at the HSP70 as well as at the18S rRNA loci. Published by Elsevier B.V.

Detection and identification of bacteria in clinical samples by 16S rRNA gene sequencing: comparison of two different approaches in clinical practice.

PubMed

Jenkins, Claire; Ling, Clare L; Ciesielczuk, Holly L; Lockwood, Julianne; Hopkins, Susan; McHugh, Timothy D; Gillespie, Stephen H; Kibbler, Christopher C

2012-04-01

Amplification and sequence analysis of the 16S rRNA gene can be applied to detect and identify bacteria in clinical samples. We examined 75 clinical samples (17 culture-positive, 58 culture-negative) prospectively by two different PCR protocols, amplifying either a single fragment (1343 bp) or two fragments (762/598 bp) of the 16S rRNA gene. The 1343 bp PCR and 762/598 bp PCRs detected and identified the bacterial 16S rRNA gene in 23 (31 %) and 38 (51 %) of the 75 samples, respectively. The 1343 bp PCR identified 19 of 23 (83 %) PCR-positive samples to species level while the 762/598 bp PCR identified 14 of 38 (37 %) bacterial 16S rRNA gene fragments to species level and 24 to the genus level only. Amplification of shorter fragments of the bacterial 16S rRNA gene (762 and 598 bp) resulted in a more sensitive assay; however, analysis of a large fragment (1343 bp) improved species discrimination. Although not statistically significant, the 762/598 bp PCR detected the bacterial 16S rRNA gene in more samples than the 1343 bp PCR, making it more likely to be a more suitable method for the primary detection of the bacterial 16S rRNA gene in the clinical setting. The 1343 bp PCR may be used in combination with the 762/598 bp PCR when identification of the bacterial rRNA gene to species level is required.

Polymerase chain reaction-based discrimination of viable from non-viable Mycoplasma gallisepticum.

PubMed

Tan, Ching Giap; Ideris, Aini; Omar, Abdul R; Yii, Chen Pei; Kleven, Stanley H

2014-09-02

The present study was based on the reverse transcription polymerase chain reaction (RT-PCR) of the 16S ribosomal nucleic acid (rRNA) of Mycoplasma for detection of viable Mycoplasma gallisepticum. To determine the stability of M. gallisepticum 16S rRNA in vitro, three inactivation methods were used and the suspensions were stored at different temperatures. The 16S rRNA of M. gallisepticum was detected up to approximately 20-25 h at 37 °C, 22-25 h at 16 °C, and 23-27 h at 4 °C. The test, therefore, could detect viable or recently dead M. gallisepticum (< 20 h). The RT-PCR method was applied during an in vivo study of drug efficacy under experimental conditions, where commercial broiler-breeder eggs were inoculated with M. gallisepticum into the yolk. Hatched chicks that had been inoculated in ovo were treated with Macrolide 1. The method was then applied in a flock of day 0 chicks with naturally acquired vertical transmission of M. gallisepticum, treated with Macrolide 2. Swabs of the respiratory tract were obtained for PCR and RT-PCR evaluations to determine the viability of M. gallisepticum. This study proved that the combination of both PCR and RT-PCR enables detection and differentiation of viable from non-viable M. gallisepticum.

Common bacterial responses in six ecosystems exposed to 10 years of elevated atmospheric carbon dioxide.

PubMed

Dunbar, John; Eichorst, Stephanie A; Gallegos-Graves, La Verne; Silva, Shannon; Xie, Gary; Hengartner, N W; Evans, R David; Hungate, Bruce A; Jackson, Robert B; Megonigal, J Patrick; Schadt, Christopher W; Vilgalys, Rytas; Zak, Donald R; Kuske, Cheryl R

2012-05-01

Six terrestrial ecosystems in the USA were exposed to elevated atmospheric CO(2) in single or multifactorial experiments for more than a decade to assess potential impacts. We retrospectively assessed soil bacterial community responses in all six-field experiments and found ecosystem-specific and common patterns of soil bacterial community response to elevated CO(2) . Soil bacterial composition differed greatly across the six ecosystems. No common effect of elevated atmospheric CO(2) on bacterial biomass, richness and community composition across all of the ecosystems was identified, although significant responses were detected in individual ecosystems. The most striking common trend across the sites was a decrease of up to 3.5-fold in the relative abundance of Acidobacteria Group 1 bacteria in soils exposed to elevated CO(2) or other climate factors. The Acidobacteria Group 1 response observed in exploratory 16S rRNA gene clone library surveys was validated in one ecosystem by 100-fold deeper sequencing and semi-quantitative PCR assays. Collectively, the 16S rRNA gene sequencing approach revealed influences of elevated CO(2) on multiple ecosystems. Although few common trends across the ecosystems were detected in the small surveys, the trends may be harbingers of more substantive changes in less abundant, more sensitive taxa that can only be detected by deeper surveys. Representative bacterial 16S rRNA gene clone sequences were deposited in GenBank with Accession No. JQ366086–JQ387568. Published 2012. This article is a U.S. Government work and is in the public domain in the USA.

Quantitative phenotyping of X-disease resistance in chokecherry using real-time PCR.

PubMed

Huang, Danqiong; Walla, James A; Dai, Wenhao

2014-03-01

A quantitative real-time SYBR Green PCR (qPCR) assay has been developed to detect and quantify X-disease phytoplasmas in chokecherry. An X-disease phytoplasma-specific and high sensitivity primer pair was designed based on the 16S rRNA gene sequence of X-disease phytoplasmas. This primer pair was specific to the 16SrIII group (X-disease) phytoplasmas. The qPCR method can quantify phytoplasmas from a DNA mix (a mix of both chokecherry and X-disease phytoplasma DNA) at as low as 0.001 ng, 10-fold lower than conventional PCR using the same primer pair. A significant correlation between the copy number of phytoplasmas and visual phenotypic rating scores of X-disease resistance in chokecherry plants was observed. Disease resistant chokecherries had a significantly lower titer of X-disease phytoplasmas than susceptible plants. This suggests that the qPCR assay provides a more objective tool to phenotype phytoplasma disease severity, particularly for early evaluation of host resistance; therefore, this method will facilitate quantitative phenotyping of disease resistance and has great potential in enhancing plant breeding. Copyright © 2013 Elsevier B.V. All rights reserved.

Microbiota associated with the migration and transformation of chlorinated aliphatic hydrocarbons in groundwater.

PubMed

Guan, Xiangyu; Liu, Fei; Xie, Yuxuan; Zhu, Lingling; Han, Bin

2013-08-01

Pollution of groundwater with chlorinated aliphatic hydrocarbons (CAHs) is a serious environmental problem which is threatening human health. Microorganisms are the major participants in degrading these contaminants. Here, groundwater contaminated for a decade with CAHs was investigated. Numerical simulation and field measurements were used to track and forecast the migration and transformation of the pollutants. The diversity, abundance, and possible activity of groundwater microbial communities at CAH-polluted sites were characterized by molecular approaches. The number of microorganisms was between 5.65E+05 and 1.49E+08 16S rRNA gene clone numbers per liter according to quantitative real-time PCR analysis. In 16S rRNA gene clone libraries constructed from samples along the groundwater flow, eight phyla were detected, and Proteobacteria were dominant (72.8 %). The microbial communities varied with the composition and concentration of pollutants. Meanwhile, toluene monooxygenases and methane monooxygenases capable of degradation of PCE and TCE were detected, demonstrating the major mechanism for PCE and TCE degradation and possibility for in situ remediation by addition of oxygen in this study.

Environmental distribution, abundance and activity of the Miscellaneous Crenarchaeotal Group

NASA Astrophysics Data System (ADS)

Lloyd, K. G.; Biddle, J.; Teske, A.

2011-12-01

Many marine sedimentary microbes have only been identified by 16S rRNA sequences. Consequently, little is known about the types of metabolism, activity levels, or relative abundance of these groups in marine sediments. We found that one of these uncultured groups, called the Miscellaneous Crenarchaeotal Group (MCG), dominated clone libraries made from reverse transcribed 16S rRNA, and 454 pyrosequenced 16S rRNA genes, in the White Oak River estuary. Primers suitable for quantitative PCR were developed for MCG and used to show that 16S rRNA DNA copy numbers from MCG account for nearly all the archaeal 16S rRNA genes present. RT-qPCR shows much less MCG rRNA than total archaeal rRNA, but comparisons of different primers for each group suggest bias in the RNA-based work relative to the DNA-based work. There is no evidence of a population shift with depth below the sulfate-methane transition zone, suggesting that the metabolism of MCG may not be tied to sulfur or methane cycles. We classified 2,771 new sequences within the SSU Silva 106 database that, along with the classified sequences in the Silva database was used to make an MCG database of 4,646 sequences that allowed us to increase the named subgroups of MCG from 7 to 19. Percent terrestrial sequences in each subgroup is positively correlated with percent of the marine sequences that are nearshore, suggesting that membership in the different subgroups is not random, but dictated by environmental selective pressures. Given their high phylogenetic diversity, ubiquitous distribution in anoxic environments, and high DNA copy number relative to total archaea, members of MCG are most likely anaerobic heterotrophs who are integral to the post-depositional marine carbon cycle.

Detection of bacterial 16S rRNA using a molecular beacon-based X sensor

PubMed Central

Gerasimova, Yulia V.; Kolpashchikov, Dmitry M.

2012-01-01

We demonstrate how a long structurally constrained RNA can be analyzed in homogeneous solution at ambient temperatures with high specificity using a sophisticated biosensor. The sensor consists of a molecular beacon probe as a signal reporter and two DNA adaptor strands, which have fragments complementary to the reporter and to the analyzed RNA. One adaptor strand uses its long RNA-binding arm to unwind the RNA secondary structure. Second adaptor strand with a short RNA-binding arm hybridizes only to a fully complementary site, thus providing high recognition specificity. Overall the three-component sensor and the target RNA form a four-stranded DNA crossover (X) structure. Using this sensor, E.coli 16S rRNA was detected in real time with the detection limit of ~ 0.17 nM. The high specificity of the analysis was proven by differentiating B.subtilus from E.coli 16S rRNA sequences. The sensor responds to the presence of the analyte within seconds. PMID:23021850

Rapid Differentiation and In Situ Detection of 16 Sourdough Lactobacillus Species by Multiplex PCR

PubMed Central

Settanni, Luca; van Sinderen, Douwe; Rossi, Jone; Corsetti, Aldo

2005-01-01

A two-step multiplex PCR-based method was designed for the rapid detection of 16 species of lactobacilli known to be commonly present in sourdough. The first step of multiplex PCR was developed with a mixture of group-specific primers, while the second step included three multiplex PCR assays with a mixture of species-specific primers. Primers were derived from sequences that specify the 16S rRNA, the 16S-23S rRNA intergenic spacer region, and part of the 23S rRNA gene. The primer pairs designed were shown to exclusively amplify the targeted rrn operon fragment of the corresponding species. Due to the reliability of simultaneously identifying Lactobacillus plantarum, Lactobacillus pentosus, and Lactobacillus paraplantarum, a previously described multiplex PCR method employing recA gene-derived primers was included in the multiplex PCR system. The combination of a newly developed, quick bacterial DNA extraction method from sourdough and this multiplex PCR assay allows the rapid in situ detection of several sourdough-associated lactobacilli, including the recently described species Lactobacillus rossii, and thus represents a very useful alternative to culture-based methodologies. PMID:15933001

Assessment of Fecal Indicator Bacteria and Potential Pathogen Co-Occurrence at a Shellfish Growing Area

PubMed Central

Leight, Andrew K.; Crump, Byron C.; Hood, Raleigh R.

2018-01-01

Routine monitoring of shellfish growing waters for bacteria indicative of human sewage pollution reveals little about the bacterial communities that co-occur with these indicators. This study investigated the bacterial community, potential pathogens, and fecal indicator bacteria in 40 water samples from a shellfish growing area in the Chesapeake Bay, USA. Bacterial community composition was quantified with deep sequencing of 16S rRNA gene amplicons, and absolute gene abundances were estimated with an internal standard (Thermus thermophilus genomes). Fecal coliforms were quantified by culture, and Vibrio vulnificus and V. parahaemolyticus with quantitative PCR. Fecal coliforms and V. vulnificus were detected in most samples, and a diverse assemblage of potential human pathogens were detected in all samples. These taxa followed two general patterns of abundance. Fecal coliforms and 16S rRNA genes for Enterobacteriaceae, Aeromonas, Arcobacter, Staphylococcus, and Bacteroides increased in abundance after a 1.3-inch rain event in May, and, for some taxa, after smaller rain events later in the season, suggesting that these are allochthonous organisms washed in from land. Clostridiaceae and Mycobacterium 16S rRNA gene abundances increased with day of the year and were not positively related to rainfall, suggesting that these are autochthonous organisms. Other groups followed both patterns, such as Legionella. Fecal coliform abundance did not correlate with most other taxa, but were extremely high following the large rainstorm in May when they co-occurred with a broad range of potential pathogen groups. V. vulnificus were absent during the large rainstorm, and did not correlate with 16S rRNA abundances of Vibrio spp. or most other taxa. These results highlight the complex nature of bacterial communities and the limited utility of using specific bacterial groups as indicators of pathogen presence. PMID:29593669

A Simultaneous Analytical Method for Duplex Identification of Porcine and Horse in the Meat Products by EvaGreen based Real-time PCR.

PubMed

Sakalar, Ergün; Ergün, Seyma Özçirak; Akar, Emine

2015-01-01

A duplex real-time polymerase chain reaction (PCR) based assay for the detection of porcine and horse meat in sausages was designed by using EvaGreen fluorescent dye. Primers were selected from mitochondrial 12S rRNA and 16S rRNA genes which are powerful regions for identification of horse and porcine meat. DNA from reference samples and industrial products was successfully extracted using the GIDAGEN® Multi-Fast DNA Isolation Kit. Genomes were identified based on their specific melting peaks (Mp) which are 82.5℃ and 78℃ for horse and porcine, respectively. The assay used in this study allowed the detection of as little as 0.0001% level of horse meat and 0.001% level of porcine meat in the experimental admixtures. These findings indicate that EvaGreen based duplex real-time PCR is a potentially sensitive, reliable, rapid and accurate assay for the detection of meat species adulterated with porcine and horse meats.

Advances in methods for detection of anaerobic ammonium oxidizing (anammox) bacteria.

PubMed

Li, Meng; Gu, Ji-Dong

2011-05-01

Anaerobic ammonium oxidation (anammox), the biochemical process oxidizing ammonium into dinitrogen gas using nitrite as an electron acceptor, has only been recognized for its significant role in the global nitrogen cycle not long ago, and its ubiquitous distribution in a wide range of environments has changed our knowledge about the contributors to the global nitrogen cycle. Currently, several groups of methods are used in detection of anammox bacteria based on their physiological and biochemical characteristics, cellular chemical composition, and both 16S rRNA gene and selective functional genes as biomarkers, including hydrazine oxidoreductase and nitrite reductase encoding genes hzo and nirS, respectively. Results from these methods coupling with advances in quantitative PCR, reverse transcription of mRNA genes and stable isotope labeling have improved our understanding on the distribution, diversity, and activity of anammox bacteria in different environments both natural and engineered ones. In this review, we summarize these methods used in detection of anammox bacteria from various environments, highlight the strengths and weakness of these methods, and also discuss the new development potentials on the existing and new techniques in the future.

Detection and identification of Theileria infection in sika deer ( Cervus nippon ) in China.

PubMed

He, Lan; Khan, Muhanmad Kasib; Zhang, Wen-Jie; Zhang, Qing-Li; Zhou, Yan-Qin; Hu, Min; Zhao, Junlong

2012-06-01

The sika deer ( Cervus nippon ) is a first-grade state-protected animal in China and designated a threatened species by the World Conservation Union. To detect hemoparasite infection of sika deer, blood samples were collected from 24 animals in the Hubei Province Deer Center. Genomic DNA was extracted, and the V4 hypervariable region encoding 18S rRNA was analyzed by reverse line blot hybridization assay. PCR products hybridized with Babesia / Theileria genus-specific probes but failed to hybridize with any of the Babesia or Theileria species-specific probes, suggesting the presence of a novel, or variant, species. Here 18S rRNA and internal transcribed spacer (ITS) genes were amplified, cloned, and sequenced from 7 isolates. Alignment and BlastN of the cloned sequences revealed high similarities to the homologous 18S rRNA genes and ITS genes of Theileria cervi (AY735122), Theileria sp. CNY1A (AB012194), and Theileria sp. ex Yamaguchi (AF529272). Phylogenetic analysis based on the 18S rRNA gene and ITS sequences showed that all cloned sequences were grouped within the Theileria clade. Phylogeny based on the 18S rRNA gene divided the organisms into 2 groups. Group 1 was closest to Theileria sp. ex Yamaguchi (AF529272), and group 2 was distinct from all other identified Theileria and Babesia species. These results suggest the existence of Theileria sp. infection in sika deer in China. To our knowledge, this is the first report of cervine Theileria sp. in China.

Comparative analysis of bacteria associated with different mosses by 16S rRNA and 16S rDNA sequencing.

PubMed

Tian, Yang; Li, Yan Hong

2017-01-01

To understand the differences of the bacteria associated with different mosses, a phylogenetic study of bacterial communities in three mosses was carried out based on 16S rDNA and 16S rRNA sequencing. The mosses used were Hygroamblystegium noterophilum, Entodon compressus and Grimmia montana, representing hygrophyte, shady plant and xerophyte, respectively. In total, the operational taxonomic units (OTUs), richness and diversity were different regardless of the moss species and the library level. All the examined 1183 clones were assigned to 248 OTUs, 56 genera were assigned in rDNA libraries and 23 genera were determined at the rRNA level. Proteobacteria and Bacteroidetes were considered as the most dominant phyla in all the libraries, whereas abundant Actinobacteria and Acidobacteria were detected in the rDNA library of Entodon compressus and approximately 24.7% clones were assigned to Candidate division TM7 in Grimmia montana at rRNA level. The heatmap showed the bacterial profiles derived from rRNA and rDNA were partly overlapping. However, the principle component analysis of all the profiles derived from rDNA showed sharper differences between the different mosses than that of rRNA-based profiles. This suggests that the metabolically active bacterial compositions in different mosses were more phylogenetically similar and the differences of the bacteria associated with different mosses were mainly detected at the rDNA level. Obtained results clearly demonstrate that combination of 16S rDNA and 16S rRNA sequencing is preferred approach to have a good understanding on the constitution of the microbial communities in mosses. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Development of a duplex droplet digital PCR assay for absolute quantitative detection of "Candidatus Liberibacter asiaticus".

PubMed

Selvaraj, Vijayanandraj; Maheshwari, Yogita; Hajeri, Subhas; Chen, Jianchi; McCollum, Thomas Greg; Yokomi, Raymond

2018-01-01

Huanglongbing (HLB, citrus greening) is a devastating citrus disease affecting citrus production worldwide. It is associated with the bacterium "Candidatus Liberibacter asiaticus" (CLas) and is vectored by the Asian citrus psyllid (ACP). Currently, diagnosis of CLas in regulatory samples is based on real-time quantitative polymerase chain reaction (qPCR) using 16S rRNA gene specific primers/probe. The detection of CLas using qPCR is challenging due to low pathogen titer and uneven distribution in infected plants and exacerbated by sampling issues and presence of inhibitors. This study evaluated a duplex droplet digital polymerase chain reaction (ddPCR) using multi-copy gene targets, 16S and RNR, to simultaneously detect CLas DNA targets in the same sample for unambiguous detection of the HLB pathogen in DNA extracts from citrus leaves and ACP. Standard curve analyses on tenfold dilution series with plasmid, citrus leaf and ACP DNA showed that both ddPCR and qPCR exhibited good linearity and efficiency in the duplex assay. CLas-infected low titer samples were used to validate the duplex ddPCR and qPCR performance and demonstrated that detection rate is higher when both 16S and RNR primers were used in duplex assay. However, the receiver operating characteristic analysis indicated that area under the curve for RNR primer was significantly broader, compared to 16S primers for CLas detection at low target titer. The absolute quantification of CLas at variable titers was reproducible and repeatable for both primer sets and the ddPCR showed higher resilience to PCR inhibitors with citrus leaf and ACP extracts. Hence, the resultant duplex ddPCR assay resulted in a significantly improved detection platform for diagnosis of CLas in samples with low pathogen titer.

Development of a duplex droplet digital PCR assay for absolute quantitative detection of "Candidatus Liberibacter asiaticus"

PubMed Central

Hajeri, Subhas; Chen, Jianchi; McCollum, Thomas Greg

2018-01-01

Huanglongbing (HLB, citrus greening) is a devastating citrus disease affecting citrus production worldwide. It is associated with the bacterium “Candidatus Liberibacter asiaticus” (CLas) and is vectored by the Asian citrus psyllid (ACP). Currently, diagnosis of CLas in regulatory samples is based on real-time quantitative polymerase chain reaction (qPCR) using 16S rRNA gene specific primers/probe. The detection of CLas using qPCR is challenging due to low pathogen titer and uneven distribution in infected plants and exacerbated by sampling issues and presence of inhibitors. This study evaluated a duplex droplet digital polymerase chain reaction (ddPCR) using multi-copy gene targets, 16S and RNR, to simultaneously detect CLas DNA targets in the same sample for unambiguous detection of the HLB pathogen in DNA extracts from citrus leaves and ACP. Standard curve analyses on tenfold dilution series with plasmid, citrus leaf and ACP DNA showed that both ddPCR and qPCR exhibited good linearity and efficiency in the duplex assay. CLas-infected low titer samples were used to validate the duplex ddPCR and qPCR performance and demonstrated that detection rate is higher when both 16S and RNR primers were used in duplex assay. However, the receiver operating characteristic analysis indicated that area under the curve for RNR primer was significantly broader, compared to 16S primers for CLas detection at low target titer. The absolute quantification of CLas at variable titers was reproducible and repeatable for both primer sets and the ddPCR showed higher resilience to PCR inhibitors with citrus leaf and ACP extracts. Hence, the resultant duplex ddPCR assay resulted in a significantly improved detection platform for diagnosis of CLas in samples with low pathogen titer. PMID:29772016

Quantification of Endospore-Forming Firmicutes by Quantitative PCR with the Functional Gene spo0A

PubMed Central

Bueche, Matthieu; Wunderlin, Tina; Roussel-Delif, Ludovic; Junier, Thomas; Sauvain, Loic; Jeanneret, Nicole

2013-01-01

Bacterial endospores are highly specialized cellular forms that allow endospore-forming Firmicutes (EFF) to tolerate harsh environmental conditions. EFF are considered ubiquitous in natural environments, in particular, those subjected to stress conditions. In addition to natural habitats, EFF are often the cause of contamination problems in anthropogenic environments, such as industrial production plants or hospitals. It is therefore desirable to assess their prevalence in environmental and industrial fields. To this end, a high-sensitivity detection method is still needed. The aim of this study was to develop and evaluate an approach based on quantitative PCR (qPCR). For this, the suitability of functional genes specific for and common to all EFF were evaluated. Seven genes were considered, but only spo0A was retained to identify conserved regions for qPCR primer design. An approach based on multivariate analysis was developed for primer design. Two primer sets were obtained and evaluated with 16 pure cultures, including representatives of the genera Bacillus, Paenibacillus, Brevibacillus, Geobacillus, Alicyclobacillus, Sulfobacillus, Clostridium, and Desulfotomaculum, as well as with environmental samples. The primer sets developed gave a reliable quantification when tested on laboratory strains, with the exception of Sulfobacillus and Desulfotomaculum. A test using sediment samples with a diverse EFF community also gave a reliable quantification compared to 16S rRNA gene pyrosequencing. A detection limit of about 104 cells (or spores) per gram of initial material was calculated, indicating this method has a promising potential for the detection of EFF over a wide range of applications. PMID:23811505

Use of 16S rRNA sequencing and quantitative PCR to correlate venous leg ulcer bacterial bioburden dynamics with wound expansion, antibiotic therapy, and healing

PubMed Central

Sprockett, Daniel D.; Ammons, Christine G.; Tuttle, Marie S.

2016-01-01

Clinical diagnosis of infection in chronic wounds is currently limited to subjective clinical signs and culture-based methods that underestimate the complexity of wound microbial bioburden as revealed by DNA-based microbial identification methods. Here, we use 16S rRNA next generation sequencing and quantitative polymerase chain reaction to characterize weekly changes in bacterial load, community structure, and diversity associated with a chronic venous leg ulcer over the 15-week course of treatment and healing. Our DNA-based methods and detailed sampling scheme reveal that the bacterial bioburden of the wound is unexpectedly dynamic, including changes in the bacterial load and community structure that correlate with wound expansion, antibiotic therapy, and healing. We demonstrate that these multidimensional changes in bacterial bioburden can be summarized using swabs taken prior to debridement, and therefore, can be more easily collected serially than debridement or biopsy samples. Overall, this case illustrates the importance of detailed clinical indicators and longitudinal sampling to determine the pathogenic significance of chronic wound microbial dynamics and guide best use of antimicrobials for improvement of healing outcomes. PMID:25902876

Comparison of MI, Chromocult® coliform, and Compass CC chromogenic culture-based methods to detect Escherichia coli and total coliforms in water using 16S rRNA sequencing for colony identification.

PubMed

Maheux, Andrée F; Bouchard, Sébastien; Bérubé, Ève; Bergeron, Michel G

2017-06-01

The MI, Chromocult ® coliform, and Compass CC chromogenic culture-based methods used to assess water quality by the detection of Escherichia coli and total coliforms were compared in terms of their specificity and sensitivity, using 16S rRNA sequencing for colony identification. A sewage water sample was divided in 2-μL subsamples for testing by all three culture-based methods. All growing colonies were harvested and subjected to 16S rRNA sequencing. Test results showed that all E. coli colonies were correctly identified by all three methods, for a specificity and a sensitivity of 100%. However, for the total coliform detection, the MI agar, Chromocult ® coliform agar, and Compass CC agar were specific for only 69.2% (9/13), 47.2% (25/53), and 40.5% (17/42), whereas sensitive for 97.8% (45/46), 97.5% (39/40), and 85.7% (24/28), respectively. Thus, given the low level of specificity of these methods for the detection of total coliforms, confirming the identity of total coliform colonies could help to take public health decisions, in particular for cities connected to a public drinking water distribution system since the growth of few putative total coliform colonies on chromogenic agar is problematic and can lead to unnecessary and costly boiling notices from public health authorities.

Genetic and functional properties of uncultivated thermophilic crenarchaeotes from a subsurface gold mine as revealed by analysis of genome fragments.

PubMed

Nunoura, Takuro; Hirayama, Hisako; Takami, Hideto; Oida, Hanako; Nishi, Shinro; Shimamura, Shigeru; Suzuki, Yohey; Inagaki, Fumio; Takai, Ken; Nealson, Kenneth H; Horikoshi, Koki

2005-12-01

Within a phylum Crenarchaeota, only some members of the hyperthermophilic class Thermoprotei, have been cultivated and characterized. In this study, we have constructed a metagenomic library from a microbial mat formation in a subsurface hot water stream of the Hishikari gold mine, Japan, and sequenced genome fragments of two different phylogroups of uncultivated thermophilic Crenarchaeota: (i) hot water crenarchaeotic group (HWCG) I (41.2 kb), and (ii) HWCG III (49.3 kb). The genome fragment of HWCG I contained a 16S rRNA gene, two tRNA genes and 35 genes encoding proteins but no 23S rRNA gene. Among the genes encoding proteins, several genes for putative aerobic-type carbon monoxide dehydrogenase represented a potential clue with regard to the yet unknown metabolism of HWCG I Archaea. The genome fragment of HWCG III contained a 16S/23S rRNA operon and 44 genes encoding proteins. In the 23S rRNA gene, we detected a homing-endonuclease encoding a group I intron similar to those detected in hyperthermophilic Crenarchaeota and Bacteria, as well as eukaryotic organelles. The reconstructed phylogenetic tree based on the 23S rRNA gene sequence reinforced the intermediate phylogenetic affiliation of HWCG III bridging the hyperthermophilic and non-thermophilic uncultivated Crenarchaeota.

Evaluation of 16S rRNA qPCR for detection of Mycobacterium leprae DNA in nasal secretion and skin biopsy samples from multibacillary and paucibacillary leprosy cases.

PubMed

Marques, Lívia Érika Carlos; Frota, Cristiane Cunha; Quetz, Josiane da Silva; Bindá, Alexandre Havt; Mota, Rosa Maria Salane; Pontes, Maria Araci de Andrade; Gonçalves, Heitor de Sá; Kendall, Carl; Kerr, Ligia Regina Franco Sansigolo

2017-12-26

Mycobacterium leprae bacilli are mainly transmitted by the dissemination of nasal aerosols from multibacillary (MB) patients to susceptible individuals through inhalation. The upper respiratory tract represents the main entry and exit routes of M. leprae. Therefore, this study aimed to evaluate the sensitivity and specificity of real-time quantitative polymerase chain reaction (qPCR) in detecting M. leprae in nasal secretion (NS) and skin biopsy (SB) samples from MB and paucibacillary (PB) cases. Fifty-four NS samples were obtained from leprosy patients at the Dona Libânia National Reference Centre for Sanitary Dermatology in Ceará, Brazil. Among them, 19 MB cases provided both NS and SB samples. Bacilloscopy index assays were conducted and qPCR amplification was performed using specific primers for M. leprae 16S rRNA gene, generating a 124-bp fragment. Primer specificity was verified by determining the amplicon melting temperature (T m  = 79.5 °C) and detection limit of qPCR was 20 fg of M. leprae DNA. Results were positive for 89.7 and 73.3% of NS samples from MB and PB cases, respectively. SB samples from MB patients were 100% positive. The number of bacilli detected in NS samples were 1.39 × 10 3 -8.02 × 10 5 , and in SB samples from MB patients were 1.87 × 10 3 -1.50 × 10 6 . Therefore, qPCR assays using SYBR Green targeting M. leprae 16S rRNA region can be employed in detecting M. leprae in nasal swabs from leprosy patients, validating this method for epidemiological studies aiming to identify healthy carriers among household contacts or within populations of an endemic area.

Automated detection and quantitation of bacterial RNA by using electrical microarrays.

PubMed

Elsholz, B; Wörl, R; Blohm, L; Albers, J; Feucht, H; Grunwald, T; Jürgen, B; Schweder, T; Hintsche, Rainer

2006-07-15

Low-density electrical 16S rRNA specific oligonucleotide microarrays and an automated analysis system have been developed for the identification and quantitation of pathogens. The pathogens are Escherichia coli, Pseudomonas aeruginosa, Enterococcus faecalis, Staphylococcus aureus, and Staphylococcus epidermidis, which are typically involved in urinary tract infections. Interdigitated gold array electrodes (IDA-electrodes), which have structures in the nanometer range, have been used for very sensitive analysis. Thiol-modified oligonucleotides are immobilized on the gold IDA as capture probes. They mediate the specific recognition of the target 16S rRNA by hybridization. Additionally three unlabeled oligonucleotides are hybridized in close proximity to the capturing site. They are supporting molecules, because they improve the RNA hybridization at the capturing site. A biotin labeled detector oligonucleotide is also allowed to hybridize to the captured RNA sequence. The biotin labels enable the binding of avidin alkaline phophatase conjugates. The phosphatase liberates the electrochemical mediator p-aminophenol from its electrically inactive phosphate derivative. The electrical signals were generated by amperometric redox cycling and detected by a unique multipotentiostat. The read out signals of the microarray are position specific current and change over time in proportion to the analyte concentration. If two additional biotins are introduced into the affinity binding complex via the supporting oligonucleotides, the sensitivity of the assays increase more than 60%. The limit of detection of Escherichia coli total RNA has been determined to be 0.5 ng/microL. The control of fluidics for variable assay formats as well as the multichannel electrical read out and data handling have all been fully automated. The fast and easy procedure does not require any amplification of the targeted nucleic acids by PCR.

Anaerobic Ammonium-Oxidizing Bacteria in Cow Manure Composting.

PubMed

Wang, Tingting; Cheng, Lijun; Zhang, Wenhao; Xu, Xiuhong; Meng, Qingxin; Sun, Xuewei; Liu, Huajing; Li, Hongtao; Sun, Yu

2017-07-28

Composting is widely used to transform waste into valuable agricultural organic fertilizer. Anaerobic ammonium-oxidizing (anammox) bacteria play an important role in the global nitrogen cycle, but their role in composting remains poorly understood. In the present study, the community structure, diversity, and abundance of anammox bacteria were analyzed using cloning and sequencing methods by targeting the 16S rRNA gene and the hydrazine oxidase gene ( hzo ) in samples isolated from compost produced from cow manure and rice straw. A total of 25 operational taxonomic units were classified based on 16S rRNA gene clone libraries, and 14 operational taxonomic units were classified based on hzo gene clone libraries. The phylogenetic tree analysis of the 16S rRNA gene and deduced HZO protein sequences from the corresponding encoding genes indicated that the majority of the obtained clones were related to the known anammox bacteria Candidatus "Brocadia," Candidatus "Kuenenia," and Candidatus "Scalindua." The abundances of anammox bacteria were determined by quantitative PCR, and between 2.13 × 10 5 and 1.15 × 10 6 16S rRNA gene copies per gram of compost were found. This study provides the first demonstration of the existence of anammox bacteria with limited diversity in cow manure composting.

Phylogenetic analysis of Fusobacterium prausnitzii based upon the 16S rRNA gene sequence and PCR confirmation.

PubMed

Wang, R F; Cao, W W; Cerniglia, C E

1996-01-01

In order to develop a PCR method to detect Fusobacterium prausnitzii in human feces and to clarify the phylogenetic position of this species, its 16S rRNA gene sequence was determined. The sequence described in this paper is different from the 16S rRNA gene sequence is specific for F. prausnitzii, and the results of this assay confirmed that F. prausnitzii is the most common species in human feces. However, a PCR assay based on the original GenBank sequence was negative when it was performed with two strains of F. prausnitzii obtained from the American Type Culture Collection. A phylogenetic tree based on the new 16S rRNA gene sequence was constructed. On this tree F. prausnitzii was not a member of the Fusobacterium group but was closer to some Eubacterium spp. and located between Clostridium "clusters III and IV" (M.D. Collins, P.A. Lawson, A. Willems, J.J. Cordoba, J. Fernandez-Garayzabal, P. Garcia, J. Cai, H. Hippe, and J.A.E. Farrow, Int. J. Syst. Bacteriol. 44:812-826, 1994).

Detection of novel strains genetically related to Anaplasma platys in Tunisian one-humped camels (Camelus dromedarius).

PubMed

Belkahia, Hanène; Ben Said, Mourad; Sayahi, Lotfi; Alberti, Alberto; Messadi, Lilia

2015-10-29

Little information is currently available regarding the presence of Anaplasma species in North African dromedaries. To fill this gap in knowledge, the prevalence, risk factors, and genetic diversity of Anaplasma species were investigated in Tunisian dromedary camels. A total of 226 camels from three different bioclimatic areas were sampled and tested for the presence of Anaplasma species by quantitative polymerase chain reaction (qPCR) and nested polymerase chain reaction (nPCR) assays. Detected Anaplasma strains were characterized by 16S rRNA sequence analysis. Overall infection rate of Anaplasma spp. was 17.7%, and was significantly higher in females. Notably, A. marginale, A. centrale, A. bovis, and A. phagocytophilum were not detected. Animals were severely infested by three tick species belonging to the genus Hyalomma (H. dromedarii, H. impeltatum, and H. excavatum). Alignment, similarity comparison, and phylogenetic analysis of the 16S rRNA sequence variants obtained in this study suggest that Tunisian dromedaries are infected by more than one novel Anaplasma strain genetically related to A. platys. This study reports the presence of novel Anaplasma sp. strains genetically related to A. platys in dromedaries from various bioclimatic areas of Tunisia. Findings raise new concerns about the specificity of the direct and indirect diagnostic tests routinely used to detect different Anaplasma species in ruminants and provide useful molecular information to elucidate the evolutionary history of bacterial species related to A. platys.

Development and evaluation of a 28S rRNA gene-based nested PCR assay for P. falciparum and P. vivax

PubMed Central

Pakalapati, Deepak; Garg, Shilpi; Middha, Sheetal; Acharya, Jyoti; Subudhi, Amit K; Boopathi, Arunachalam P; Saxena, Vishal; Kochar, Sanjay K; Kochar, Dhanpat K; Das, Ashis

2013-01-01

The 28S rRNA gene was amplified and sequenced from P. falciparum and P. vivax isolates collected from northwest India. Based upon the sequence diversity of the Plasmodium 28SrRNA gene in comparison with its human counterpart, various nested polymerase chain reaction (PCR) primers were designed from the 3R region of the 28SrRNA gene and evaluated on field isolates. This is the first report demonstrating the utility of this gene for species-specific diagnosis of malaria for these two species, prevalent in India. The initial evaluation on 363 clinical isolates indicated that, in comparison with microscopy, which showed sensitivity and specificity of 85.39% and 100% respectively, the sensitivity and specificity of the nested PCR assay was found to be 99.08% and 100% respectively. This assay was also successful in detecting mixed infections that are undetected by microscopy. Our results demonstrate the utility of the 28S rRNA gene as a diagnostic target for the detection of the major plasmodial species infecting humans. PMID:23816509

Electrokinetic Stringency Control in Self-Assembled Monolayer-based Biosensors for Multiplex Urinary Tract Infection Diagnosis

PubMed Central

Liu, Tingting; Sin, Mandy L. Y.; Pyne, Jeff D.; Gau, Vincent; Liao, Joseph C.; Wong, Pak Kin

2013-01-01

Rapid detection of bacterial pathogens is critical toward judicious management of infectious diseases. Herein, we demonstrate an in situ electrokinetic stringency control approach for a self-assembled monolayer-based electrochemical biosensor toward urinary tract infection diagnosis. The in situ electrokinetic stringency control technique generates Joule heating induced temperature rise and electrothermal fluid motion directly on the sensor to improve its performance for detecting bacterial 16S rRNA, a phylogenetic biomarker. The dependence of the hybridization efficiency reveals that in situ electrokinetic stringency control is capable of discriminating single-base mismatches. With electrokinetic stringency control, the background noise due to the matrix effects of clinical urine samples can be reduced by 60%. The applicability of the system is demonstrated by multiplex detection of three uropathogenic clinical isolates with similar 16S rRNA sequences. The results demonstrate that electrokinetic stringency control can significantly improve the signal-to-noise ratio of the biosensor for multiplex urinary tract infection diagnosis. PMID:23891989

Biodegradation of 2,4-dichlorophenoxyacetic acid by bacteria with highly antibiotic-resistant pattern isolated from wheat field soils in Kurdistan, Iran.

PubMed

Karami, Solmaz; Maleki, Afshin; Karimi, Ebrahim; Poormazaheri, Helen; Zandi, Shiva; Davari, Behrooz; Salimi, Yahya Zand; Gharibi, Fardin; Kalantar, Enayatollah

2016-12-01

Recently, there has been increasing interest to clean up the soils contaminated with herbicide. Our aim was to determine the bioremediation of 2,4-dichlorophenoxyacetic acid (2,4-D) from wheat fields which have a long history of herbicide in Sanandaj. Based on our literature survey, this study is the first report to isolate and identify antimicrobial resistant bacteria from polluted wheat field soils in Sanandaj which has the capacity to degrade 2,4-D. From 150 2,4-D-exposed soil samples, five different bacteria were isolated and identified based on biochemical tests and 16S ribosomal RNA (rRNA). Pseudomonas has been the most frequently isolated genus. By sequencing the 16S rRNA gene of the isolated bacteria, the strains were detected and identified as a member of the genus Pseudomonas sp, Entrobacter sp, Bacillus sp, Seratia sp, and Staphylococcus sp. The sequence of Sanandaj 1 isolate displayed 87% similarity with the 16S rRNA gene of a Pseudomonas sp (HE995788). Similarly, all the isolates were compared to standard strains based on 16S rRNA. Small amounts of 2,4-D could be transmitted to a depth of 10-20 cm; however, in the depth of 20-40 cm, we could not detect the 2,4-D. The isolates were resistant to various antibiotics particularly, penicillin, ampicillin, and amoxicillin.

Correlation of quantitative PCR for a poultry-specific brevibacterium marker gene with bacterial and chemical indicators of water pollution in a watershed impacted by land application of poultry litter.

PubMed

Weidhaas, Jennifer L; Macbeth, Tamzen W; Olsen, Roger L; Harwood, Valerie J

2011-03-01

The impact of fecal contamination from human and agricultural animal waste on water quality is a major public health concern. Identification of the dominant source(s) of fecal pollution in a watershed is necessary for assessing the safety of recreational water and protecting water resources. A field study was conducted using quantitative PCR (qPCR) for the 16S rRNA gene of Brevibacterium sp. LA35 to track feces-contaminated poultry litter in environmental samples. Based on sensitivity and specificity characteristics of the qPCR method, the Bayesian conditional probability that detection of the LA35 marker gene in a water sample represented a true-positive result was 93%. The marker's covariance with fecal indicator bacteria (FIB) and metals associated with poultry litter was also assessed in litter, runoff, surface water, and groundwater samples. LA35 was detected in water and soil samples collected throughout the watershed, and its concentration covaried with concentrations of Escherichia coli, enterococci, As, Cu, P, and Zn. Significantly greater concentrations of FIB, As, Cu, P, and Zn were observed in edge-of-field runoff samples in which LA35 was detected, compared to samples in which it was not detected. Furthermore, As, Cu, P, and Zn concentrations covaried in environmental samples in which LA35 was detected and typically did not in samples in which the marker gene was not detected. The covariance of the poultry-specific LA35 marker gene with these known contaminants from poultry feces provides further evidence that it is a useful tool for assessing the impact of poultry-derived fecal pollution in environmental waters.

How Much Do rRNA Gene Surveys Underestimate Extant Bacterial Diversity?

PubMed

Rodriguez-R, Luis M; Castro, Juan C; Kyrpides, Nikos C; Cole, James R; Tiedje, James M; Konstantinidis, Konstantinos T

2018-03-15

The most common practice in studying and cataloguing prokaryotic diversity involves the grouping of sequences into operational taxonomic units (OTUs) at the 97% 16S rRNA gene sequence identity level, often using partial gene sequences, such as PCR-generated amplicons. Due to the high sequence conservation of rRNA genes, organisms belonging to closely related yet distinct species may be grouped under the same OTU. However, it remains unclear how much diversity has been underestimated by this practice. To address this question, we compared the OTUs of genomes defined at the 97% or 98.5% 16S rRNA gene identity level against OTUs of the same genomes defined at the 95% whole-genome average nucleotide identity (ANI), which is a much more accurate proxy for species. Our results show that OTUs resulting from a 98.5% 16S rRNA gene identity cutoff are more accurate than 97% compared to 95% ANI (90.5% versus 89.9% accuracy) but indistinguishable from any other threshold in the 98.29 to 98.78% range. Even with the more stringent thresholds, however, the 16S rRNA gene-based approach commonly underestimates the number of OTUs by ∼12%, on average, compared to the ANI-based approach (∼14% underestimation when using the 97% identity threshold). More importantly, the degree of underestimation can become 50% or more for certain taxa, such as the genera Pseudomonas , Burkholderia , Escherichia , Campylobacter , and Citrobacter These results provide a quantitative view of the degree of underestimation of extant prokaryotic diversity by 16S rRNA gene-defined OTUs and suggest that genomic resolution is often necessary. IMPORTANCE Species diversity is one of the most fundamental pieces of information for community ecology and conservational biology. Therefore, employing accurate proxies for what a species or the unit of diversity is are cornerstones for a large set of microbial ecology and diversity studies. The most common proxies currently used rely on the clustering of 16S rRNA gene sequences at some threshold of nucleotide identity, typically 97% or 98.5%. Here, we explore how well this strategy reflects the more accurate whole-genome-based proxies and determine the frequency with which the high conservation of 16S rRNA sequences masks substantial species-level diversity. Copyright © 2018 American Society for Microbiology.

Specific detection and identification of [Actinobacillus] muris by PCR using primers targeting the 16S-23S rRNA internal transcribed spacer regions.

PubMed

Benga, Laurentiu; Benten, W Peter M; Engelhardt, Eva; Gougoula, Christina; Sager, Martin

2013-08-01

[Actinobacillus] muris represents along with [Pasteurella] pneumotropica the most prevalent Pasteurellaceae species isolated from the laboratory mouse. Despite the biological and economic importance of Pasteurellaceae in relation to experimental animals, no molecular based methods for the identification of [A.] muris are available. The aim of the present investigation was to develop a PCR method allowing detection and identification of [A.] muris. In this assay, a Pasteurellaceae common forward primer based on a conserved region of the 16S rRNA gene was used in conjunction with two different reverse primers specific for [A.] muris, targeting the 16S-23S internal transcribed spacer sequences. The specificity of the assay was tested against 78 reference and clinical isolates of Pasteurellaceae, including 37 strains of [A.] muris. In addition, eight other mice associated bacterial species which could pose a diagnostic problem were included. The assay showed 100% sensitivity and 97.95% specificity. Identification of the clinical isolates was validated by ITS profiling and when necessary by 16S rRNA sequencing. This multiplex PCR represents the first molecular tool able to detect [A.] muris and may become a reliable alternative to the present diagnostic methods. Copyright © 2013 Elsevier B.V. All rights reserved.

Lessons from an evolving rRNA: 16S and 23S rRNA structures from a comparative perspective

NASA Technical Reports Server (NTRS)

Gutell, R. R.; Larsen, N.; Woese, C. R.

1994-01-01

The 16S and 23S rRNA higher-order structures inferred from comparative analysis are now quite refined. The models presented here differ from their immediate predecessors only in minor detail. Thus, it is safe to assert that all of the standard secondary-structure elements in (prokaryotic) rRNAs have been identified, with approximately 90% of the individual base pairs in each molecule having independent comparative support, and that at least some of the tertiary interactions have been revealed. It is interesting to compare the rRNAs in this respect with tRNA, whose higher-order structure is known in detail from its crystal structure (36) (Table 2). It can be seen that rRNAs have as great a fraction of their sequence in established secondary-structure elements as does tRNA. However, the fact that the former show a much lower fraction of identified tertiary interactions and a greater fraction of unpaired nucleotides than the latter implies that many of the rRNA tertiary interactions remain to be located. (Alternatively, the ribosome might involve protein-rRNA rather than intramolecular rRNA interactions to stabilize three-dimensional structure.) Experimental studies on rRNA are consistent to a first approximation with the structures proposed here, confirming the basic assumption of comparative analysis, i.e., that bases whose compositions strictly covary are physically interacting. In the exhaustive study of Moazed et al. (45) on protection of the bases in the small-subunit rRNA against chemical modification, the vast majority of bases inferred to pair by covariation are found to be protected from chemical modification, both in isolated small-subunit rRNA and in the 30S subunit. The majority of the tertiary interactions are reflected in the chemical protection data as well (45). On the other hand, many of the bases not shown as paired in Fig. 1 are accessible to chemical attack (45). However, in this case a sizeable fraction of them are also protected against chemical modification (in the isolated rRNA), which suggests that considerable higher-order structure remains to be found (although all of it may not involve base-base interactions and so may not be detectable by comparative analysis). The agreement between the higher-order structure of the small-subunit rRNA and protection against chemical modification is not perfect, however; some bases shown to covary canonically are accessible to chemical modification (45).(ABSTRACT TRUNCATED AT 400 WORDS).

Oligonucleotide Microarray for 16S rRNA Gene-Based Detection of All Recognized Lineages of Sulfate-Reducing Prokaryotes in the Environment

PubMed Central

Loy, Alexander; Lehner, Angelika; Lee, Natuschka; Adamczyk, Justyna; Meier, Harald; Ernst, Jens; Schleifer, Karl-Heinz; Wagner, Michael

2002-01-01

For cultivation-independent detection of sulfate-reducing prokaryotes (SRPs) an oligonucleotide microarray consisting of 132 16S rRNA gene-targeted oligonucleotide probes (18-mers) having hierarchical and parallel (identical) specificity for the detection of all known lineages of sulfate-reducing prokaryotes (SRP-PhyloChip) was designed and subsequently evaluated with 41 suitable pure cultures of SRPs. The applicability of SRP-PhyloChip for diversity screening of SRPs in environmental and clinical samples was tested by using samples from periodontal tooth pockets and from the chemocline of a hypersaline cyanobacterial mat from Solar Lake (Sinai, Egypt). Consistent with previous studies, SRP-PhyloChip indicated the occurrence of Desulfomicrobium spp. in the tooth pockets and the presence of Desulfonema- and Desulfomonile-like SRPs (together with other SRPs) in the chemocline of the mat. The SRP-PhyloChip results were confirmed by several DNA microarray-independent techniques, including specific PCR amplification, cloning, and sequencing of SRP 16S rRNA genes and the genes encoding the dissimilatory (bi)sulfite reductase (dsrAB). PMID:12324358

Q-PCR based bioburden assessment of drinking water throughout treatment and delivery to the International Space Station

NASA Technical Reports Server (NTRS)

Newcombe, David; Stuecker, Tara; La Duc, Myron; Venkateswaran, Kasthuri

2005-01-01

Previous studies indicated evidence of opportunistic pathogens samples obtained during missions to the International Space Station (ISS). This study utilized TaqMan quantitative PCR to determine specific gene abundance in potable and non-potable ISS waters. Probe and primer sets specific to the small subunit rRNA genes were used to elucidate overall bacterial rRNA gene numbers. while those specific for Burkholderia cepacia and Stenotrophomonas maltophilia were optimized and used to probe for the presence of these two opportunistic pathogens. This research builds upon previous microbial diversity studies of ISS water and demonstrates the utility of Q-PCR tool to examine water quality.

Comparative evaluation of rRNA depletion procedures for the improved analysis of bacterial biofilm and mixed pathogen culture transcriptomes

PubMed Central

Petrova, Olga E.; Garcia-Alcalde, Fernando; Zampaloni, Claudia; Sauer, Karin

2017-01-01

Global transcriptomic analysis via RNA-seq is often hampered by the high abundance of ribosomal (r)RNA in bacterial cells. To remove rRNA and enrich coding sequences, subtractive hybridization procedures have become the approach of choice prior to RNA-seq, with their efficiency varying in a manner dependent on sample type and composition. Yet, despite an increasing number of RNA-seq studies, comparative evaluation of bacterial rRNA depletion methods has remained limited. Moreover, no such study has utilized RNA derived from bacterial biofilms, which have potentially higher rRNA:mRNA ratios and higher rRNA carryover during RNA-seq analysis. Presently, we evaluated the efficiency of three subtractive hybridization-based kits in depleting rRNA from samples derived from biofilm, as well as planktonic cells of the opportunistic human pathogen Pseudomonas aeruginosa. Our results indicated different rRNA removal efficiency for the three procedures, with the Ribo-Zero kit yielding the highest degree of rRNA depletion, which translated into enhanced enrichment of non-rRNA transcripts and increased depth of RNA-seq coverage. The results indicated that, in addition to improving RNA-seq sensitivity, efficient rRNA removal enhanced detection of low abundance transcripts via qPCR. Finally, we demonstrate that the Ribo-Zero kit also exhibited the highest efficiency when P. aeruginosa/Staphylococcus aureus co-culture RNA samples were tested. PMID:28117413

A method for release and multiple strand amplification of small quantities of DNA from endospores of the fastidious bacterium Pasteuria penetrans.

PubMed

Mauchline, T H; Mohan, S; Davies, K G; Schaff, J E; Opperman, C H; Kerry, B R; Hirsch, P R

2010-05-01

To establish a reliable protocol to extract DNA from Pasteuria penetrans endospores for use as template in multiple strand amplification, thus providing sufficient material for genetic analyses. To develop a highly sensitive PCR-based diagnostic tool for P. penetrans. An optimized method to decontaminate endospores, release and purify DNA enabled multiple strand amplification. DNA purity was assessed by cloning and sequencing gyrB and 16S rRNA gene fragments obtained from PCR using generic primers. Samples indicated to be 100%P. penetrans by the gyrB assay were estimated at 46% using the 16S rRNA gene. No bias was detected on cloning and sequencing 12 housekeeping and sporulation gene fragments from amplified DNA. The detection limit by PCR with Pasteuria-specific 16S rRNA gene primers following multiple strand amplification of DNA extracted using the method was a single endospore. Generation of large quantities DNA will facilitate genomic sequencing of P. penetrans. Apparent differences in sample purity are explained by variations in 16S rRNA gene copy number in Eubacteria leading to exaggerated estimations of sample contamination. Detection of single endospores will facilitate investigations of P. penetrans molecular ecology. These methods will advance studies on P. penetrans and facilitate research on other obligate and fastidious micro-organisms where it is currently impractical to obtain DNA in sufficient quantity and quality.

Molecular Diagnosis of Invasive Aspergillosis and Detection of Azole Resistance by a Newly Commercialized PCR Kit

PubMed Central

Gabriel, Frédéric; Gaboyard, Manuel; Lagardere, Gaëlle; Audebert, Lucile; Quesne, Gilles; Godichaud, Sandrine; Verweij, Paul E.; Accoceberry, Isabelle

2017-01-01

ABSTRACT Aspergillus fumigatus is the main species responsible for aspergillosis in humans. The diagnosis of aspergillosis remains difficult, and the rapid emergence of azole resistance in A. fumigatus is worrisome. The aim of this study was to validate the new MycoGENIE A. fumigatus real-time PCR kit and to evaluate its performance on clinical samples for the detection of A. fumigatus and its azole resistance. This multiplex assay detects DNA from the A. fumigatus species complex by targeting the multicopy 28S rRNA gene and specific TR34 and L98H mutations in the single-copy-number cyp51A gene of A. fumigatus. The specificity of cyp51A mutation detection was assessed by testing DNA samples from 25 wild-type or mutated clinical A. fumigatus isolates. Clinical validation was performed on 88 respiratory samples obtained from 62 patients and on 69 serum samples obtained from 16 patients with proven or probable aspergillosis and 13 patients without aspergillosis. The limit of detection was <1 copy for the Aspergillus 28S rRNA gene and 6 copies for the cyp51A gene harboring the TR34 and L98H alterations. No cross-reactivity was detected with various fungi and bacteria. All isolates harboring the TR34 and L98H mutations were accurately detected by quantitative PCR (qPCR) analysis. With respiratory samples, qPCR results showed a sensitivity and specificity of 92.9% and 90.1%, respectively, while with serum samples, the sensitivity and specificity were 100% and 84.6%, respectively. Our study demonstrated that this new real-time PCR kit enables sensitive and rapid detection of A. fumigatus DNA and azole resistance due to TR34 and L98H mutations in clinical samples. PMID:28814586

16S rRNA gene-based phylogenetic microarray for simultaneous identification of members of the genus Burkholderia.

PubMed

Schönmann, Susan; Loy, Alexander; Wimmersberger, Céline; Sobek, Jens; Aquino, Catharine; Vandamme, Peter; Frey, Beat; Rehrauer, Hubert; Eberl, Leo

2009-04-01

For cultivation-independent and highly parallel analysis of members of the genus Burkholderia, an oligonucleotide microarray (phylochip) consisting of 131 hierarchically nested 16S rRNA gene-targeted oligonucleotide probes was developed. A novel primer pair was designed for selective amplification of a 1.3 kb 16S rRNA gene fragment of Burkholderia species prior to microarray analysis. The diagnostic performance of the microarray for identification and differentiation of Burkholderia species was tested with 44 reference strains of the genera Burkholderia, Pandoraea, Ralstonia and Limnobacter. Hybridization patterns based on presence/absence of probe signals were interpreted semi-automatically using the novel likelihood-based strategy of the web-tool Phylo- Detect. Eighty-eight per cent of the reference strains were correctly identified at the species level. The evaluated microarray was applied to investigate shifts in the Burkholderia community structure in acidic forest soil upon addition of cadmium, a condition that selected for Burkholderia species. The microarray results were in agreement with those obtained from phylogenetic analysis of Burkholderia 16S rRNA gene sequences recovered from the same cadmiumcontaminated soil, demonstrating the value of the Burkholderia phylochip for determinative and environmental studies.

Discrimination of Bacillus anthracis from closely related microorganisms by analysis of 16S and 23S rRNA with oligonucleotide microchips

DOEpatents

Bavykin, Sergei G.; Mirzabekova, legal representative, Natalia V.; Mirzabekov, deceased, Andrei D.

2007-12-04

The present invention relates to methods and compositions for using nucleotide sequence variations of 16S and 23S rRNA within the B. cereus group to discriminate a highly infectious bacterium B. anthracis from closely related microorganisms. Sequence variations in the 16S and 23S rRNA of the B. cereus subgroup including B. anthracis are utilized to construct an array that can detect these sequence variations through selective hybridizations and discriminate B. cereus group that includes B. anthracis. Discrimination of single base differences in rRNA was achieved with a microchip during analysis of B. cereus group isolates from both single and in mixed samples, as well as identification of polymorphic sites. Successful use of a microchip to determine the appropriate subgroup classification using eight reference microorganisms from the B. cereus group as a study set, was demonstrated.

Real-Time PCR Assay for Detection and Enumeration of Dekkera bruxellensis in Wine

PubMed Central

Phister, Trevor G.; Mills, David A.

2003-01-01

Traditional methods to detect the spoilage yeast Dekkera bruxellensis from wine involve lengthy enrichments. To overcome this difficulty, we developed a quantitative real-time PCR method to directly detect and enumerate D. bruxellensis in wine. Specific PCR primers to D. bruxellensis were designed to the 26S rRNA gene, and nontarget yeast and bacteria common to the winery environment were not amplified. The assay was linear over a range of cell concentrations (6 log units) and could detect as little as 1 cell per ml in wine. The addition of large amounts of nontarget yeasts did not impact the efficiency of the assay. This method will be helpful to identify possible routes of D. bruxellensis infection in winery environments. Moreover, the time involved in performing the assay (3 h) should enable winemakers to more quickly make wine processing decisions in order to reduce the threat of spoilage by D. bruxellensis. PMID:14660395

Phylogenetic diversity and in situ detection of eukaryotes in anaerobic sludge digesters.

PubMed

Matsubayashi, Miri; Shimada, Yusuke; Li, Yu-You; Harada, Hideki; Kubota, Kengo

2017-01-01

Eukaryotic communities in aerobic wastewater treatment processes are well characterized, but little is known about them in anaerobic processes. In this study, abundance, diversity and morphology of eukaryotes in anaerobic sludge digesters were investigated by quantitative real-time PCR (qPCR), 18S rRNA gene clone library construction and catalyzed reporter deposition-fluorescence in situ hybridization (CARD-FISH). Samples were taken from four different anaerobic sludge digesters in Japan. Results of qPCR of rRNA genes revealed that Eukarya accounted from 0.1% to 1.4% of the total number of microbial rRNA gene copy numbers. The phylogenetic affiliations of a total of 251 clones were Fungi, Alveolata, Viridiplantae, Amoebozoa, Rhizaria, Stramenopiles and Metazoa. Eighty-five percent of the clones showed less than 97.0% sequence identity to described eukaryotes, indicating most of the eukaryotes in anaerobic sludge digesters are largely unknown. Clones belonging to the uncultured lineage LKM11 in Cryptomycota of Fungi were most abundant in anaerobic sludge, which accounted for 50% of the total clones. The most dominant OTU in each library belonged to either the LKM11 lineage or the uncultured lineage A31 in Alveolata. Principal coordinate analysis indicated that the eukaryotic and prokaryotic community structures were related. The detection of anaerobic eukaryotes, including the members of the LKM11 and A31 lineages in anaerobic sludge digesters, by CARD-FISH revealed their sizes in the range of 2-8 μm. The diverse and uncultured eukaryotes in the LKM11 and the A31 lineages are common and ecologically relevant members in anaerobic sludge digester.

Detection of a mixed infection in a culture-negative brain abscess by broad-spectrum bacterial 16S rRNA gene PCR.

PubMed

Keller, Peter M; Rampini, Silvana K; Bloemberg, Guido V

2010-06-01

We describe the identification of two bacterial pathogens from a culture-negative brain abscess by the use of broad-spectrum 16S rRNA gene PCR. Simultaneous detection of Fusobacterium nucleatum and Porphyromonas endodontalis was possible due to a 24-bp length difference of their partially amplified 16S rRNA genes, which allowed separation by high-resolution polyacrylamide gel electrophoresis.

A broad range assay for rapid detection and etiologic characterization of bacterial meningitis: performance testing in samples from sub-Sahara.

PubMed

Won, Helen; Yang, Samuel; Gaydos, Charlotte; Hardick, Justin; Ramachandran, Padmini; Hsieh, Yu-Hsiang; Kecojevic, Alexander; Njanpop-Lafourcade, Berthe-Marie; Mueller, Judith E; Tameklo, Tsidi Agbeko; Badziklou, Kossi; Gessner, Bradford D; Rothman, Richard E

2012-09-01

This study aimed to conduct a pilot evaluation of broad-based multiprobe polymerase chain reaction (PCR) in clinical cerebrospinal fluid (CSF) samples compared to local conventional PCR/culture methods used for bacterial meningitis surveillance. A previously described PCR consisting of initial broad-based detection of Eubacteriales by a universal probe, followed by Gram typing, and pathogen-specific probes was designed targeting variable regions of the 16S rRNA gene. The diagnostic performance of the 16S rRNA assay in "127 CSF samples was evaluated in samples from patients from Togo, Africa, by comparison to conventional PCR/culture methods. Our probes detected Neisseria meningitidis, Streptococcus pneumoniae, and Haemophilus influenzae. Uniprobe sensitivity and specificity versus conventional PCR were 100% and 54.6%, respectively. Sensitivity and specificity of uniprobe versus culture methods were 96.5% and 52.5%, respectively. Gram-typing probes correctly typed 98.8% (82/83) and pathogen-specific probes identified 96.4% (80/83) of the positives. This broad-based PCR algorithm successfully detected and provided species level information for multiple bacterial meningitis agents in clinical samples. Copyright © 2012 Elsevier Inc. All rights reserved.

A broad range assay for rapid detection and etiologic characterization of bacterial meningitis: performance testing in samples from sub-Sahara☆, ☆☆,★

PubMed Central

Won, Helen; Yang, Samuel; Gaydos, Charlotte; Hardick, Justin; Ramachandran, Padmini; Hsieh, Yu-Hsiang; Kecojevic, Alexander; Njanpop-Lafourcade, Berthe-Marie; Mueller, Judith E.; Tameklo, Tsidi Agbeko; Badziklou, Kossi; Gessner, Bradford D.; Rothman, Richard E.

2012-01-01

This study aimed to conduct a pilot evaluation of broad-based multiprobe polymerase chain reaction (PCR) in clinical cerebrospinal fluid (CSF) samples compared to local conventional PCR/culture methods used for bacterial meningitis surveillance. A previously described PCR consisting of initial broad-based detection of Eubacteriales by a universal probe, followed by Gram typing, and pathogen-specific probes was designed targeting variable regions of the 16S rRNA gene. The diagnostic performance of the 16S rRNA assay in “”127 CSF samples was evaluated in samples from patients from Togo, Africa, by comparison to conventional PCR/culture methods. Our probes detected Neisseria meningitidis, Streptococcus pneumoniae, and Haemophilus influenzae. Uniprobe sensitivity and specificity versus conventional PCR were 100% and 54.6%, respectively. Sensitivity and specificity of uniprobe versus culture methods were 96.5% and 52.5%, respectively. Gram-typing probes correctly typed 98.8% (82/83) and pathogen-specific probes identified 96.4% (80/83) of the positives. This broad-based PCR algorithm successfully detected and provided species level information for multiple bacterial meningitis agents in clinical samples. PMID:22809694

Design and Evaluation of a Lactobacillus manihotivorans Species-Specific rRNA-Targeted Hybridization Probe and Its Application to the Study of Sour Cassava Fermentation

PubMed Central

Ampe, Frédéric

2000-01-01

Based on 16S rRNA sequence comparison, we have designed a 20-mer oligonucleotide that targets a region specific to the species Lactobacillus manihotivorans recently isolated from sour cassava fermentation. The probe recognized the rRNA obtained from all the L. manihotivorans strains tested but did not recognize 56 strains of microorganisms from culture collections or directly isolated from sour cassava, including 29 species of lactic acid bacteria. This probe was then successfully used in quantitative RNA blots and demonstrated the importance of L. manihotivorans in the fermentation of sour cassava starch, which could represent up to 20% of total lactic acid bacteria. PMID:10788405

Oligonucleotide primers, probes and molecular methods for the environmental monitoring of methanogenic archaea

PubMed Central

Narihiro, Takashi; Sekiguchi, Yuji

2011-01-01

Summary For the identification and quantification of methanogenic archaea (methanogens) in environmental samples, various oligonucleotide probes/primers targeting phylogenetic markers of methanogens, such as 16S rRNA, 16S rRNA gene and the gene for the α‐subunit of methyl coenzyme M reductase (mcrA), have been extensively developed and characterized experimentally. These oligonucleotides were designed to resolve different groups of methanogens at different taxonomic levels, and have been widely used as hybridization probes or polymerase chain reaction primers for membrane hybridization, fluorescence in situ hybridization, rRNA cleavage method, gene cloning, DNA microarray and quantitative polymerase chain reaction for studies in environmental and determinative microbiology. In this review, we present a comprehensive list of such oligonucleotide probes/primers, which enable us to determine methanogen populations in an environment quantitatively and hierarchically, with examples of the practical applications of the probes and primers. PMID:21375721

Microbial community profiling of fresh basil and pitfalls in taxonomic assignment of enterobacterial pathogenic species based upon 16S rRNA amplicon sequencing.

PubMed

Ceuppens, Siele; De Coninck, Dieter; Bottledoorn, Nadine; Van Nieuwerburgh, Filip; Uyttendaele, Mieke

2017-09-18

Application of 16S rRNA (gene) amplicon sequencing on food samples is increasingly applied for assessing microbial diversity but may as unintended advantage also enable simultaneous detection of any human pathogens without a priori definition. In the present study high-throughput next-generation sequencing (NGS) of the V1-V2-V3 regions of the 16S rRNA gene was applied to identify the bacteria present on fresh basil leaves. However, results were strongly impacted by variations in the bioinformatics analysis pipelines (MEGAN, SILVAngs, QIIME and MG-RAST), including the database choice (Greengenes, RDP and M5RNA) and the annotation algorithm (best hit, representative hit and lowest common ancestor). The use of pipelines with default parameters will lead to discrepancies. The estimate of microbial diversity of fresh basil using 16S rRNA (gene) amplicon sequencing is thus indicative but subject to biases. Salmonella enterica was detected at low frequencies, between 0.1% and 0.4% of bacterial sequences, corresponding with 37 to 166 reads. However, this result was dependent upon the pipeline used: Salmonella was detected by MEGAN, SILVAngs and MG-RAST, but not by QIIME. Confirmation of Salmonella sequences by real-time PCR was unsuccessful. It was shown that taxonomic resolution obtained from the short (500bp) sequence reads of the 16S rRNA gene containing the hypervariable regions V1-V3 cannot allow distinction of Salmonella with closely related enterobacterial species. In conclusion 16S amplicon sequencing, getting the status of standard method in microbial ecology studies of foods, needs expertise on both bioinformatics and microbiology for analysis of results. It is a powerful tool to estimate bacterial diversity but amenable to biases. Limitations concerning taxonomic resolution for some bacterial species or its inability to detect sub-dominant (pathogenic) species should be acknowledged in order to avoid overinterpretation of results. Copyright © 2017 Elsevier B.V. All rights reserved.

Molecular analysis of the rRNA genes of Babesia spp and Ehrlichia canis detected in dogs from RibeirÃo Preto, Brazil

PubMed Central

Oliveira, L.P.; Cardozo, G.P.; Santos, E.V.; Mansur, M.A.B.; Donini, I.A.N.; Zissou, V.G.; Roberto, P.G.; Marins, M.

2009-01-01

The partial DNA sequences of the 18S rRNA gene of Babesia canis and the 16S rRNA gene of Ehrlichia canis detected in dogs from Ribeirão Preto, Brazil, were compared to sequences from other strains deposited in GenBank. The E. canis strain circulating in Ribeirão Preto is identical to other strains previously detected in the region, whereas the subspecies Babesia canis vogeli is the main Babesia strain circulating in dogs from Ribeirão Preto. PMID:24031351

Detection of Methanotroph Diversity on Roots of Submerged Rice Plants by Molecular Retrieval of pmoA, mmoX, mxaF, and 16S rRNA and Ribosomal DNA, Including pmoA-Based Terminal Restriction Fragment Length Polymorphism Profiling

PubMed Central

Horz, Hans-Peter; Yimga, Merlin Tchawa; Liesack, Werner

2001-01-01

The diversity of methanotrophic bacteria associated with roots of submerged rice plants was assessed using cultivation-independent techniques. The research focused mainly on the retrieval of pmoA, which encodes the α subunit of the particulate methane monooxygenase. A novel methanotroph-specific community-profiling method was established using the terminal restriction fragment length polymorphism (T-RFLP) technique. The T-RFLP profiles clearly revealed a more complex root-associated methanotrophic community than did banding patterns obtained by pmoA-based denaturing gradient gel electrophoresis. The comparison of pmoA-based T-RFLP profiles obtained from rice roots and bulk soil of flooded rice microcosms suggested that there was a substantially higher abundance of type I methanotrophs on rice roots than in the bulk soil. These were affiliated to the genera Methylomonas, Methylobacter, Methylococcus, and to a novel type I methanotroph sublineage. By contrast, type II methanotrophs of the Methylocystis-Methylosinus group could be detected with high relative signal intensity in both soil and root compartments. Phylogenetic treeing analyses and a set of substrate-diagnostic amino acid residues provided evidence that a novel pmoA lineage was detected. This branched distinctly from all currently known methanotrophs. To examine whether the retrieval of pmoA provided a complete view of root-associated methanotroph diversity, we also assessed the diversity detectable by recovery of genes coding for subunits of soluble methane monooxygenase (mmoX) and methanol dehydrogenase (mxaF). In addition, both 16S rRNA and 16S ribosomal DNA (rDNA) were retrieved using a PCR primer set specific to type I methanotrophs. The overall methanotroph diversity detected by recovery of mmoX, mxaF, and 16S rRNA and 16S rDNA corresponded well to the diversity detectable by retrieval of pmoA. PMID:11526021

Detection of a Mixed Infection in a Culture-Negative Brain Abscess by Broad-Spectrum Bacterial 16S rRNA Gene PCR ▿ †

PubMed Central

Keller, Peter M.; Rampini, Silvana K.; Bloemberg, Guido V.

2010-01-01

We describe the identification of two bacterial pathogens from a culture-negative brain abscess by the use of broad-spectrum 16S rRNA gene PCR. Simultaneous detection of Fusobacterium nucleatum and Porphyromonas endodontalis was possible due to a 24-bp length difference of their partially amplified 16S rRNA genes, which allowed separation by high-resolution polyacrylamide gel electrophoresis. PMID:20392909

Detection and characterization of Pasteuria 16S rRNA gene sequences from nematodes and soils.

PubMed

Duan, Y P; Castro, H F; Hewlett, T E; White, J H; Ogram, A V

2003-01-01

Various bacterial species in the genus Pasteuria have great potential as biocontrol agents against plant-parasitic nematodes, although study of this important genus is hampered by the current inability to cultivate Pasteuria species outside their host. To aid in the study of this genus, an extensive 16S rRNA gene sequence phylogeny was constructed and this information was used to develop cultivation-independent methods for detection of Pasteuria in soils and nematodes. Thirty new clones of Pasteuria 16S rRNA genes were obtained directly from nematodes and soil samples. These were sequenced and used to construct an extensive phylogeny of this genus. These sequences were divided into two deeply branching clades within the low-G + C, Gram-positive division; some sequences appear to represent novel species within the genus Pasteuria. In addition, a surprising degree of 16S rRNA gene sequence diversity was observed within what had previously been designated a single strain of Pasteuria penetrans (P-20). PCR primers specific to Pasteuria 16S rRNA for detection of Pasteuria in soils were also designed and evaluated. Detection limits for soil DNA were 100-10,000 Pasteuria endospores (g soil)(-1).

Development of a quantitative PCR assay for monitoring Streptococcus agalactiae colonization and tissue tropism in experimentally infected tilapia.

PubMed

Su, Y-L; Feng, J; Li, Y-W; Bai, J-S; Li, A-X

2016-02-01

Streptococcus agalactiae has become one of the most important emerging pathogens in the aquaculture industry and has resulted in large economic losses for tilapia farms in China. In this study, three pairs of specific primers were designed and tested for their specificities and sensitivities in quantitative real-time polymerase chain reactions (qPCRs) after optimization of the annealing temperature. The primer pair IGS-s/IGS-a, which targets the 16S-23S rRNA intergenic spacer region, was finally chosen, having a detection limit of 8.6 copies of S. agalactiae DNA in a 20 μL reaction mixture. Bacterial tissue tropism was demonstrated by qPCR in Oreochromis niloticus 5 days post-injection with a virulent S. agalactiae strain. Bacterial loads were detected at the highest level in brain, followed by moderately high levels in kidney, heart, spleen, intestines, and eye. Significantly lower bacterial loads were observed in muscle, gill and liver. In addition, significantly lower bacterial loads were observed in the brain of convalescent O. niloticus 14 days post-injection with several different S. agalactiae strains. The qPCR for the detection of S. agalactiae developed in this study provides a quantitative tool for investigating bacterial tissue tropism in infected fish, as well as for monitoring bacterial colonization in convalescent fish. © 2015 John Wiley & Sons Ltd.

Benchmarking taxonomic assignments based on 16S rRNA gene profiling of the microbiota from commonly sampled environments.

PubMed

Almeida, Alexandre; Mitchell, Alex L; Tarkowska, Aleksandra; Finn, Robert D

2018-05-01

Taxonomic profiling of ribosomal RNA (rRNA) sequences has been the accepted norm for inferring the composition of complex microbial ecosystems. Quantitative Insights Into Microbial Ecology (QIIME) and mothur have been the most widely used taxonomic analysis tools for this purpose, with MAPseq and QIIME 2 being two recently released alternatives. However, no independent and direct comparison between these four main tools has been performed. Here, we compared the default classifiers of MAPseq, mothur, QIIME, and QIIME 2 using synthetic simulated datasets comprised of some of the most abundant genera found in the human gut, ocean, and soil environments. We evaluate their accuracy when paired with both different reference databases and variable sub-regions of the 16S rRNA gene. We show that QIIME 2 provided the best recall and F-scores at genus and family levels, together with the lowest distance estimates between the observed and simulated samples. However, MAPseq showed the highest precision, with miscall rates consistently <2%. Notably, QIIME 2 was the most computationally expensive tool, with CPU time and memory usage almost 2 and 30 times higher than MAPseq, respectively. Using the SILVA database generally yielded a higher recall than using Greengenes, while assignment results of different 16S rRNA variable sub-regions varied up to 40% between samples analysed with the same pipeline. Our results support the use of either QIIME 2 or MAPseq for optimal 16S rRNA gene profiling, and we suggest that the choice between the two should be based on the level of recall, precision, and/or computational performance required.

Use of Blood Smears and Dried Blood Spots for Polymerase Chain Reaction-Based Detection and Quantification of Bacterial Infection and Plasmodium falciparum in Severely Ill Febrile African Children.

PubMed

Wihokhoen, Benchawan; Dondorp, Arjen M; Turner, Paul; Woodrow, Charles J; Imwong, Mallika

2016-02-01

Molecular approaches offer a means of testing archived samples stored as dried blood spots in settings where standard blood cultures are not possible. Peripheral blood films are one suggested source of material, although the sensitivity of this approach has not been well defined. Thin blood smears and dried blood spots from a severe pediatric malaria study were assessed using specific polymerase chain reaction (PCR) primers to detect non-typhoidal Salmonella (NTS; MisL gene), Streptococcus pneumoniae (lytA), and Plasmodium falciparum (18S rRNA). Of 16 cases of NTS and S. pneumoniae confirmed on blood culture, none were positive by PCR using DNA extracts from blood films or dried blood spots. In contrast, four of 36 dried blood spots and two of 178 plasma samples were PCR positive for S. pneumoniae, despite negative bacterial blood cultures, suggesting false positives. Quantitative assessment revealed that the effective concentration of P. falciparum DNA in blood films was three log orders of magnitude lower than for dried blood spots. The P. falciparum kelch13 gene could not be amplified from blood films. These findings question the value of blood PCR-based approaches for detection of NTS and S. pneumoniae, and show that stored blood films are an inefficient method of studying P. falciparum. © The American Society of Tropical Medicine and Hygiene.

Use of Blood Smears and Dried Blood Spots for Polymerase Chain Reaction–Based Detection and Quantification of Bacterial Infection and Plasmodium falciparum in Severely Ill Febrile African Children

PubMed Central

Wihokhoen, Benchawan; Dondorp, Arjen M.; Turner, Paul; Woodrow, Charles J.; Imwong, Mallika

2016-01-01

Molecular approaches offer a means of testing archived samples stored as dried blood spots in settings where standard blood cultures are not possible. Peripheral blood films are one suggested source of material, although the sensitivity of this approach has not been well defined. Thin blood smears and dried blood spots from a severe pediatric malaria study were assessed using specific polymerase chain reaction (PCR) primers to detect non-typhoidal Salmonella (NTS; MisL gene), Streptococcus pneumoniae (lytA), and Plasmodium falciparum (18S rRNA). Of 16 cases of NTS and S. pneumoniae confirmed on blood culture, none were positive by PCR using DNA extracts from blood films or dried blood spots. In contrast, four of 36 dried blood spots and two of 178 plasma samples were PCR positive for S. pneumoniae, despite negative bacterial blood cultures, suggesting false positives. Quantitative assessment revealed that the effective concentration of P. falciparum DNA in blood films was three log orders of magnitude lower than for dried blood spots. The P. falciparum kelch13 gene could not be amplified from blood films. These findings question the value of blood PCR-based approaches for detection of NTS and S. pneumoniae, and show that stored blood films are an inefficient method of studying P. falciparum. PMID:26711525

Detection and molecular status of Isospora sp. from the domestic pigeon (Columba livia domestica).

PubMed

Matsubara, Ryuma; Fukuda, Yasuhiro; Murakoshi, Fumi; Nomura, Osamu; Suzuki, Toru; Tada, Chika; Nakai, Yutaka

2017-10-01

The domestic pigeon, Columba livia domestica, is reared for meat production, as a pet, or for racing. Few reports have characterized the parasitic protists from the genus Isospora isolated from Columbiformes. We detected Isospora-like oocysts from C. livia reared for racing. The oocyst contained two sporocysts, and each sporocyst included four sporozoites. The sporulated oocysts (n=4) were spherical; their mean diameters were 25.6 (24.0-27.2)×24.7 (23.4-26.0) μm. Micropyles, polar granules, and oocyst residuum were absent. The mean length and width of the sporocysts (n=8) were 19.5 (18.5-20.5) and 11.2 (10.2-12.1) μm, respectively. Stieda and sub-Stieda bodies were observed. Single-oocyst PCR revealed two different 18S rRNA gene sequences and one 28S rRNA gene sequence in a single oocyst of Isospora sp. Based on a phylogenetic analysis of the 18S rRNA gene, the two sequences made a group which fell within a cluster of known avian Isospora species. A tree based on the 28S rRNA gene sequence indicated that sequences from the pigeon Isospora sp. fell within a cluster of avian Isospora species. Both trees failed to clarify the phylogenetic relationships among the avian Isospora species due to limited resolution. Because the morphological description of Isospora sp. is based on only four oocysts, Isospora sp. is not proposed as a novel species here. This is the first description of Isospora sp. isolated from the domestic pigeon C. livia. Copyright © 2017 Elsevier B.V. All rights reserved.

Evolutionary Origin and Conserved Structural Building Blocks of Riboswitches and Ribosomal RNAs: Riboswitches as Probable Target Sites for Aminoglycosides Interaction.

PubMed

Mehdizadeh Aghdam, Elnaz; Barzegar, Abolfazl; Hejazi, Mohammad Saeid

2014-01-01

Riboswitches, as noncoding RNA sequences, control gene expression through direct ligand binding. Sporadic reports on the structural relation of riboswitches with ribosomal RNAs (rRNA), raises an interest in possible similarity between riboswitches and rRNAs evolutionary origins. Since aminoglycoside antibiotics affect microbial cells through binding to functional sites of the bacterial rRNA, finding any conformational and functional relation between riboswitches/rRNAs is utmost important in both of medicinal and basic research. Analysis of the riboswitches structures were carried out using bioinformatics and computational tools. The possible functional similarity of riboswitches with rRNAs was evaluated based on the affinity of paromomycin antibiotic (targeting "A site" of 16S rRNA) to riboswitches via docking method. There was high structural similarity between riboswitches and rRNAs, but not any particular sequence based similarity between them was found. The building blocks including "hairpin loop containing UUU", "peptidyl transferase center conserved hairpin A loop"," helix 45" and "S2 (G8) hairpin" as high identical rRNA motifs were detected in all kinds of riboswitches. Surprisingly, binding energies of paromomycin with different riboswitches are considerably better than the binding energy of paromomycin with "16S rRNA A site". Therefore the high affinity of paromomycin to bind riboswitches in comparison with rRNA "A site" suggests a new insight about riboswitches as possible targets for aminoglycoside antibiotics. These findings are considered as a possible supporting evidence for evolutionary origin of riboswitches/rRNAs and also their role in the exertion of antibiotics effects to design new drugs based on the concomitant effects via rRNA/riboswitches.

Ammonia oxidation driven by archaea rather than bacteria in the hot spring at Tengchong geothermal field, China.

NASA Astrophysics Data System (ADS)

Chen, Shun; Peng, Xiaotong; Xu, Hengchao; Li, Jiwei; Ta, Kaiwen

2015-04-01

The occurrence of microbial mediated ammonia oxidation and these organisms are present in large numbers in natural environments indicated a potential biogeochemical role for them in the global nitrogen cycle. However, very little is understood about their role and contribution to nitrification in the high temperature extreme environments. Here we explore the ammonia oxidation rates and abundance of potential ammonia-oxidizing archaea (AOA) in upper and bottom sediments from Gongxiaoshe hot spring, Tengchong, Yunnan, China. The 15N-incorporating AOA cells and cell aggregated were detected with Fluorescence in situ hybridization (FISH) and Nano secondary ion mass spectrometry (Nano-SIMS). Ammonia oxidation rates measured using 15N-NO3- pool dilution in upper and bottom sediments (without NH4+ stimulated) were 4.8 and 5.3 nmol N g-1h-1, respectively. Close relatives of the autotrophic, ammonia-oxidizing archaeon 'Candidatus Nitrosocaldus yellowstonii' represented the most abundant OTU in both of the two spring sediments by 16S rRNA gene analysis. Furthermore, it should be noted that no ammonia-oxidizing bacterial clones detected in this study. Quantitative PCR (qPCR) indicated that AOA and 16S rRNA genes were present at 2.75-9.80×105 and 0.128-1.96×108 gene copies g-1 sediment. Based on the reaction rates and AOA abundance, we estimated the cell-specific nitrification rates were 0.41 to 0.79 fmol N archaeal cell-1 h-1, which are comparable to those observed in estuary environment. We suggest that AOA have the responsibility in nitrification in this hot spring, and these archaea rather than bacteria may be considered as a driver in nitrogen cycling in terrestrial hot ecosystems. Key words: ammonia-oxidizing archaea (AOA); nitrification; ammonia-oxidizing rate; hot spring;

Primers to block the amplification of symbiotic apostome ciliate 18S rRNA gene in a PCR-based copepod diet study

NASA Astrophysics Data System (ADS)

Yi, Xiaoyan; Zhang, Huan; Liu, Guangxing

2014-05-01

Pelagic copepods play an important role in the marine food web. However, a full understanding of the ecological status of this zooplankton group depends on the careful study of their natural diets. In previous PCR-based copepod diet studies, we found many apostome ciliates that live symbiotically under the exoskeleton of the copepods, and their sequences were often over-represented in the 18S rRNA gene (18S rDNA) libraries. As a first step to address this issue, we designed three apostome ciliate 18S rDNA blocking primers, and tested their blocking efficiency against apostome ciliate 18s rDNA under various PCR conditions. Using a semi-quantitative PCR method, we optimized the conditions to efficiently amplify the 18S rDNA of the prey while simultaneously excluding the symbiotic apostome ciliates. This technique will facilitate PCR-based diet studies of copepods and other zooplankton in their natural environments.

Bacterial community structure in the rhizosphere of a Cry1Ac Bt-brinjal crop and comparison to its non-transgenic counterpart in the tropical soil.

PubMed

Singh, Amit Kishore; Rai, Govind Kumar; Singh, Major; Dubey, Suresh Kumar

2013-11-01

To elucidate whether the transgenic crop alters the rhizospheric bacterial community structure, a 2-year study was performed with Cry1Ac gene-inserted brinjal crop (Bt) and their near isogenic non-transformed trait (non-Bt). The event of Bt crop (VRBT-8) was screened using an insect bioassay and enzyme-linked immunosorbent assay. Soil moisture, NH4 (+)-N, NO3 (-)-N, and PO4 (-)-P level had non-significant variation. Quantitative polymerase chain reaction revealed that abundance of bacterial 16S rRNA gene copies were lower in soils associated with Bt brinjal. Microbial biomass carbon (MBC) showed slight reduction in Bt brinjal soils. Higher MBC values in the non-Bt crop soil may be attributed to increased root activity and availability of readily metabolizable carbon compounds. The restriction fragment length polymorphism of PCR-amplified rRNA gene fragments detected 13 different bacterial groups with the exclusive presence of β-Proteobacteria, Chloroflexus, Planctomycetes, and Fusobacteria in non-Bt, and Cyanobacteria and Bacteroidetes in Bt soils, respectively, reflecting minor changes in the community structure. Despite the detection of Cry1Ac protein in the rhizospheric soil, the overall impact of Cry1Ac expressing Bt brinjal was less compared to that due to seasonal changes.

Quantitation of base substitutions in eukaryotic 5S rRNA: selection for the maintenance of RNA secondary structure.

PubMed

Curtiss, W C; Vournakis, J N

1984-01-01

Eukaryotic 5S rRNA sequences from 34 diverse species were compared by the following method: (1) The sequences were aligned; (2) the positions of substitutions were located by comparison of all possible pairs of sequences; (3) the substitution sites were mapped to an assumed general base pairing model; and (4) the R-Y model of base stacking was used to study stacking pattern relationships in the structure. An analysis of the sequence and structure variability in each region of the molecule is presented. It was found that the degree of base substitution varies over a wide range, from absolute conservation to occurrence of over 90% of the possible observable substitutions. The substitutions are located primarily in stem regions of the 5S rRNA secondary structure. More than 88% of the substitutions in helical regions maintain base pairing. The disruptive substitutions are primarily located at the edges of helical regions, resulting in shortening of the helical regions and lengthening of the adjacent nonpaired regions. Base stacking patterns determined by the R-Y model are mapped onto the general secondary structure. Intrastrand and interstrand stacking could stabilize alternative coaxial structures and limit the conformational flexibility of nonpaired regions. Two short contiguous regions are 100% conserved in all species. This may reflect evolutionary constraints imposed at the DNA level by the requirement for binding of a 5S gene transcription initiation factor during gene expression.

Efficient Fluorescence Resonance Energy Transfer between Quantum Dots and Gold Nanoparticles Based on Porous Silicon Photonic Crystal for DNA Detection.

PubMed

Zhang, Hongyan; Lv, Jie; Jia, Zhenhong

2017-05-10

A novel assembled biosensor was prepared for detecting 16S rRNA, a small-size persistent specific for Actinobacteria. The mechanism of the porous silicon (PS) photonic crystal biosensor is based on the fluorescence resonance energy transfer (FRET) between quantum dots (QDs) and gold nanoparticles (AuNPs) through DNA hybridization, where QDs act as an emission donor and AuNPs serve as a fluorescence quencher. Results showed that the photoluminescence (PL) intensity of PS photonic crystal was drastically increased when the QDs-conjugated probe DNA was adhered to the PS layer by surface modification using a standard cross-link chemistry method. The PL intensity of QDs was decreased when the addition of AuNPs-conjugated complementary 16S rRNA was dropped onto QDs-conjugated PS. Based on the analysis of different target DNA concentration, it was found that the decrease of the PL intensity showed a good linear relationship with complementary DNA concentration in a range from 0.25 to 10 μM, and the detection limit was 328.7 nM. Such an optical FRET biosensor functions on PS-based photonic crystal for DNA detection that differs from the traditional FRET, which is used only in liquid. This method will benefit the development of a new optical FRET label-free biosensor on Si substrate and has great potential in biochips based on integrated optical devices.

16S rRNA based microarray analysis of ten periodontal bacteria in patients with different forms of periodontitis.

PubMed

Topcuoglu, Nursen; Kulekci, Guven

2015-10-01

DNA microarray analysis is a computer based technology, that a reverse capture, which targets 10 periodontal bacteria (ParoCheck) is available for rapid semi-quantitative determination. The aim of this three-year retrospective study was to display the microarray analysis results for the subgingival biofilm samples taken from patient cases diagnosed with different forms of periodontitis. A total of 84 patients with generalized aggressive periodontitis (GAP,n:29), generalized chronic periodontitis (GCP, n:25), peri-implantitis (PI,n:14), localized aggressive periodontitis (LAP,n:8) and refractory chronic periodontitis (RP,n:8) were consecutively selected from the archives of the Oral Microbiological Diagnostic Laboratory. The subgingival biofilm samples were analyzed by the microarray-based identification of 10 selected species. All the tested species were detected in the samples. The red complex bacteria were the most prevalent with very high levels in all groups. Fusobacterium nucleatum was detected in all samples at high levels. The green and blue complex bacteria were less prevalent compared with red and orange complex, except Aggregatibacter actinomycetemcomitas was detected in all LAP group. Positive correlations were found within all the red complex bacteria and between red and orange complex bacteria especially in GCP and GAP groups. Parocheck enables to monitoring of periodontal pathogens in all forms of periodontal disease and can be alternative to other guiding and reliable microbiologic tests. Copyright © 2015 Elsevier Ltd. All rights reserved.

Miniprimer PCR, a New Lens for Viewing the Microbial World▿ †

PubMed Central

Isenbarger, Thomas A.; Finney, Michael; Ríos-Velázquez, Carlos; Handelsman, Jo; Ruvkun, Gary

2008-01-01

Molecular methods based on the 16S rRNA gene sequence are used widely in microbial ecology to reveal the diversity of microbial populations in environmental samples. Here we show that a new PCR method using an engineered polymerase and 10-nucleotide “miniprimers” expands the scope of detectable sequences beyond those detected by standard methods using longer primers and Taq polymerase. After testing the method in silico to identify divergent ribosomal genes in previously cloned environmental sequences, we applied the method to soil and microbial mat samples, which revealed novel 16S rRNA gene sequences that would not have been detected with standard primers. Deeply divergent sequences were discovered with high frequency and included representatives that define two new division-level taxa, designated CR1 and CR2, suggesting that miniprimer PCR may reveal new dimensions of microbial diversity. PMID:18083877

A sensible technique to detect mollicutes impurities in human cells cultured in GMP condition.

PubMed

Ugolotti, Elisabetta; Vanni, Irene

2014-01-01

In therapeutic trials the use of manipulated cell cultures for clinical applications is often required. Mollicutes microorganism contamination of tissue cultures is a major problem because it can determine various and severe alterations in cellular function. Thus methods able to detect and trace cell cultures with Mollicutes contamination are needed in the monitoring of cells grown under good manufacturing practice conditions, and cell lines in continuous culture must be tested at regular intervals. We here describe a multiplex quantitative polymerase chain reaction assay able to detect contaminant Mollicutes species in a single-tube reaction through analysis of 16S-23S rRNA intergenic spacer regions and Tuf and P1 cytoadhesin genes. The method shows a sensitivity, specificity, and robustness comparable with the culture and the indicator cell culture as required by the European Pharmacopoeia guidelines and was validated following International Conference on Harmonization guidelines and Food and Drug Administration requirements.

On the Stability of Deinoxanthin Exposed to Mars Conditions during a Long-Term Space Mission and Implications for Biomarker Detection on Other Planets

PubMed Central

Leuko, Stefan; Bohmeier, Maria; Hanke, Franziska; Böettger, Ute; Rabbow, Elke; Parpart, Andre; Rettberg, Petra; de Vera, Jean-Pierre P.

2017-01-01

Outer space, the final frontier, is a hostile and unforgiving place for any form of life as we know it. The unique environment of space allows for a close simulation of Mars surface conditions that cannot be simulated as accurately on the Earth. For this experiment, we tested the resistance of Deinococcus radiodurans to survive exposure to simulated Mars-like conditions in low-Earth orbit for a prolonged period of time as part of the Biology and Mars experiment (BIOMEX) project. Special focus was placed on the integrity of the carotenoid deinoxanthin, which may serve as a potential biomarker to search for remnants of life on other planets. Survival was investigated by evaluating colony forming units, damage inflicted to the 16S rRNA gene by quantitative PCR, and the integrity and detectability of deinoxanthin by Raman spectroscopy. Exposure to space conditions had a strong detrimental effect on the survival of the strains and the 16S rRNA integrity, yet results show that deinoxanthin survives exposure to conditions as they prevail on Mars. Solar radiation is not only strongly detrimental to the survival and 16S rRNA integrity but also to the Raman signal of deinoxanthin. Samples not exposed to solar radiation showed only minuscule signs of deterioration. To test whether deinoxanthin is able to withstand the tested parameters without the protection of the cell, it was extracted from cell homogenate and exposed to high/low temperatures, vacuum, germicidal UV-C radiation, and simulated solar radiation. Results obtained by Raman investigations showed a strong resistance of deinoxanthin against outer space and Mars conditions, with the only exception of the exposure to simulated solar radiation. Therefore, deinoxanthin proved to be a suitable easily detectable biomarker for the search of Earth-like organic pigment-containing life on other planets. PMID:28966605

On the Stability of Deinoxanthin Exposed to Mars Conditions during a Long-Term Space Mission and Implications for Biomarker Detection on Other Planets.

PubMed

Leuko, Stefan; Bohmeier, Maria; Hanke, Franziska; Böettger, Ute; Rabbow, Elke; Parpart, Andre; Rettberg, Petra; de Vera, Jean-Pierre P

2017-01-01

Outer space, the final frontier, is a hostile and unforgiving place for any form of life as we know it. The unique environment of space allows for a close simulation of Mars surface conditions that cannot be simulated as accurately on the Earth. For this experiment, we tested the resistance of Deinococcus radiodurans to survive exposure to simulated Mars-like conditions in low-Earth orbit for a prolonged period of time as part of the Biology and Mars experiment (BIOMEX) project. Special focus was placed on the integrity of the carotenoid deinoxanthin, which may serve as a potential biomarker to search for remnants of life on other planets. Survival was investigated by evaluating colony forming units, damage inflicted to the 16S rRNA gene by quantitative PCR, and the integrity and detectability of deinoxanthin by Raman spectroscopy. Exposure to space conditions had a strong detrimental effect on the survival of the strains and the 16S rRNA integrity, yet results show that deinoxanthin survives exposure to conditions as they prevail on Mars. Solar radiation is not only strongly detrimental to the survival and 16S rRNA integrity but also to the Raman signal of deinoxanthin. Samples not exposed to solar radiation showed only minuscule signs of deterioration. To test whether deinoxanthin is able to withstand the tested parameters without the protection of the cell, it was extracted from cell homogenate and exposed to high/low temperatures, vacuum, germicidal UV-C radiation, and simulated solar radiation. Results obtained by Raman investigations showed a strong resistance of deinoxanthin against outer space and Mars conditions, with the only exception of the exposure to simulated solar radiation. Therefore, deinoxanthin proved to be a suitable easily detectable biomarker for the search of Earth-like organic pigment-containing life on other planets.

Variation in spatial and temporal incidence of the crustacean pathogen Hematodinium perezi in environmental samples from Atlantic Coastal Bays

PubMed Central

2013-01-01

Background Hematodinium perezi, a parasitic dinoflagellate, infects and kills blue crabs, Callinectes sapidus, along the Atlantic and Gulf coasts of the United States. The parasite proliferates within host hemolymph and tissues, and also produces free-swimming biflagellated dinospores that emerge from infected crabs. Infections in C. sapidus recur annually, and it is not known if biotic or environmental reservoirs contribute to reinfection and outbreaks. To address this data gap, a quantitative PCR assay based on the internal transcribed spacer 2 (ITS2) region of H. perezi rRNA genes was developed to asses the temporal and spatial incidence of the parasite in Delaware and Maryland coastal bays. Results A previously-used PCR assay for H. perezi, based on the small subunit rRNA gene sequence, was found to lack adequate species specificity to discriminate non-Hematodinium sp. dinoflagellate species in environmental samples. A new ITS2-targeted assay was developed and validated to detect H. perezi DNA in sediment and water samples using E. coli carrying the H. perezi rDNA genes. Application of the method to environmental samples identified potential hotspots in sediment in Indian River Inlet, DE and Chincoteague Bay, MD and VA. H. perezi DNA was not detected in co-occurring shrimp or snails, even during an outbreak of the parasite in C. sapidus. Conclusions H. perezi is present in water and sediment samples in Maryland and Delaware coastal bays from April through November with a wide spatial and temporal variability in incidence. Sampling sites with high levels of H. perezi DNA in both bays share characteristics of silty, organic sediments and low tidal currents. The environmental detection of H. perezi in spring, ahead of peak prevalence in crabs, points to gaps in our understanding of the parasite’s life history prior to infection in crabs as well as the mode of environmental transmission. To better understand the H. perezi life cycle will require further monitoring of the parasite in habitats as well as hosts. Improved understanding of potential environmental transmission to crabs will facilitate the development of disease forecasting. PMID:23641869

A novel multiplex PCR assay for simultaneous detection of nine clinically significant bacterial pathogens associated with bovine mastitis.

PubMed

Ashraf, Aqeela; Imran, Muhammad; Yaqub, Tahir; Tayyab, Muhammad; Shehzad, Wasim; Thomson, Peter C

2017-06-01

For rapid and simultaneous detection of nine bovine mastitic pathogens, a sensitive and specific multiplex PCR assay was developed. The assay was standardized using reference strains and validated on mastitic milk cultures which were identified to species level based on 16S rRNA sequencing. Multiplex PCR assay also efficiently detected the target bacterial strains directly from milk. The detection limit of the assay was up to 50 pg for DNA isolated from pure cultures and 10 4  CFU/ml for spiked milk samples. As estimated by latent class analysis, the assay was sensitive up to 88% and specific up to 98% for targeted mastitic pathogens, compared with the bacterial culture method and the 16S rRNA sequence analysis. This novel molecular assay could be useful for monitoring and maintaining the bovine udder health, ensuring the bacteriological safety of milk, and conducting epidemiological studies. Copyright © 2017 Elsevier Ltd. All rights reserved.

Multiple identification of most important waterborne protozoa in surface water used for irrigation purposes by 18S rRNA amplicon-based metagenomics.

PubMed

Moreno, Y; Moreno-Mesonero, L; Amorós, I; Pérez, R; Morillo, J A; Alonso, J L

2018-01-01

Understanding waterborne protozoan parasites (WPPs) diversity has important implications in public health. In this study, we evaluated a NGS-based method as a detection approach to identify simultaneously most important WPPs using 18S rRNA high-throughput sequencing. A set of primers to target the V4 18S rRNA region of WPPs such as Cryptosporidium spp., Giardia sp., Blastocystis sp., Entamoeba spp, Toxoplasma sp. and free-living amoebae (FLA) was designed. In order to optimize PCR conditions before sequencing, both a mock community with a defined composition of representative WPPs and a real water sample inoculated with specific WPPs DNA were prepared. Using the method proposed in this study, we have detected the presence of Giardia intestinalis, Acanthamoeba castellanii, Toxoplasma gondii, Entamoeba histolytica and Blastocystis sp. at species level in real irrigation water samples. Our results showed that untreated surface irrigation water in open fields can provide an important source of WPPs. Therefore, the methodology proposed in this study can establish a basis for an accurate and effective diagnostic of WPPs to provide a better understanding of the risk associated to irrigation water. Copyright © 2017 The Authors. Published by Elsevier GmbH.. All rights reserved.

Monitoring the dynamics of syntrophic β-oxidizing bacteria during anaerobic degradation of oleic acid by quantitative PCR.

PubMed

Ziels, Ryan M; Beck, David A C; Martí, Magalí; Gough, Heidi L; Stensel, H David; Svensson, Bo H

2015-04-01

The ecophysiology of long-chain fatty acid-degrading syntrophic β-oxidizing bacteria has been poorly understood due to a lack of quantitative abundance data. Here, TaqMan quantitative PCR (qPCR) assays targeting the 16S rRNA gene of the known mesophilic syntrophic β-oxidizing bacterial genera Syntrophomonas and Syntrophus were developed and validated. Microbial community dynamics were followed using qPCR and Illumina-based high-throughput amplicon sequencing in triplicate methanogenic bioreactors subjected to five consecutive batch feedings of oleic acid. With repeated oleic acid feeding, the initial specific methane production rate significantly increased along with the relative abundances of Syntrophomonas and methanogenic archaea in the bioreactor communities. The novel qPCR assays showed that Syntrophomonas increased from 7 to 31% of the bacterial community 16S rRNA gene concentration, whereas that of Syntrophus decreased from 0.02 to less than 0.005%. High-throughput amplicon sequencing also revealed that Syntrophomonas became the dominant genus within the bioreactor microbiomes. These results suggest that increased specific mineralization rates of oleic acid were attributed to quantitative shifts within the microbial communities toward higher abundances of syntrophic β-oxidizing bacteria and methanogenic archaea. The novel qPCR assays targeting syntrophic β-oxidizing bacteria may thus serve as monitoring tools to indicate the fatty acid β-oxidization potential of anaerobic digester communities. © FEMS 2015. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Comparison of PCR-Electrospray Ionization Mass Spectrometry with 16S rRNA PCR and Amplicon Sequencing for Detection of Bacteria in Excised Heart Valves

PubMed Central

Peeters, Bart; Herijgers, Paul; Beuselinck, Kurt; Peetermans, Willy E.; Herregods, Marie-Christin

2016-01-01

Identification of the causative pathogen of infective endocarditis (IE) is crucial for adequate management and therapy. A broad-range PCR-electrospray ionization mass spectrometry (PCR-ESI-MS) technique was compared with broad-spectrum 16S rRNA PCR and amplicon sequencing (16S rRNA PCR) for the detection of bacterial pathogens in 40 heart valves obtained from 34 definite infective endocarditis patients according to the modified Duke criteria and six nonendocarditis patients. Concordance between the two molecular techniques was 98% for being positive or negative, 97% for concordant identification up to the genus level, and 77% for concordant identification up to the species level. Sensitivity for detecting the causative pathogen (up to the genus level) in excised heart valves was 88% for 16S rRNA PCR and 85% for PCR-ESI-MS; the specificity was 83% for both methods. The two molecular techniques were significantly more sensitive than valve culture (18%) and accurately identified bacteria in excised heart valves. In eight patients with culture-negative IE, the following results were obtained: concordant detection of Coxiella burnetii (n = 2), Streptococcus gallolyticus (n = 1), Propionibacterium acnes (n = 1), and viridans group streptococci (n = 1) by both molecular tests, detection of P. acnes by PCR-ESI-MS whereas the 16S rRNA PCR was negative (n = 1), and a false-negative result by both molecular techniques (n = 2). In one case of IE caused by viridans streptococci, PCR-ESI-MS was positive for Enterococcus spp. The advantages of PCR-ESI-MS compared to 16S rRNA PCR are its automated workflow and shorter turnaround times. PMID:27629895

CHARACTERIZATION OF PRECURSOR 165 RRNA FOR AEROMONAS HYDROPHILA

EPA Science Inventory

Current strategies for monitoring drinking water quality involve culture-based methods to detect the presence of microbial indicators. However, these methods are insensitive when the organisms have undergone physiological changes such as injury and starvation that can occur in h...

CATCh, an Ensemble Classifier for Chimera Detection in 16S rRNA Sequencing Studies

PubMed Central

Mysara, Mohamed; Saeys, Yvan; Leys, Natalie; Raes, Jeroen

2014-01-01

In ecological studies, microbial diversity is nowadays mostly assessed via the detection of phylogenetic marker genes, such as 16S rRNA. However, PCR amplification of these marker genes produces a significant amount of artificial sequences, often referred to as chimeras. Different algorithms have been developed to remove these chimeras, but efforts to combine different methodologies are limited. Therefore, two machine learning classifiers (reference-based and de novo CATCh) were developed by integrating the output of existing chimera detection tools into a new, more powerful method. When comparing our classifiers with existing tools in either the reference-based or de novo mode, a higher performance of our ensemble method was observed on a wide range of sequencing data, including simulated, 454 pyrosequencing, and Illumina MiSeq data sets. Since our algorithm combines the advantages of different individual chimera detection tools, our approach produces more robust results when challenged with chimeric sequences having a low parent divergence, short length of the chimeric range, and various numbers of parents. Additionally, it could be shown that integrating CATCh in the preprocessing pipeline has a beneficial effect on the quality of the clustering in operational taxonomic units. PMID:25527546

The E. coli 16S rRNA binding site of ribosomal protein S15: higher-order structure in the absence and in the presence of the protein.

PubMed Central

Mougel, M; Philippe, C; Ebel, J P; Ehresmann, B; Ehresmann, C

1988-01-01

We have investigated in detail the secondary and tertiary structures of E. coli 16S rRNA binding site of protein S15 using a variety of enzymatic and chemical probes. RNase T1 and nuclease S1 were used to probe unpaired nucleotides and RNase V1 to monitor base-paired or stacked nucleotides. Bases were probed with dimethylsulfate (at A(N-1), C(N-3) and G(N-7)), with 1-cyclohexyl-3 (2-(1-methylmorpholino)-ethyl)-carboiimide-p- toluenesulfonate (at U(N-3) and G(N-1)) and with diethylpyrocarbonate (at A(N-7)). The RNA region corresponding to nucleotides 652 to 753 was tested within: (1) the complete 16S rRNA molecule; (2) a 16S rRNA fragment corresponding to nucleotides 578 to 756 obtained by transcription in vitro; (3) the S15-16S rRNA complex; (4) the S15-fragment complex. Cleavage and modification sites were detected by primer extension with reverse transcriptase. Our results show that: (1) The synthetized fragment folds into the same overall secondary structure as in the complete 16S rRNA, with the exception of the large asymmetrical internal loop (nucleotides 673-676/714-733) which is fully accessible in the fragment while it appears conformationally heterogeneous in the 16S rRNA; (2) the reactivity patterns of the S15-16S rRNA and S15-fragment complexes are identical; (3) the protein protects defined RNA regions, located in the large interior loop and in the 3'-end strand of helix [655-672]-[734-751]; (4) the protein also causes enhanced chemical reactivity and enzyme accessibility interpreted as resulting from a local conformational rearrangement, induced by S15 binding. Images PMID:2453025

[A Duplex PCR Method for Detection of Babesia caballi and Theileria equi].

PubMed

Zhang, Yang; Zhang, Yu-ting; Wang, Zhen-bao; Bolati; Li, Hai; Bayinchahan

2015-04-01

To develop a duplex PCR assay for detection of Babesia caballi and Theileria equi. Two pairs of primers were designed according to the BC48 gene of B. caballi and 18 s rRNA gene of T. equi, and a duplex PCR assay was developed by the optimization of reaction conditions. The specificity, sensitivity and reliability of the method were tested. The horse blood samples of suspected cases were collected from Yili region, and detected by the duplex PCR, microspopy, conventional PCR, and fluorescence quantitative PCR, and the results were compared. Using the duplex PCR assay, the specific fragments of 155 bp and 280 bp were amplified from DNA samples of B. caballi and T. equi, respectively. No specific fragment was amplified from DNA samples of B. bigemina, Theilerdia annulata, Theilerdia sergenti, Toxoplasma gondii, Neospora caninum, and Trypanosoma evansi. The limit of detection was 4.85 x 10(5) copies/L for B. caballi DNA and 4.85 x 10(4) copies/µl for T. equi DNA, respectively. Among the 24 blood samples, 11 were found B. caballi-positive by the duplex PCR assay, and 18 were T. equi-positive. The coincidence rate of microscopy, conventional PCR, and fluorescence quantitative PCR with duplex PCR was 91.7% (22/24), 95.8% (23/24), and 95.8% (23/24), respectively. A duplex PCR assay for simultaneous detection of B. caballi and T. equi is established.

Detection of rabbit and hare processed material in compound feeds by TaqMan real-time PCR.

PubMed

Pegels, N; López-Calleja, I; García, T; Martín, R; González, I

2013-01-01

Food and feed traceability has become a priority for governments due to consumer demand for comprehensive and integrated safety policies. In the present work, a TaqMan real-time PCR assay targeting the mitochondrial 12S rRNA gene was developed for specific detection of rabbit and hare material in animal feeds and pet foods. The technique is based on the use of three species-specific primer/probe detection systems targeting three 12S rRNA gene fragments: one from rabbit species, another one from hare species and a third fragment common to rabbit and hare (62, 102 and 75 bp length, respectively). A nuclear 18S rRNA PCR system, detecting a 77-bp amplicon, was used as positive amplification control. Assay performance and sensitivity were assessed through the analysis of a batch of laboratory-scale feeds treated at 133°C at 3 bar for 20 min to reproduce feed processing conditions dictated by European regulations. Successful detection of highly degraded rabbit and hare material was achieved at the lowest target concentration assayed (0.1%). Furthermore, the method was applied to 96 processed commercial pet food products to determine whether correct labelling had been used at the market level. The reported real-time PCR technique detected the presence of rabbit tissues in 80 of the 96 samples analysed (83.3%), indicating a possible labelling fraud in some pet foods. The real-time PCR method reported may be a useful tool for traceability purposes within the framework of feed control.

Coxiella Detection in Ticks from Wildlife and Livestock in Malaysia

PubMed Central

Khoo, Jing-Jing; Lim, Fang-Shiang; Chen, Fezshin; Phoon, Wai-Hong; Khor, Chee-Sieng; Pike, Brian L.; Chang, Li-Yen

2016-01-01

Abstract Recent studies have shown that ticks harbor Coxiella-like bacteria, which are potentially tick-specific endosymbionts. We recently described the detection of Coxiella-like bacteria and possibly Coxiella burnetii in ticks found from rural areas in Malaysia. In the present study, we collected ticks, including Haemaphysalis bispinosa, Haemaphysalis hystricis, Dermacentor compactus, Dermacentor steini, and Amblyomma sp. from wildlife and domesticated goats from four different locations in Malaysia. Coxiella 16s rRNA genomic sequences were detected by PCR in 89% of ticks tested. Similarity analysis and phylogenetic analyses of the 16s rRNA and rpoB partial sequences were performed for 10 representative samples selected based on the tick species, sex, and location. The findings here suggested the presence of C. burnetii in two samples, each from D. steini and H. hystricis. The sequences of both samples clustered with published C. burnetii sequences. The remaining eight tick samples were shown to harbor 16s rRNA sequences of Coxiella-like bacteria, which clustered phylogenetically according to the respective tick host species. The findings presented here added to the growing evidence of the association between Coxiella-like bacteria and ticks across species and geographical boundaries. The importance of C. burnetii found in ticks in Malaysia warrants further investigation. PMID:27763821

Sequence Variation in the Small-Subunit rRNA Gene of Plasmodium malariae and Prevalence of Isolates with the Variant Sequence in Sichuan, China

PubMed Central

Liu, Qing; Zhu, Shenghua; Mizuno, Sahoko; Kimura, Masatsugu; Liu, Peina; Isomura, Shin; Wang, Xingzhen; Kawamoto, Fumihiko

1998-01-01

By two PCR-based diagnostic methods, Plasmodium malariae infections have been rediscovered at two foci in the Sichuan province of China, a region where no cases of P. malariae have been officially reported for the last 2 decades. In addition, a variant form of P. malariae which has a deletion of 19 bp and seven substitutions of base pairs in the target sequence of the small-subunit (SSU) rRNA gene was detected with high frequency. Alignment analysis of Plasmodium sp. SSU rRNA gene sequences revealed that the 5′ region of the variant sequence is identical to that of P. vivax or P. knowlesi and its 3′ region is identical to that of P. malariae. The same sequence variations were also found in P. malariae isolates collected along the Thai-Myanmar border, suggesting a wide distribution of this variant form from southern China to Southeast Asia. PMID:9774600

Diversity of ribosomal 16S DNA- and RNA-based bacterial community in an office building drinking water system.

PubMed

Inkinen, J; Jayaprakash, B; Santo Domingo, J W; Keinänen-Toivola, M M; Ryu, H; Pitkänen, T

2016-06-01

Next-generation sequencing of 16S ribosomal RNA genes (rDNA) and ribosomal RNA (rRNA) was used to characterize water and biofilm microbiome collected from a drinking water distribution system of an office building after its first year of operation. The total bacterial community (rDNA) and active bacterial members (rRNA) sequencing databases were generated by Illumina MiSeq PE250 platform. As estimated by Chao1 index, species richness in cold water system was lower (180-260) in biofilms (Sphingomonas spp., Methylobacterium spp., Limnohabitans spp., Rhizobiales order) than in waters (250-580), (also Methylotenera spp.) (P = 0·005, n = 20). Similarly species richness (Chao1) was slightly higher (210-580) in rDNA libraries compared to rRNA libraries (150-400; P = 0·054, n = 24). Active Mycobacterium spp. was found in cross-linked polyethylene (PEX), but not in corresponding copper pipeline biofilm. Nonpathogenic Legionella spp. was found in rDNA libraries but not in rRNA libraries. Microbial communities differed between water and biofilms, between cold and hot water systems, locations in the building and between water rRNA and rDNA libraries, as shown by clear clusters in principal component analysis (PcoA). By using the rRNA method, we found that not all bacterial community members were active (e.g. Legionella spp.), whereas other members showed increased activity in some locations; for example, Pseudomonas spp. in hot water circulations' biofilm and order Rhizobiales and Limnohabitans spp. in stagnated locations' water and biofilm. rRNA-based methods may be better than rDNA-based methods for evaluating human health implications as rRNA methods can be used to describe the active bacterial fraction. This study indicates that copper as a pipeline material might have an adverse impact on the occurrence of Mycobacterium spp. The activity of Legionella spp. maybe questionable when detected solely by using DNA-based methods. © 2016 The Society for Applied Microbiology.

CHARACTERIZATION OF PRECURSOR FOR 16S rRNA FOR AEROMONAS HYDROPHILA

EPA Science Inventory

Current strategies for monitoring drinking water quality involve culture-based methods to detect the presence of microbial indicators. However, these methods are insensitive when the organisms have undergone physiological changes such as injury and starvation that can occur in h...

Fluctuations and synchrony of RNA synthesis in nucleoli.

PubMed

Pliss, Artem; Kuzmin, Andrey N; Kachynski, Aliaksandr V; Baev, Alexander; Berezney, Ronald; Prasad, Paras N

2015-06-01

Ribosomal RNA (rRNA) sequences are synthesized at exceptionally high rates and, together with ribosomal proteins (r-proteins), are utilized as building blocks for the assembly of pre-ribosomal particles. Although it is widely acknowledged that tight regulation and coordination of rRNA and r-protein production are fundamentally important for the maintenance of cellular homeostasis, still little is known about the real-time kinetics of the ribosome component synthesis in individual cells. In this communication we introduce a label-free MicroRaman spectrometric approach for monitoring rRNA synthesis in live cultured cells. Remarkably high and rapid fluctuations of rRNA production rates were revealed by this technique. Strikingly, the changes in the rRNA output were synchronous for ribosomal genes located in separate nucleoli of the same cell. Our findings call for the development of new concepts to elucidate the coordination of ribosomal components production. In this regard, numerical modeling further demonstrated that the production of rRNA and r-proteins can be coordinated, regardless of the fluctuations in rRNA synthesis. Overall, our quantitative data reveal a spectacular interplay of inherently stochastic rates of RNA synthesis and the coordination of gene expression.

Determining the analytical specificity of PCR-based assays for the diagnosis of IA: What is Aspergillus?

PubMed

Morton, C Oliver; White, P Lewis; Barnes, Rosemary A; Klingspor, Lena; Cuenca-Estrella, Manuel; Lagrou, Katrien; Bretagne, Stéphane; Melchers, Willem; Mengoli, Carlo; Caliendo, Angela M; Cogliati, Massimo; Debets-Ossenkopp, Yvette; Gorton, Rebecca; Hagen, Ferry; Halliday, Catriona; Hamal, Petr; Harvey-Wood, Kathleen; Jaton, Katia; Johnson, Gemma; Kidd, Sarah; Lengerova, Martina; Lass-Florl, Cornelia; Linton, Chris; Millon, Laurence; Morrissey, C Orla; Paholcsek, Melinda; Talento, Alida Fe; Ruhnke, Markus; Willinger, Birgit; Donnelly, J Peter; Loeffler, Juergen

2017-06-01

A wide array of PCR tests has been developed to aid the diagnosis of invasive aspergillosis (IA), providing technical diversity but limiting standardisation and acceptance. Methodological recommendations for testing blood samples using PCR exist, based on achieving optimal assay sensitivity to help exclude IA. Conversely, when testing more invasive samples (BAL, biopsy, CSF) emphasis is placed on confirming disease, so analytical specificity is paramount. This multicenter study examined the analytical specificity of PCR methods for detecting IA by blind testing a panel of DNA extracted from a various fungal species to explore the range of Aspergillus species that could be detected, but also potential cross reactivity with other fungal species. Positivity rates were calculated and regression analysis was performed to determine any associations between technical specifications and performance. The accuracy of Aspergillus genus specific assays was 71.8%, significantly greater (P < .0001) than assays specific for individual Aspergillus species (47.2%). For genus specific assays the most often missed species were A. lentulus (25.0%), A. versicolor (24.1%), A. terreus (16.1%), A. flavus (15.2%), A. niger (13.4%), and A. fumigatus (6.2%). There was a significant positive association between accuracy and using an Aspergillus genus PCR assay targeting the rRNA genes (P = .0011). Conversely, there was a significant association between rRNA PCR targets and false positivity (P = .0032). To conclude current Aspergillus PCR assays are better suited for detecting A. fumigatus, with inferior detection of most other Aspergillus species. The use of an Aspergillus genus specific PCR assay targeting the rRNA genes is preferential. © The Author 2016. Published by Oxford University Press on behalf of The International Society for Human and Animal Mycology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Use of 16S rRNA gene for identification of a broad range of clinically relevant bacterial pathogens

DOE PAGES

Srinivasan, Ramya; Karaoz, Ulas; Volegova, Marina; ...

2015-02-06

According to World Health Organization statistics of 2011, infectious diseases remain in the top five causes of mortality worldwide. However, despite sophisticated research tools for microbial detection, rapid and accurate molecular diagnostics for identification of infection in humans have not been extensively adopted. Time-consuming culture-based methods remain to the forefront of clinical microbial detection. The 16S rRNA gene, a molecular marker for identification of bacterial species, is ubiquitous to members of this domain and, thanks to ever-expanding databases of sequence information, a useful tool for bacterial identification. In this study, we assembled an extensive repository of clinical isolates (n =more » 617), representing 30 medically important pathogenic species and originally identified using traditional culture-based or non-16S molecular methods. This strain repository was used to systematically evaluate the ability of 16S rRNA for species level identification. To enable the most accurate species level classification based on the paucity of sequence data accumulated in public databases, we built a Naïve Bayes classifier representing a diverse set of high-quality sequences from medically important bacterial organisms. We show that for species identification, a model-based approach is superior to an alignment based method. Overall, between 16S gene based and clinical identities, our study shows a genus-level concordance rate of 96% and a species-level concordance rate of 87.5%. We point to multiple cases of probable clinical misidentification with traditional culture based identification across a wide range of gram-negative rods and gram-positive cocci as well as common gram-negative cocci.« less

Use of 16S rRNA Gene for Identification of a Broad Range of Clinically Relevant Bacterial Pathogens

PubMed Central

Srinivasan, Ramya; Karaoz, Ulas; Volegova, Marina; MacKichan, Joanna; Kato-Maeda, Midori; Miller, Steve; Nadarajan, Rohan; Brodie, Eoin L.; Lynch, Susan V.

2015-01-01

According to World Health Organization statistics of 2011, infectious diseases remain in the top five causes of mortality worldwide. However, despite sophisticated research tools for microbial detection, rapid and accurate molecular diagnostics for identification of infection in humans have not been extensively adopted. Time-consuming culture-based methods remain to the forefront of clinical microbial detection. The 16S rRNA gene, a molecular marker for identification of bacterial species, is ubiquitous to members of this domain and, thanks to ever-expanding databases of sequence information, a useful tool for bacterial identification. In this study, we assembled an extensive repository of clinical isolates (n = 617), representing 30 medically important pathogenic species and originally identified using traditional culture-based or non-16S molecular methods. This strain repository was used to systematically evaluate the ability of 16S rRNA for species level identification. To enable the most accurate species level classification based on the paucity of sequence data accumulated in public databases, we built a Naïve Bayes classifier representing a diverse set of high-quality sequences from medically important bacterial organisms. We show that for species identification, a model-based approach is superior to an alignment based method. Overall, between 16S gene based and clinical identities, our study shows a genus-level concordance rate of 96% and a species-level concordance rate of 87.5%. We point to multiple cases of probable clinical misidentification with traditional culture based identification across a wide range of gram-negative rods and gram-positive cocci as well as common gram-negative cocci. PMID:25658760

Molecular Diagnosis of Invasive Aspergillosis and Detection of Azole Resistance by a Newly Commercialized PCR Kit.

PubMed

Dannaoui, Eric; Gabriel, Frédéric; Gaboyard, Manuel; Lagardere, Gaëlle; Audebert, Lucile; Quesne, Gilles; Godichaud, Sandrine; Verweij, Paul E; Accoceberry, Isabelle; Bougnoux, Marie-Elisabeth

2017-11-01

Aspergillus fumigatus is the main species responsible for aspergillosis in humans. The diagnosis of aspergillosis remains difficult, and the rapid emergence of azole resistance in A. fumigatus is worrisome. The aim of this study was to validate the new MycoGENIE A. fumigatus real-time PCR kit and to evaluate its performance on clinical samples for the detection of A. fumigatus and its azole resistance. This multiplex assay detects DNA from the A. fumigatus species complex by targeting the multicopy 28S rRNA gene and specific TR 34 and L98H mutations in the single-copy-number cyp51A gene of A. fumigatus The specificity of cyp51A mutation detection was assessed by testing DNA samples from 25 wild-type or mutated clinical A. fumigatus isolates. Clinical validation was performed on 88 respiratory samples obtained from 62 patients and on 69 serum samples obtained from 16 patients with proven or probable aspergillosis and 13 patients without aspergillosis. The limit of detection was <1 copy for the Aspergillus 28S rRNA gene and 6 copies for the cyp51A gene harboring the TR 34 and L98H alterations. No cross-reactivity was detected with various fungi and bacteria. All isolates harboring the TR 34 and L98H mutations were accurately detected by quantitative PCR (qPCR) analysis. With respiratory samples, qPCR results showed a sensitivity and specificity of 92.9% and 90.1%, respectively, while with serum samples, the sensitivity and specificity were 100% and 84.6%, respectively. Our study demonstrated that this new real-time PCR kit enables sensitive and rapid detection of A. fumigatus DNA and azole resistance due to TR 34 and L98H mutations in clinical samples. Copyright © 2017 American Society for Microbiology.

PCR detection of thermophilic spore-forming bacteria involved in canned food spoilage.

PubMed

Prevost, S; Andre, S; Remize, F

2010-12-01

Thermophilic bacteria that form highly heat-resistant spores constitute an important group of spoilage bacteria of low-acid canned food. A PCR assay was developed in order to rapidly trace these bacteria. Three PCR primer pairs were designed from rRNA gene sequences. These primers were evaluated for the specificity and the sensitivity of detection. Two primer pairs allowed detection at the species level of Geobacillus stearothermophilus and Moorella thermoacetica/thermoautrophica. The other pair allowed group-specific detection of anaerobic thermophilic bacteria of the genera Thermoanaerobacterium, Thermoanaerobacter, Caldanerobium and Caldanaerobacter. After a single enrichment step, these PCR assays allowed the detection of 28 thermophiles from 34 cans of spoiled low-acid food. In addition, 13 ingredients were screened for the presence of these bacteria. This PCR assay serves as a detection method for strains able to spoil low-acid canned food treated at 55°C. It will lead to better reactivity in the canning industry. Raw materials and ingredients might be qualified not only for quantitative spore contamination, but also for qualitative contamination by highly heat-resistant spores.

Detection and Identification of Gastrointestinal Lactobacillus Species by Using Denaturing Gradient Gel Electrophoresis and Species-Specific PCR Primers

PubMed Central

Walter, J.; Tannock, G. W.; Tilsala-Timisjarvi, A.; Rodtong, S.; Loach, D. M.; Munro, K.; Alatossava, T.

2000-01-01

Denaturing gradient gel electrophoresis (DGGE) of DNA fragments obtained by PCR amplification of the V2-V3 region of the 16S rRNA gene was used to detect the presence of Lactobacillus species in the stomach contents of mice. Lactobacillus isolates cultured from human and porcine gastrointestinal samples were identified to the species level by using a combination of DGGE and species-specific PCR primers that targeted 16S-23S rRNA intergenic spacer region or 16S rRNA gene sequences. The identifications obtained by this approach were confirmed by sequencing the V2-V3 region of the 16S rRNA gene and by a BLAST search of the GenBank database. PMID:10618239

Efficient Fluorescence Resonance Energy Transfer between Quantum Dots and Gold Nanoparticles Based on Porous Silicon Photonic Crystal for DNA Detection

PubMed Central

Zhang, Hongyan; Lv, Jie; Jia, Zhenhong

2017-01-01

A novel assembled biosensor was prepared for detecting 16S rRNA, a small-size persistent specific for Actinobacteria. The mechanism of the porous silicon (PS) photonic crystal biosensor is based on the fluorescence resonance energy transfer (FRET) between quantum dots (QDs) and gold nanoparticles (AuNPs) through DNA hybridization, where QDs act as an emission donor and AuNPs serve as a fluorescence quencher. Results showed that the photoluminescence (PL) intensity of PS photonic crystal was drastically increased when the QDs-conjugated probe DNA was adhered to the PS layer by surface modification using a standard cross-link chemistry method. The PL intensity of QDs was decreased when the addition of AuNPs-conjugated complementary 16S rRNA was dropped onto QDs-conjugated PS. Based on the analysis of different target DNA concentration, it was found that the decrease of the PL intensity showed a good linear relationship with complementary DNA concentration in a range from 0.25 to 10 μM, and the detection limit was 328.7 nM. Such an optical FRET biosensor functions on PS-based photonic crystal for DNA detection that differs from the traditional FRET, which is used only in liquid. This method will benefit the development of a new optical FRET label-free biosensor on Si substrate and has great potential in biochips based on integrated optical devices. PMID:28489033

Fermentation properties of isomaltooligosaccharides are affected by human fecal enterotypes.

PubMed

Wu, Qinqin; Pi, Xiong'e; Liu, Wei; Chen, Huahai; Yin, Yeshi; Yu, Hongwei D; Wang, Xin; Zhu, Liying

2017-12-01

Isomaltooligosaccharides (IMOs) are enzymatically synthesized oligosaccharides that have potential prebiotic effects. Five IMO substrates with 2-16° of polymerization (DP) were studied for their fermentation capacities using human microbiomes in an in vitro batch fermentation model. Eleven fecal slurries belonging to three enterotypes, including the Bacteroides-, Prevotella- and Mixed-type, exhibited different degradation rates for long chain IMOs (DP 7 to 16). In contrast, the degradation rates for short chain IMOs (DP 2 to 6) were not affected by enterotypes. Both 16S rRNA gene sequencing and quantitative PCR demonstrated that, after fermentation, the Bifidobacterium growth with IMOs was primarily detected in the Bacteroides- and Mixed-type (non-Prevotella-type), and to a lesser degree in the Prevotella-type. Interestingly, the Prevotella-type microbiome had higher levels of propionic acid and butyric acid production than non-Prevotella-type microbiome after IMOs fermentation. Moreover, principal coordinate analysis (PCoA) of both denaturing gradient gel electrophoresis (DGGE) profiling and 16S rRNA sequencing data demonstrated that the microbiome community compositions were separately clustered based on IMO chain length, suggesting significant impact of DP on the bacterial community structure. The current results clearly demonstrated that the IMO chain length could modulate the structure and composition of the human colonic microbiome. Different responses to short and long chain IMOs were observed from three human enterotypes, indicating that IMOs may be used as therapeutic substrates for directly altering human colonic bacteria. Copyright © 2017 Elsevier Ltd. All rights reserved.

Occurrence and molecular characterization of hemoplasmas in domestic dogs and wild mammals in a Brazilian wetland.

PubMed

de Sousa, Keyla Carstens Marques; Herrera, Heitor Miraglia; Secato, Caroline Tostes; Oliveira, André do Vale; Santos, Filipe Martins; Rocha, Fabiana Lopes; Barreto, Wanessa Teixeira Gomes; Macedo, Gabriel Carvalho; de Andrade Pinto, Pedro Cordeiro Estrela; Machado, Rosangela Zacarias; Costa, Mirela Tinucci; André, Marcos Rogério

2017-07-01

Hemotropic mycoplasmas are known to cause anemia in several mammalian species. The present work aimed to investigate the occurrence of Mycoplasma spp. in wild mammals, domestic dogs and their respective ectoparasites, in southern Pantanal region, central-western Brazil. Between August 2013 and March 2015, 31 Nasua nasua, 78 Cerdocyon thous, seven Leopardus pardalis, 42 dogs, 110 wild rodents, and 30 marsupials were trapped and ectoparasites (ticks and fleas) found parasitizing the animals were collected. Mammals and ectoparasites DNA samples were submitted to conventional PCR assays for Mycoplasma spp. targeting 16S rRNA and RnaseP genes. Twenty-four N. nasua, three C. thous, two domestic dogs, one L. pardalis and one wild rodent were positive for 16S rRNA PCR protocols. Fourteen N. nasua samples were also positive in RnaseP PCR. No marsupial or arthropod showed positivity for Mycoplasma spp. The phylogenetic analyses based on 16S rRNA gene showed that all sequences obtained from dogs, two sequences obtained from C. thous and ten sequences obtained from N. nasua showed to be closely related to Mycoplasma haemocanis/Mycoplasma haemofelis species. Genotypes closely related to 'Candidatus Mycoplasma haemominutum' and Mycoplasma haemomuris were detected in the L. pardalis and in the wild rodent, respectively. Probably a novel Mycoplasma genotype, closely related to a sequence obtained from a Brazilian capybara was detected in 14 N. nasua, based on a concatenated phylogenetic analysis of 16S rRNA and RnaseP genes. The present study revealed that wild animals in southern Pantanal region, Brazil, are exposed to different species of hemoplasmas. Copyright © 2017 Elsevier B.V. All rights reserved.

Evaluation of FRET real-time PCR assay for rapid detection and differentiation of Plasmodium species in returning travellers and migrants

PubMed Central

Safeukui, Innocent; Millet, Pascal; Boucher, Sébastien; Melinard, Laurence; Fregeville, Frédéric; Receveur, Marie-Catherine; Pistone, Thierry; Fialon, Pierre; Vincendeau, Philippe; Fleury, Hervé; Malvy, Denis

2008-01-01

Background A simple real-time PCR assay using one set of primer and probe for rapid, sensitive and quantitative detection of Plasmodium species, with simultaneous differentiation of Plasmodium falciparum from the three other Plasmodium species (Plasmodium vivax, Plasmodium ovale and Plasmodium malariae) in febrile returning travellers and migrants was developed and evaluated. Methods Consensus primers were used to amplify a species-specific region of the multicopy 18S rRNA gene, and fluorescence resonance energy transfer hybridization probes were used for detection in a LightCycler platform (Roche). The anchor probe sequence was designed to be perfect matches to the 18S rRNA gene of the fourth Plasmodium species, while the acceptor probe sequence was designed for P. falciparum over a region containing one mismatched, which allowed differentiation of the three other Plasmodium species. The performance characteristics of the real-time PCR assay were compared with those of conventional PCR and microscopy-based diagnosis from 119 individuals with a suspected clinical diagnostic of imported malaria. Results Blood samples with parasite densities less than 0.01% were all detected, and analytical sensitivity was 0.5 parasite per PCR reaction. The melt curve means Tms (standard deviation) in clinical isolates were 60.5°C (0.6°C) for P. falciparum infection and 64.6°C (1.8°C) for non-P. falciparum species. These Tms values of the P. falciparum or non-P. falciparum species did not vary with the geographic origin of the parasite. The real-time PCR results correlated with conventional PCR using both genus-specific (Kappa coefficient: 0.95, 95% confidence interval: 0.9 – 1) or P. falciparum-specific (0.91, 0.8 – 1) primers, or with the microscopy results (0.70, 0.6 – 0.8). The real-time assay was 100% sensitive and specific for differentiation of P. falciparum to non-P. falciparum species, compared with conventional PCR or microscopy. The real-time PCR assay can also detect individuals with mixed infections (P. falciparum and non-P. falciparum sp.) in the same sample. Conclusion This real-time PCR assay with melting curve analysis is rapid, and specific for the detection and differentiation of P. falciparum to other Plasmodium species. The suitability for routine use of this assay in clinical diagnostic laboratories is discussed. PMID:18442362

Microbial diversity in an Armenian geothermal spring assessed by molecular and culture-based methods.

PubMed

Panosyan, Hovik; Birkeland, Nils-Kåre

2014-11-01

The phylogenetic diversity of the prokaryotic community thriving in the Arzakan hot spring in Armenia was studied using molecular and culture-based methods. A sequence analysis of 16S rRNA gene clone libraries demonstrated the presence of a diversity of microorganisms belonging to the Alphaproteobacteria, Betaproteobacteria, Gammaproteobacteria, Epsilonproteobacteria, Firmicutes, Bacteroidetes phyla, and Cyanobacteria. Proteobacteria was the dominant group, representing 52% of the bacterial clones. Denaturing gradient gel electrophoresis profiles of the bacterial 16S rRNA gene fragments also indicated the abundance of Proteobacteria, Bacteroidetes, and Cyanobacteria populations. Most of the sequences were most closely related to uncultivated microorganisms and shared less than 96% similarity with their closest matches in GenBank, indicating that this spring harbors a unique community of novel microbial species or genera. The majority of the sequences of an archaeal 16S rRNA gene library, generated from a methanogenic enrichment, were close relatives of members of the genus Methanoculleus. Aerobic endospore-forming bacteria mainly belonging to Bacillus and Geobacillus were detected only by culture-dependent methods. Three isolates were successfully obtained having 99, 96, and 96% 16S rRNA gene sequence similarities to Arcobacter sp., Methylocaldum sp., and Methanoculleus sp., respectively. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Mycobacterial RNA isolation optimized for non-coding RNA: high fidelity isolation of 5S rRNA from Mycobacterium bovis BCG reveals novel post-transcriptional processing and a complete spectrum of modified ribonucleosides.

PubMed

Hia, Fabian; Chionh, Yok Hian; Pang, Yan Ling Joy; DeMott, Michael S; McBee, Megan E; Dedon, Peter C

2015-03-11

A major challenge in the study of mycobacterial RNA biology is the lack of a comprehensive RNA isolation method that overcomes the unusual cell wall to faithfully yield the full spectrum of non-coding RNA (ncRNA) species. Here, we describe a simple and robust procedure optimized for the isolation of total ncRNA, including 5S, 16S and 23S ribosomal RNA (rRNA) and tRNA, from mycobacteria, using Mycobacterium bovis BCG to illustrate the method. Based on a combination of mechanical disruption and liquid and solid-phase technologies, the method produces all major species of ncRNA in high yield and with high integrity, enabling direct chemical and sequence analysis of the ncRNA species. The reproducibility of the method with BCG was evident in bioanalyzer electrophoretic analysis of isolated RNA, which revealed quantitatively significant differences in the ncRNA profiles of exponentially growing and non-replicating hypoxic bacilli. The method also overcame an historical inconsistency in 5S rRNA isolation, with direct sequencing revealing a novel post-transcriptional processing of 5S rRNA to its functional form and with chemical analysis revealing seven post-transcriptional ribonucleoside modifications in the 5S rRNA. This optimized RNA isolation procedure thus provides a means to more rigorously explore the biology of ncRNA species in mycobacteria. © The Author(s) 2014. Published by Oxford University Press on behalf of Nucleic Acids Research.

Assessing hog lagoon waste contamination in the Cape Fear Watershed using Bacteroidetes 16S rRNA gene pyrosequencing.

PubMed

Arfken, Ann M; Song, Bongkeun; Mallin, Michael A

2015-09-01

Hog lagoons can be major sources of waste and nutrient contamination to watersheds adjacent to pig farms. Fecal source tracking methods targeting Bacteroidetes 16S rRNA genes in pig fecal matter may underestimate or fail to detect hog lagoon contamination in riverine environments. In order to detect hog lagoon wastewater contamination in the Cape Fear Watershed, where a large number of hog farms are present, we conducted pyrosequencing analyses of Bacteroidetes 16S rRNA genes in hog lagoon waste and identified new hog lagoon-specific marker sequences. Additional pyrosequencing analyses of Bacteroidetes 16S rRNA genes were conducted with surface water samples collected at 4 sites during 5 months in the Cape Fear Watershed. Using an operational taxonomic unit (OTU) identity cutoff value of 97 %, these newly identified hog lagoon markers were found in 3 of the river samples, while only 1 sample contained the pig fecal marker. In the sample containing the pig fecal marker, there was a relatively high percentage (14.1 %) of the hog lagoon markers and a low pig fecal marker relative abundance of 0.4 % in the Bacteroidetes 16S rRNA gene sequences. This suggests that hog lagoon contamination must be somewhat significant in order for pig fecal markers to be detected, and low levels of hog lagoon contamination cannot be detected targeting only pig-specific fecal markers. Thus, new hog lagoon markers have a better detection capacity for lagoon waste contamination, and in conjunction with a pig fecal marker, provide a more comprehensive and accurate detection of hog lagoon waste contamination in susceptible watersheds.

Inhabitancy of active Nitrosopumilus-like ammonia-oxidizing archaea and Nitrospira nitrite-oxidizing bacteria in the sponge Theonella swinhoei

PubMed Central

Feng, Guofang; Sun, Wei; Zhang, Fengli; Karthik, Loganathan; Li, Zhiyong

2016-01-01

Nitrification directly contributes to the ammonia removal in sponges, and it plays an indispensable role in sponge-mediated nitrogen cycle. Previous studies have demonstrated genomic evidences of nitrifying lineages in the sponge Theonella swinhoei. However, little is known about the transcriptional activity of nitrifying community in this sponge. In this study, combined DNA- and transcript-based analyses were performed to reveal the composition and transcriptional activity of the nitrifiers in T. swinhoei from the South China Sea. Transcriptional activity of ammonia-oxidizing archaea (AOA) and nitrite-oxidizing bacteria (NOB) in this sponge were confirmed by targeting their nitrifying genes,16S rRNA genes and their transcripts. Phylogenetic analysis coupled with RDP rRNA classification indicated that archaeal 16S rRNA genes, amoA (the subunit of ammonia monooxygenase) genes and their transcripts were closely related to Nitrosopumilus-like AOA; whereas nitrifying bacterial 16S rRNA genes, nxrB (the subunit of nitrite oxidoreductase) genes and their transcripts were closely related to Nitrospira NOB. Quantitative assessment demonstrated relative higher abundances of nitrifying genes and transcripts of Nitrosopumilus-like AOA than those of Nitrospira NOB in this sponge. This study illustrated the transcriptional potentials of Nitrosopumilus-like archaea and Nitrospira bacteria that would predominantly contribute to the nitrification functionality in the South China Sea T. swinhoei. PMID:27113140

Rapid Detection of Pathogens

DOE Office of Scientific and Technical Information (OSTI.GOV)

David Perlin

2005-08-14

Pathogen identification is a crucial first defense against bioterrorism. A major emphasis of our national biodefense strategy is to establish fast, accurate and sensitive assays for diagnosis of infectious diseases agents. Such assays will ensure early and appropriate treatment of infected patients. Rapid diagnostics can also support infection control measures, which monitor and limit the spread of infectious diseases agents. Many select agents are highly transmissible in the early stages of disease, and it is critical to identify infected patients and limit the risk to the remainder of the population and to stem potential panic in the general population. Nucleicmore » acid-based molecular approaches for identification overcome many of the deficiencies associated with conventional culture methods by exploiting both large- and small-scale genomic differences between organisms. PCR-based amplification of highly conserved ribosomal RNA (rRNA) genes, intergenic sequences, and specific toxin genes is currently the most reliable approach for bacterial, fungal and many viral pathogenic agents. When combined with fluorescence-based oligonucleotide detection systems, this approach provides real-time, quantitative, high fidelity analysis capable of single nucleotide allelic discrimination (4). These probe systems offer rapid turn around time (<2 h) and are suitable for high throughput, automated multiplex operations that are critical for clinical diagnostic laboratories. In this pilot program, we have used molecular beacon technology invented at the Public health Research Institute to develop a new generation of molecular probes to rapidly detect important agents of infectious diseases. We have also developed protocols to rapidly extract nucleic acids from a variety of clinical specimen including and blood and tissue to for detection in the molecular assays. This work represented a cooperative research development program between the Kramer-Tyagi/Perlin labs on probe development and the Perlin lab in sample preparation and testing in animal models.« less

O-5S quantitative real-time PCR: a new diagnostic tool for laboratory confirmation of human onchocerciasis.

PubMed

Mekonnen, Solomon A; Beissner, Marcus; Saar, Malkin; Ali, Solomon; Zeynudin, Ahmed; Tesfaye, Kassahun; Adbaru, Mulatu G; Battke, Florian; Poppert, Sven; Hoelscher, Michael; Löscher, Thomas; Bretzel, Gisela; Herbinger, Karl-Heinz

2017-10-02

Onchocerciasis is a parasitic disease caused by the filarial nematode Onchocerca volvulus. In endemic areas, the diagnosis is commonly confirmed by microscopic examination of skin snip samples, though this technique is considered to have low sensitivity. The available melting-curve based quantitative real-time PCR (qPCR) using degenerated primers targeting the O-150 repeat of O. volvulus was considered insufficient for confirming the individual diagnosis, especially in elimination studies. This study aimed to improve detection of O. volvulus DNA in clinical samples through the development of a highly sensitive qPCR assay. A novel hydrolysis probe based qPCR assay was designed targeting the specific sequence of the O. volvulus O-5S rRNA gene. A total of 200 clinically suspected onchocerciasis cases were included from Goma district in South-west Ethiopia, from October 2012 through May 2013. Skin snip samples were collected and subjected to microscopy, O-150 qPCR, and the novel O-5S qPCR. Among the 200 individuals, 133 patients tested positive (positivity rate of 66.5%) and 67 negative by O-5S qPCR, 74 tested positive by microscopy (37.0%) and 78 tested positive by O-150 qPCR (39.0%). Among the 133 O-5S qPCR positive individuals, microscopy and O-150 qPCR detected 55.6 and 59.4% patients, respectively, implying a higher sensitivity of O-5S qPCR than microscopy and O-150 qPCR. None of the 67 individuals who tested negative by O-5S qPCR tested positive by microscopy or O-150 qPCR, implying 100% specificity of the newly designed O-5S qPCR assay. The novel O-5S qPCR assay is more sensitive than both microscopic examination and the existing O-150 qPCR for the detection of O. volvulus from skin snip samples. The newly designed assay is an important step towards appropriate individual diagnosis and control of onchocerciasis.

Rapid detection of rRNA group I pseudomonads in contaminated metalworking fluids and biofilm formation by fluorescent in situ hybridization.

PubMed

Saha, Ratul; Donofrio, Robert S; Goeres, Darla M; Bagley, Susan T

2012-05-01

Metalworking fluids (MWFs), used in different machining operations, are highly prone to microbial degradation. Microbial communities present in MWFs lead to biofilm formation in the MWF systems, which act as a continuous source of contamination. Species of rRNA group I Pseudomonas dominate in contaminated MWFs. However, their actual distribution is typically underestimated when using standard culturing techniques as most fail to grow on the commonly used Pseudomonas Isolation Agar. To overcome this, fluorescent in situ hybridization (FISH) was used to study their abundance along with biofilm formation by two species recovered from MWFs, Pseudomonas fluorescens MWF-1 and the newly described Pseudomonas oleovorans subsp. lubricantis. Based on 16S rRNA sequences, a unique fluorescent molecular probe (Pseudo120) was designed targeting a conserved signature sequence common to all rRNA group I Pseudomonas. The specificity of the probe was evaluated using hybridization experiments with whole cells of different Pseudomonas species. The probe's sensitivity was determined to be 10(3) cells/ml. It successfully detected and enumerated the abundance and distribution of Pseudomonas indicating levels between 3.2 (± 1.1) × 10(6) and 5.0 (± 2.3) × 10(6) cells/ml in four different industrial MWF samples collected from three different locations. Biofilm formation was visualized under stagnant conditions using high and low concentrations of cells for both P. fluorescens MWF-1 and P. oleovorans subsp. lubricantis stained with methylene blue and Pseudo120. On the basis of these observations, this molecular probe can be successfully be used in the management of MWF systems to monitor the levels and biofilm formation of rRNA group I pseudomonads.

Pyrosequencing of mcrA and Archaeal 16S rRNA Genes Reveals Diversity and Substrate Preferences of Methanogen Communities in Anaerobic Digesters

PubMed Central

Wilkins, David; Lu, Xiao-Ying; Shen, Zhiyong; Chen, Jiapeng

2014-01-01

Methanogenic archaea play a key role in biogas-producing anaerobic digestion and yet remain poorly taxonomically characterized. This is in part due to the limitations of low-throughput Sanger sequencing of a single (16S rRNA) gene, which in the past may have undersampled methanogen diversity. In this study, archaeal communities from three sludge digesters in Hong Kong and one wastewater digester in China were examined using high-throughput pyrosequencing of the methyl coenzyme M reductase (mcrA) and 16S rRNA genes. Methanobacteriales, Methanomicrobiales, and Methanosarcinales were detected in each digester, indicating that both hydrogenotrophic and acetoclastic methanogenesis was occurring. Two sludge digesters had similar community structures, likely due to their similar design and feedstock. Taxonomic classification of the mcrA genes suggested that these digesters were dominated by acetoclastic methanogens, particularly Methanosarcinales, while the other digesters were dominated by hydrogenotrophic Methanomicrobiales. The proposed euryarchaeotal order Methanomassiliicoccales and the uncultured WSA2 group were detected with the 16S rRNA gene, and potential mcrA genes for these groups were identified. 16S rRNA gene sequencing also recovered several crenarchaeotal groups potentially involved in the initial anaerobic digestion processes. Overall, the two genes produced different taxonomic profiles for the digesters, while greater methanogen richness was detected using the mcrA gene, supporting the use of this functional gene as a complement to the 16S rRNA gene to better assess methanogen diversity. A significant positive correlation was detected between methane production and the abundance of mcrA transcripts in digesters treating sludge and wastewater samples, supporting the mcrA gene as a biomarker for methane yield. PMID:25381241

Simultaneous separation of five major ribonucleic acids by capillary electrophoresis with laser-induced fluorescence in the presence of electroosmotic flow: application to the rapid screening of 5S rRNA from ovarian cancer cells.

PubMed

Shih, Ya-Chu; Liao, Ching-Ru; Chung, I-Che; Chang, Yu-Sun; Chang, Po-Ling

2014-10-17

RNA integrity is important in RNA studies because poor RNA quality may impact downstream methodologies. This study proposes a rapid and cost-effective method for the determination of RNA integrity based on CE-LIF in the presence of electroosmotic flow. The proposed method uses poly(ethylene) oxide (Mavg=4,000,000 Da) as a sieving matrix for total RNA separation. Ethidium bromide (μg mL(-1)) was dissolved in a polymer solution as an interchelating dye for on-column fluorescent labeling. The 28S rRNA, 18S rRNA, 5.8S rRNA, 5S rRNA and tRNA from the total human RNA extracted from the cells were fully separated using the proposed method. The lowest detectable concentration of total RNA achieved was 100 pg μL(-1) with a 6 min sample injection followed by on-column concentration. In addition, the temperature-induced degradation of total RNA was observed by CE-LIF. The electropherograms revealed more fragmentation of 28S and 18S rRNAs by temperature-induced hydrolysis compared with the 5.8S rRNA, 5S rRNA and tRNA. Therefore, the results indicated that RNA degradation should be considered for long-term, high-temperature incubations in RNA-related experiments involving RNA hybridization. The proposed method is furthermore, applied to the determination of 5S rRNA overexpressed in ovarian cancer cells as compared to the cervical cancer cells. Overall, CE-LIF is highly promising for rapid screening of ovarian cancers without tedious pre-amplification steps. Copyright © 2014 Elsevier B.V. All rights reserved.

An intergenic non-coding rRNA correlated with expression of the rRNA and frequency of an rRNA single nucleotide polymorphism in lung cancer cells.

PubMed

Shiao, Yih-Horng; Lupascu, Sorin T; Gu, Yuhan D; Kasprzak, Wojciech; Hwang, Christopher J; Fields, Janet R; Leighty, Robert M; Quiñones, Octavio; Shapiro, Bruce A; Alvord, W Gregory; Anderson, Lucy M

2009-10-19

Ribosomal RNA (rRNA) is a central regulator of cell growth and may control cancer development. A cis noncoding rRNA (nc-rRNA) upstream from the 45S rRNA transcription start site has recently been implicated in control of rRNA transcription in mouse fibroblasts. We investigated whether a similar nc-rRNA might be expressed in human cancer epithelial cells, and related to any genomic characteristics. Using quantitative rRNA measurement, we demonstrated that a nc-rRNA is transcribed in human lung epithelial and lung cancer cells, starting from approximately -1000 nucleotides upstream of the rRNA transcription start site (+1) and extending at least to +203. This nc-rRNA was significantly more abundant in the majority of lung cancer cell lines, relative to a nontransformed lung epithelial cell line. Its abundance correlated negatively with total 45S rRNA in 12 of 13 cell lines (P = 0.014). During sequence analysis from -388 to +306, we observed diverse, frequent intercopy single nucleotide polymorphisms (SNPs) in rRNA, with a frequency greater than predicted by chance at 12 sites. A SNP at +139 (U/C) in the 5' leader sequence varied among the cell lines and correlated negatively with level of the nc-rRNA (P = 0.014). Modelling of the secondary structure of the rRNA 5'-leader sequence indicated a small increase in structural stability due to the +139 U/C SNP and a minor shift in local configuration occurrences. The results demonstrate occurrence of a sense nc-rRNA in human lung epithelial and cancer cells, and imply a role in regulation of the rRNA gene, which may be affected by a +139 SNP in the 5' leader sequence of the primary rRNA transcript.

Evaluation of quantitative polymerase chain reaction assays targeting Mycobacterium avium, M. intracellulare, and M. avium subspecies paratuberculosis in drinking water biofilms.

PubMed

Chern, Eunice C; King, Dawn; Haugland, Richard; Pfaller, Stacy

2015-03-01

Mycobacterium avium (MA), Mycobacterium intracellulare (MI), and Mycobacterium avium subsp. paratuberculosis (MAP) are difficult to culture due to their slow growing nature. A quantitative polymerase chain reaction (qPCR) method for the rapid detection of MA, MI, and MAP can be used to provide data supporting drinking water biofilms as potential sources of human exposure. The aim of this study was to characterize two qPCR assays targeting partial 16S rRNA gene sequences of MA and MI and use these assays, along with two previously reported MAP qPCR assays (IS900 and Target 251), to investigate Mycobacterium occurrence in kitchen faucet biofilms. MA and MI qPCR assays demonstrated 100% specificity and sensitivity when evaluated against 18 non-MA complex, 76 MA, and 17 MI isolates. Both assays detected approximately 1,000 cells from a diluted cell stock inoculated on a sampling swab 100% of the time. DNA analysis by qPCR indicated that 35.3, 56.9 and 11.8% of the 51 kitchen faucet biofilm samples collected contained MA, MI, and MAP, respectively. This study introduces novel qPCR assays designed to specifically detect MA and MI in biofilm. Results support the use of qPCR as an alternative to culture for detection and enumeration of MA, MI, and MAP in microbiologically complex samples.

Automated Broad-Range Molecular Detection of Bacteria in Clinical Samples

PubMed Central

Hoogewerf, Martine; Vandenbroucke-Grauls, Christina M. J. E.; Savelkoul, Paul H. M.

2016-01-01

Molecular detection methods, such as quantitative PCR (qPCR), have found their way into clinical microbiology laboratories for the detection of an array of pathogens. Most routinely used methods, however, are directed at specific species. Thus, anything that is not explicitly searched for will be missed. This greatly limits the flexibility and universal application of these techniques. We investigated the application of a rapid universal bacterial molecular identification method, IS-pro, to routine patient samples received in a clinical microbiology laboratory. IS-pro is a eubacterial technique based on the detection and categorization of 16S-23S rRNA gene interspace regions with lengths that are specific for each microbial species. As this is an open technique, clinicians do not need to decide in advance what to look for. We compared routine culture to IS-pro using 66 samples sent in for routine bacterial diagnostic testing. The samples were obtained from patients with infections in normally sterile sites (without a resident microbiota). The results were identical in 20 (30%) samples, IS-pro detected more bacterial species than culture in 31 (47%) samples, and five of the 10 culture-negative samples were positive with IS-pro. The case histories of the five patients from whom these culture-negative/IS-pro-positive samples were obtained suggest that the IS-pro findings are highly clinically relevant. Our findings indicate that an open molecular approach, such as IS-pro, may have a high added value for clinical practice. PMID:26763956

Detecting 16S rRNA Methyltransferases in Enterobacteriaceae by Use of Arbekacin

PubMed Central

Chahine, Sarah; Okafor, Darius; Ong, Ana C.; Maybank, Rosslyn; Kwak, Yoon I.; Wilson, Kerry; Zapor, Michael; Lesho, Emil; Hinkle, Mary

2015-01-01

16S rRNA methyltransferases confer resistance to most aminoglycosides, but discriminating their activity from that of aminoglycoside-modifying enzymes (AMEs) is challenging using phenotypic methods. We demonstrate that arbekacin, an aminoglycoside refractory to most AMEs, can rapidly detect 16S methyltransferase activity in Enterobacteriaceae with high specificity using the standard disk susceptibility test. PMID:26537447

Microbial detection with low molecular weight RNA.

PubMed

Kourentzi, K D; Fox, G E; Willson, R C

2001-12-01

The need to monitor microorganisms in the environment has increased interest in assays based on hybridization probes that target nucleic acids (e.g., rRNA). We report the development of liquid-phase assays for specific bacterial 5S rRNA sequences or similarly sized artificial RNAs (aRNAs) using molecular beacon technology. These beacons fluoresce only in the presence of specific target sequences, rendering as much as a 27-fold fluorescence enhancement. The assays can be used with both crude cell lysates and purified total RNA preparations. Minimal sample preparation (e.g., heating to promote leakage from cells) is sufficient to detect many Gram-negative bacteria. Using this approach it was possible to detect an aRNA-labeled Escherichia coli strain in the presence of a large background of an otherwise identical E. coli strain. Finally, by using a longer wavelength carboxytetramethylrhodamine beacon it was possible to reduce the fraction of the signal due to cellular autofluorescence to below 0.5%.

Microbial detection with low molecular weight RNA

NASA Technical Reports Server (NTRS)

Kourentzi, K. D.; Fox, G. E.; Willson, R. C.

2001-01-01

The need to monitor microorganisms in the environment has increased interest in assays based on hybridization probes that target nucleic acids (e.g., rRNA). We report the development of liquid-phase assays for specific bacterial 5S rRNA sequences or similarly sized artificial RNAs (aRNAs) using molecular beacon technology. These beacons fluoresce only in the presence of specific target sequences, rendering as much as a 27-fold fluorescence enhancement. The assays can be used with both crude cell lysates and purified total RNA preparations. Minimal sample preparation (e.g., heating to promote leakage from cells) is sufficient to detect many Gram-negative bacteria. Using this approach it was possible to detect an aRNA-labeled Escherichia coli strain in the presence of a large background of an otherwise identical E. coli strain. Finally, by using a longer wavelength carboxytetramethylrhodamine beacon it was possible to reduce the fraction of the signal due to cellular autofluorescence to below 0.5%.

Discrimination of Bacillus anthracis from closely related microorganisms by analysis of 16S and 23S rRNA with oligonucleotide microchips

DOEpatents

Bavykin, Sergei G.; Mirzabekov, Andrei D.

2007-10-30

The present invention is directed to a novel method of discriminating a highly infectious bacterium Bacillus anthracis from a group of closely related microorganisms. Sequence variations in the 16S and 23S rRNA of the B. cereus subgroup including B. anthracis are utilized to construct an array that can detect these sequence variations through selective hybridizations. The identification and analysis of these sequence variations enables positive discrimination of isolates of the B. cereus group that includes B. anthracis. Discrimination of single base differences in rRNA was achieved with a microchip during analysis of B. cereus group isolates from both single and in mixed probes, as well as identification of polymorphic sites. Successful use of a microchip to determine the appropriate subgroup classification using eight reference microorganisms from the B. cereus group as a study set, was demonstrated.

Development and application of absolute quantitative detection by duplex chamber-based digital PCR of genetically modified maize events without pretreatment steps.

PubMed

Zhu, Pengyu; Fu, Wei; Wang, Chenguang; Du, Zhixin; Huang, Kunlun; Zhu, Shuifang; Xu, Wentao

2016-04-15

The possibility of the absolute quantitation of GMO events by digital PCR was recently reported. However, most absolute quantitation methods based on the digital PCR required pretreatment steps. Meanwhile, singleplex detection could not meet the demand of the absolute quantitation of GMO events that is based on the ratio of foreign fragments and reference genes. Thus, to promote the absolute quantitative detection of different GMO events by digital PCR, we developed a quantitative detection method based on duplex digital PCR without pretreatment. Moreover, we tested 7 GMO events in our study to evaluate the fitness of our method. The optimized combination of foreign and reference primers, limit of quantitation (LOQ), limit of detection (LOD) and specificity were validated. The results showed that the LOQ of our method for different GMO events was 0.5%, while the LOD is 0.1%. Additionally, we found that duplex digital PCR could achieve the detection results with lower RSD compared with singleplex digital PCR. In summary, the duplex digital PCR detection system is a simple and stable way to achieve the absolute quantitation of different GMO events. Moreover, the LOQ and LOD indicated that this method is suitable for the daily detection and quantitation of GMO events. Copyright © 2016 Elsevier B.V. All rights reserved.

Hepatozoon caimani in Caiman crocodilus yacare (Crocodylia, Alligatoridae) from North Pantanal, Brazil.

PubMed

Bouer, Andréa; André, Marcos Rogério; Gonçalves, Luiz Ricardo; Luzzi, Mayara de Cássia; Oliveira, Juliana Paula de; Rodrigues, Adriana Carlos; Varani, Alessandro de Melo; Miranda, Vitor Fernandes Oliveira de; Perles, Lívia; Werther, Karin; Machado, Rosangela Zacarias

2017-01-01

Hepatozoon species are the most common intracellular hemoparasite found in reptiles. Hepatozoon caimani, whose vectors are Culex mosquitoes, has been detected in a high prevalence among caimans in Brazil by blood smears examinations. The present work aimed to detect and characterize the Hepatozoon spp. found in 33 caimans (24 free-ranging and 9 captive; 28 males and 5 females) (Caiman crocodilus yacare) sampled at Poconé, North Pantanal, state of Mato Grosso, Brazil, using blood smears examinations and molecular techniques. Hepatozoon spp.-gametocytes were found in 70.8% (17/24) and 88.8% (8/9) of blood smears from free-ranging and captive caimans, respectively. Hepatozoon spp. 18S rRNA DNA was found in 79.2% (19/24) and 88.8% (8/9) of free-ranging and captive caimans, respectively. Comparative analysis of parasitized and non-parasitized erythrocytes showed that all analyzed features were significantly different (P<0.05) for both linear and area dimensions. Phylogenetic analysis based on 18S rRNA sequences grouped the Hepatozoon spp. sequences detected in the present study together with H. caimani, recently detected in caimans in southern Pantanal.

Rapid Detection of the Chlamydiaceae and Other Families in the Order Chlamydiales: Three PCR Tests

PubMed Central

Everett, Karin D. E.; Hornung, Linda J.; Andersen, Arthur A.

1999-01-01

Few identification methods will rapidly or specifically detect all bacteria in the order Chlamydiales, family Chlamydiaceae. In this study, three PCR tests based on sequence data from over 48 chlamydial strains were developed for identification of these bacteria. Two tests exclusively recognized the Chlamydiaceae: a multiplex test targeting the ompA gene and the rRNA intergenic spacer and a TaqMan test targeting the 23S ribosomal DNA. The multiplex test was able to detect as few as 200 inclusion-forming units (IFU), while the TaqMan test could detect 2 IFU. The amplicons produced in these tests ranged from 132 to 320 bp in length. The third test, targeting the 23S rRNA gene, produced a 600-bp amplicon from strains belonging to several families in the order Chlamydiales. Direct sequence analysis of this amplicon has facilitated the identification of new chlamydial strains. These three tests permit ready identification of chlamydiae for diagnostic and epidemiologic study. The specificity of these tests indicates that they might also be used to identify chlamydiae without culture or isolation. PMID:9986815

Rapid polymerase chain reaction-based screening assay for bacterial biothreat agents.

PubMed

Yang, Samuel; Rothman, Richard E; Hardick, Justin; Kuroki, Marcos; Hardick, Andrew; Doshi, Vishal; Ramachandran, Padmini; Gaydos, Charlotte A

2008-04-01

To design and evaluate a rapid polymerase chain reaction (PCR)-based assay for detecting Eubacteria and performing early screening for selected Class A biothreat bacterial pathogens. The authors designed a two-step PCR-based algorithm consisting of an initial broad-based universal detection step, followed by specific pathogen identification targeted for identification of the Class A bacterial biothreat agents. A region in the bacterial 16S rRNA gene containing a highly variable sequence flanked by clusters of conserved sequences was chosen as the target for the PCR assay design. A previously described highly conserved region located within the 16S rRNA amplicon was selected as the universal probe (UniProbe, Integrated DNA Technology, Coralville, IA). Pathogen-specific TaqMan probes were designed for Bacillus anthracis, Yersinia pestis, and Francisella tularensis. Performance of the assay was assessed using genomic DNA extracted from the aforementioned biothreat-related organisms (inactivated or surrogate) and other common bacteria. The UniProbe detected the presence of all tested Eubacteria (31/31) with high analytical sensitivity. The biothreat-specific probes accurately identified organisms down to the closely related species and genus level, but were unable to discriminate between very close surrogates, such as Yersinia philomiragia and Bacillus cereus. A simple, two-step PCR-based assay proved capable of both universal bacterial detection and identification of select Class A bacterial biothreat and biothreat-related pathogens. Although this assay requires confirmatory testing for definitive species identification, the method has great potential for use in ED-based settings for rapid diagnosis in cases of suspected Category A bacterial biothreat agents.

Evidence for rRNA 2'-O-methylation plasticity: Control of intrinsic translational capabilities of human ribosomes.

PubMed

Erales, Jenny; Marchand, Virginie; Panthu, Baptiste; Gillot, Sandra; Belin, Stéphane; Ghayad, Sandra E; Garcia, Maxime; Laforêts, Florian; Marcel, Virginie; Baudin-Baillieu, Agnès; Bertin, Pierre; Couté, Yohann; Adrait, Annie; Meyer, Mélanie; Therizols, Gabriel; Yusupov, Marat; Namy, Olivier; Ohlmann, Théophile; Motorin, Yuri; Catez, Frédéric; Diaz, Jean-Jacques

2017-12-05

Ribosomal RNAs (rRNAs) are main effectors of messenger RNA (mRNA) decoding, peptide-bond formation, and ribosome dynamics during translation. Ribose 2'-O-methylation (2'-O-Me) is the most abundant rRNA chemical modification, and displays a complex pattern in rRNA. 2'-O-Me was shown to be essential for accurate and efficient protein synthesis in eukaryotic cells. However, whether rRNA 2'-O-Me is an adjustable feature of the human ribosome and a means of regulating ribosome function remains to be determined. Here we challenged rRNA 2'-O-Me globally by inhibiting the rRNA methyl-transferase fibrillarin in human cells. Using RiboMethSeq, a nonbiased quantitative mapping of 2'-O-Me, we identified a repertoire of 2'-O-Me sites subjected to variation and demonstrate that functional domains of ribosomes are targets of 2'-O-Me plasticity. Using the cricket paralysis virus internal ribosome entry site element, coupled to in vitro translation, we show that the intrinsic capability of ribosomes to translate mRNAs is modulated through a 2'-O-Me pattern and not by nonribosomal actors of the translational machinery. Our data establish rRNA 2'-O-Me plasticity as a mechanism providing functional specificity to human ribosomes.

Multicolor microRNA FISH effectively differentiates tumor types

PubMed Central

Renwick, Neil; Cekan, Pavol; Masry, Paul A.; McGeary, Sean E.; Miller, Jason B.; Hafner, Markus; Li, Zhen; Mihailovic, Aleksandra; Morozov, Pavel; Brown, Miguel; Gogakos, Tasos; Mobin, Mehrpouya B.; Snorrason, Einar L.; Feilotter, Harriet E.; Zhang, Xiao; Perlis, Clifford S.; Wu, Hong; Suárez-Fariñas, Mayte; Feng, Huichen; Shuda, Masahiro; Moore, Patrick S.; Tron, Victor A.; Chang, Yuan; Tuschl, Thomas

2013-01-01

MicroRNAs (miRNAs) are excellent tumor biomarkers because of their cell-type specificity and abundance. However, many miRNA detection methods, such as real-time PCR, obliterate valuable visuospatial information in tissue samples. To enable miRNA visualization in formalin-fixed paraffin-embedded (FFPE) tissues, we developed multicolor miRNA FISH. As a proof of concept, we used this method to differentiate two skin tumors, basal cell carcinoma (BCC) and Merkel cell carcinoma (MCC), with overlapping histologic features but distinct cellular origins. Using sequencing-based miRNA profiling and discriminant analysis, we identified the tumor-specific miRNAs miR-205 and miR-375 in BCC and MCC, respectively. We addressed three major shortcomings in miRNA FISH, identifying optimal conditions for miRNA fixation and ribosomal RNA (rRNA) retention using model compounds and high-pressure liquid chromatography (HPLC) analyses, enhancing signal amplification and detection by increasing probe-hapten linker lengths, and improving probe specificity using shortened probes with minimal rRNA sequence complementarity. We validated our method on 4 BCC and 12 MCC tumors. Amplified miR-205 and miR-375 signals were normalized against directly detectable reference rRNA signals. Tumors were classified using predefined cutoff values, and all were correctly identified in blinded analysis. Our study establishes a reliable miRNA FISH technique for parallel visualization of differentially expressed miRNAs in FFPE tumor tissues. PMID:23728175

High abundance of JS-1- and Chloroflexi-related Bacteria in deeply buried marine sediments revealed by quantitative, real-time PCR.

PubMed

Blazejak, Anna; Schippers, Axel

2010-05-01

Sequences of members of the bacterial candidate division JS-1 and the classes Anaerolineae and Caldilineae of the phylum Chloroflexi are frequently found in 16S rRNA gene clone libraries obtained from marine sediments. Using a newly designed quantitative, real-time PCR assay, these bacterial groups were jointly quantified in samples from near-surface and deeply buried marine sediments from the Peru margin, the Black Sea, and a forearc basin off the island of Sumatra. In near-surface sediments, sequences of the JS-1 as well as Anaerolineae- and Caldilineae-related Bacteria were quantified with significantly lower 16S rRNA gene copy numbers than the sequences of total Bacteria. In contrast, in deeply buried sediments below approximately 1 m depth, similar quantities of the 16S rRNA gene copies of these specific groups and Bacteria were found. This finding indicates that JS-1 and Anaerolineae- and Caldilineae-related Bacteria might dominate the bacterial community in deeply buried marine sediments and thus seem to play an important ecological role in the deep biosphere.

Evaluation of the Bacterial Diversity in the Human Tongue Coating Based on Genus-Specific Primers for 16S rRNA Sequencing.

PubMed

Sun, Beili; Zhou, Dongrui; Tu, Jing; Lu, Zuhong

2017-01-01

The characteristics of tongue coating are very important symbols for disease diagnosis in traditional Chinese medicine (TCM) theory. As a habitat of oral microbiota, bacteria on the tongue dorsum have been proved to be the cause of many oral diseases. The high-throughput next-generation sequencing (NGS) platforms have been widely applied in the analysis of bacterial 16S rRNA gene. We developed a methodology based on genus-specific multiprimer amplification and ligation-based sequencing for microbiota analysis. In order to validate the efficiency of the approach, we thoroughly analyzed six tongue coating samples from lung cancer patients with different TCM types, and more than 600 genera of bacteria were detected by this platform. The results showed that ligation-based parallel sequencing combined with enzyme digestion and multiamplification could expand the effective length of sequencing reads and could be applied in the microbiota analysis.

Expression stability of two housekeeping genes (18S rRNA and G3PDH) during in vitro maturation of follicular oocytes in buffalo (Bubalus bubalis).

PubMed

Aswal, Ajay Pal Singh; Raghav, Sarvesh; De, Sachinandan; Thakur, Manish; Goswami, Surender Lal; Datta, Tirtha Kumar

2008-01-15

The present study was undertaken to evaluate the expression stability of two housekeeping genes (HKGs), 18S rRNA and G3PDH during in vitro maturation (IVM) of oocytes in buffalo, which qualifies their use as internal controls for valid qRT-PCR estimation of other oocyte transcripts. A semi quantitative RT-PCR system was used with optimised qRT-PCR parameters at exponential PCR cycle for evaluation of temporal expression pattern of these genes over 24 h of IVM. 18S rRNA was found more stable in its expression pattern than G3PDH.

Development and validation of a multiplex quantitative polymerase chain reaction assay for the detection of Mollicutes impurities in human cells, cultured under good manufacturing practice conditions, and following European Pharmacopoeia requirements and the International Conference on Harmonization guidelines.

PubMed

Vanni, Irene; Ugolotti, Elisabetta; Raso, Alessandro; Di Marco, Eddi; Melioli, Giovanni; Biassoni, Roberto

2012-07-01

The clinical applications of in vitro manipulated cultured cells and their precursors are often made use of in therapeutic trials. However, tissue cultures can be easily contaminated by the ubiquitous Mollicutes micro-organisms, which can cause various and severe alterations in cellular function. Thus methods able to detect and trace Mollicutes impurities contaminating cell cultures are required before starting any attempt to grow cells under good manufacturing practice (GMP) conditions. We developed a multiplex quantitative polymerase chain reaction (qPCR) assay specific for the 16S-23S rRNA intergenic spacer regions, for the Tuf and P1 cytoadhesin genes, able to detect contaminant Mollicutes species in a single tube reaction. The system was validated by analyzing different cell lines and the positive samples were confirmed by 16S and P1 cytoadhesin gene dideoxy sequencing. Our multiplex qPCR detection system was able to reach a sensitivity, specificity and robustness comparable with the culture and the indicator cell culture method, as required by the European Pharmacopoeia guidelines. We have developed a multiplex qPCR method, validated following International Conference on Harmonization (ICH) guidelines, as a qualitative limit test for impurities, assessing the validation characteristics of limit of detection and specificity. It also follows the European Pharmacopoeia guidelines and Food and Drug Administration (FDA) requirements.

IDENTIFICATION OF SPECIES AND SOURCES OF CRYPTOSPORIDIUM OOCYSTS IN STORM WATERS BY A SMALL SUBUNIT RRNA-BASED DIAGNOSTIC AND GENOTYPING TOOL

EPA Science Inventory

The identification of Cryptosporidium oocysts in environmental samples is largely made by the use of immunofluorescent assay (IFA). because IFA detects oocysts from all Cryptosporidium parasites, the species distribution and source of Cryptosporidium parasites in environmental sa...

Molecular survey of the occurrence of Legionella spp., Mycobacterium spp., Pseudomonas aeruginosa, and amoeba hosts in two chloraminated drinking water distribution systems.

PubMed

Wang, Hong; Edwards, Marc; Falkinham, Joseph O; Pruden, Amy

2012-09-01

The spread of opportunistic pathogens via public water systems is of growing concern. The purpose of this study was to identify patterns of occurrence among three opportunistic pathogens (Legionella pneumophila, Mycobacterium avium, and Pseudomonas aeruginosa) relative to biotic and abiotic factors in two representative chloraminated drinking water distribution systems using culture-independent methods. Generally, a high occurrence of Legionella (≥69.0%) and mycobacteria (100%), lower occurrence of L. pneumophila (≤20%) and M. avium (≤33.3%), and rare detection of Pseudomonas aeruginosa (≤13.3%) were observed in both systems according to quantitative PCR. Also, Hartmanella vermiformis was more prevalent than Acanthamoeba, both of which are known hosts for opportunistic pathogen amplification, the latter itself containing pathogenic members. Three-minute flushing served to distinguish distribution system water from plumbing in buildings (i.e., premise plumbing water) and resulted in reduced numbers of copies of Legionella, mycobacteria, H. vermiformis, and 16S rRNA genes (P < 0.05) while yielding distinct terminal restriction fragment polymorphism (T-RFLP) profiles of 16S rRNA genes. Within certain subgroups of samples, some positive correlations, including correlations of numbers of mycobacteria and total bacteria (16S rRNA genes), H. vermiformis and total bacteria, mycobacteria and H. vermiformis, and Legionella and H. vermiformis, were noted, emphasizing potential microbial ecological relationships. Overall, the results provide insight into factors that may aid in controlling opportunistic pathogen proliferation in real-world water systems.

Molecular Survey of the Occurrence of Legionella spp., Mycobacterium spp., Pseudomonas aeruginosa, and Amoeba Hosts in Two Chloraminated Drinking Water Distribution Systems

PubMed Central

Wang, Hong; Edwards, Marc; Falkinham, Joseph O.

2012-01-01

The spread of opportunistic pathogens via public water systems is of growing concern. The purpose of this study was to identify patterns of occurrence among three opportunistic pathogens (Legionella pneumophila, Mycobacterium avium, and Pseudomonas aeruginosa) relative to biotic and abiotic factors in two representative chloraminated drinking water distribution systems using culture-independent methods. Generally, a high occurrence of Legionella (≥69.0%) and mycobacteria (100%), lower occurrence of L. pneumophila (≤20%) and M. avium (≤33.3%), and rare detection of Pseudomonas aeruginosa (≤13.3%) were observed in both systems according to quantitative PCR. Also, Hartmanella vermiformis was more prevalent than Acanthamoeba, both of which are known hosts for opportunistic pathogen amplification, the latter itself containing pathogenic members. Three-minute flushing served to distinguish distribution system water from plumbing in buildings (i.e., premise plumbing water) and resulted in reduced numbers of copies of Legionella, mycobacteria, H. vermiformis, and 16S rRNA genes (P < 0.05) while yielding distinct terminal restriction fragment polymorphism (T-RFLP) profiles of 16S rRNA genes. Within certain subgroups of samples, some positive correlations, including correlations of numbers of mycobacteria and total bacteria (16S rRNA genes), H. vermiformis and total bacteria, mycobacteria and H. vermiformis, and Legionella and H. vermiformis, were noted, emphasizing potential microbial ecological relationships. Overall, the results provide insight into factors that may aid in controlling opportunistic pathogen proliferation in real-world water systems. PMID:22752174

Archaeal homologs of eukaryotic methylation guide small nucleolar RNAs: lessons from the Pyrococcus genomes.

PubMed

Gaspin, C; Cavaillé, J; Erauso, G; Bachellerie, J P

2000-04-07

Ribose methylation is a prevalent type of nucleotide modification in rRNA. Eukaryotic rRNAs display a complex pattern of ribose methylations, amounting to 55 in yeast Saccharomyces cerevisiae and about 100 in vertebrates. Ribose methylations of eukaryotic rRNAs are each guided by a cognate small RNA, belonging to the family of box C/D antisense snoRNAs, through transient formation of a specific base-pairing at the rRNA modification site. In prokaryotes, the pattern of rRNA ribose methylations has been fully characterized in a single species so far, Escherichia coli, which contains only four ribose methylated rRNA nucleotides. However, the hyperthermophile archaeon Sulfolobus solfataricus contains, like eukaryotes, a large number of (yet unmapped) rRNA ribose methylations and homologs of eukaryotic box C/D small nucleolar ribonuclear proteins have been identified in archaeal genomes. We have therefore searched archaeal genomes for potential homologs of eukaryotic methylation guide small nucleolar RNAs, by combining searches for structured motifs with homology searches. We have identified a family of 46 small RNAs, conserved in the genomes of three hyperthermophile Pyrococcus species, which we have experimentally characterized in Pyrococcus abyssi. The Pyrococcus small RNAs, the first reported homologs of methylation guide small nucleolar RNAs in organisms devoid of a nucleus, appear as a paradigm of minimalist box C/D antisense RNAs. They differ from their eukaryotic homologs by their outstanding structural homogeneity, extended consensus box motifs and the quasi-systematic presence of two (instead of one) rRNA antisense elements. Remarkably, for each small RNA the two antisense elements always match rRNA sequences close to each other in rRNA structure, suggesting an important role in rRNA folding. Only a few of the predicted P. abyssi rRNA ribose methylations have been detected so far. Further analysis of these archaeal small RNAs could provide new insights into the origin and functions of methylation guide small nucleolar RNAs and illuminate the still elusive role of rRNA ribose methylations. Copyright 2000 Academic Press.

Anaplasma phagocytophilum in questing Ixodes ricinus ticks: comparison of prevalences and partial 16S rRNA gene variants in urban, pasture, and natural habitats.

PubMed

Overzier, Evelyn; Pfister, Kurt; Thiel, Claudia; Herb, Ingrid; Mahling, Monia; Silaghi, Cornelia

2013-03-01

Urban, natural, and pasture areas were investigated for prevalences and 16S rRNA gene variants of Anaplasma phagocytophilum in questing Ixodes ricinus ticks. The prevalences differed significantly between habitat types, and year-to-year variations in prevalence and habitat-dependent occurrence of 16S rRNA gene variants were detected.

Development of a genus-specific next generation sequencing approach for sensitive and quantitative determination of the Legionella microbiome in freshwater systems.

PubMed

Pereira, Rui P A; Peplies, Jörg; Brettar, Ingrid; Höfle, Manfred G

2017-03-31

Next Generation Sequencing (NGS) has revolutionized the analysis of natural and man-made microbial communities by using universal primers for bacteria in a PCR based approach targeting the 16S rRNA gene. In our study we narrowed primer specificity to a single, monophyletic genus because for many questions in microbiology only a specific part of the whole microbiome is of interest. We have chosen the genus Legionella, comprising more than 20 pathogenic species, due to its high relevance for water-based respiratory infections. A new NGS-based approach was designed by sequencing 16S rRNA gene amplicons specific for the genus Legionella using the Illumina MiSeq technology. This approach was validated and applied to a set of representative freshwater samples. Our results revealed that the generated libraries presented a low average raw error rate per base (<0.5%); and substantiated the use of high-fidelity enzymes, such as KAPA HiFi, for increased sequence accuracy and quality. The approach also showed high in situ specificity (>95%) and very good repeatability. Only in samples in which the gammabacterial clade SAR86 was present more than 1% non-Legionella sequences were observed. Next-generation sequencing read counts did not reveal considerable amplification/sequencing biases and showed a sensitive as well as precise quantification of L. pneumophila along a dilution range using a spiked-in, certified genome standard. The genome standard and a mock community consisting of six different Legionella species demonstrated that the developed NGS approach was quantitative and specific at the level of individual species, including L. pneumophila. The sensitivity of our genus-specific approach was at least one order of magnitude higher compared to the universal NGS approach. Comparison of quantification by real-time PCR showed consistency with the NGS data. Overall, our NGS approach can determine the quantitative abundances of Legionella species, i. e. the complete Legionella microbiome, without the need for species-specific primers. The developed NGS approach provides a new molecular surveillance tool to monitor all Legionella species in qualitative and quantitative terms if a spiked-in genome standard is used to calibrate the method. Overall, the genus-specific NGS approach opens up a new avenue to massive parallel diagnostics in a quantitative, specific and sensitive way.

A quantitative TaqMan PCR assay for the detection of Ureaplasma diversum.

PubMed

Marques, Lucas M; Amorim, Aline T; Martins, Hellen Braga; Rezende, Izadora Souza; Barbosa, Maysa Santos; Lobão, Tassia Neves; Campos, Guilherme B; Timenetsky, Jorge

2013-12-27

Ureaplasma diversum in veterinary studies is an undesirable microbe, which may cause infection in bulls and may result in seminal vesiculitis, balanopostitis, and alterations in spermatozoids, whereas in cows, it may cause placentitis, fetal alveolitis, abortion, and birth of weak calves. U. diversum is released through organic secretions, especially semen, preputial and vaginal mucus, conjunctival secretion, and milk. The aim of the present study was to develop a TaqMan probe, highly sensitive and specific quantitative PCR (qPCR) assay for the detection and quantification of U. diversum from genital swabs of bovines. Primers and probes specific to U. diversum 16S rRNA gene were designed. The specificity, detection limit, intra- and inter-assay variability of qPCR to detect this ureaplasma was compared with the results of the conventional PCR assay (cPCR). Swabs of vaginal mucus from 169 cows were tested. The qPCR assay detected as few as 10 copies of U. diversum and was 100-fold more sensitive than the cPCR. No cross-reactivity with other Mollicutes or eubacteria was observed. U. diversum was detected in 79 swabs (46.42%) by qPCR, while using cPCR it was detected in 42 (25%) samples. The difference in cPCR and qPCR ureaplasma detection between healthy and sick animals was not statistically significant. But the U. diversum load in samples from animals with genital disorders was higher than in healthy animals. The qPCR assay developed herein is highly sensitive and specific for the detection and quantification of U. diversum in vaginal bovine samples. Copyright © 2013. Published by Elsevier B.V.

Electrochemical detection of synthetic DNA and native 16S rRNA fragments on a microarray using a biotinylated intercalator as coupling site for an enzyme label.

PubMed

Zimdars, Andreas; Gebala, Magdalena; Hartwich, Gerhard; Neugebauer, Sebastian; Schuhmann, Wolfgang

2015-10-01

The direct electrochemical detection of synthetic DNA and native 16S rRNA fragments isolated from Escherichia coli is described. Oligonucleotides are detected via selective post-labeling of double stranded DNA and DNA-RNA duplexes with a biotinylated intercalator that enables high-specific binding of a streptavidin/alkaline phosphatase conjugate. The alkaline phosphatase catalyzes formation of p-aminophenol that is subsequently oxidized at the underlying gold electrode and hence enables the detection of complementary hybridization of the DNA capture strands due to the enzymatic signal amplification. The hybridization assay was performed on microarrays consisting of 32 individually addressable gold microelectrodes. Synthetic DNA strands with sequences representing six different pathogens which are important for the diagnosis of urinary tract infections could be detected at concentrations of 60 nM. Native 16S rRNA isolated from the different pathogens could be detected at a concentration of 30 fM. Optimization of the sensing surface is described and influences on the assay performance are discussed. Copyright © 2015 Elsevier B.V. All rights reserved.

Culture-Independent Techniques for Rapid Detection of Bacteria Associated with Loss of Chloramine Residual in a Drinking Water System

PubMed Central

Hoefel, Daniel; Monis, Paul T.; Grooby, Warwick L.; Andrews, Stuart; Saint, Christopher P.

2005-01-01

Chloramination is often the disinfection regimen of choice for extended drinking water systems. However, this process is prone to instability due to the growth of nitrifying bacteria. This is the first study to use alternative approaches for rapid investigation of chloraminated drinking water system instability in which flow cytometric cell sorting of bacteria with intact membranes (membrane-intact fraction) (BacLight kit) or with active esterases (esterase-active fraction) (carboxyfluorescein diacetate) was combined with 16S rRNA gene-directed PCR and denaturing gradient gel electrophoresis (DGGE). No active bacteria were detected when water left the water treatment plant (WTP), but 12 km downstream the chloramine residual had diminished and the level of active bacteria in the bulk water had increased to more than 1 × 105 bacteria ml−1. The bacterial diversity in the system was represented by six major DGGE bands for the membrane-intact fraction and 10 major DGGE bands for the esterase-active fraction. PCR targeting of the 16S rRNA gene of chemolithotrophic ammonia-oxidizing bacteria (AOB) and subsequent DGGE and DNA sequence analysis revealed the presence of an active Nitrosospira-related species and Nitrosomonas cryotolerans in the system, but no AOB were detected in the associated WTP. The abundance of active AOB was then determined by quantitative real-time PCR (qPCR) targeting the amoA gene; 3.43 × 103 active AOB ml−1 were detected in the membrane-intact fraction, and 1.40 × 104 active AOB ml−1 were detected in the esterase-active fraction. These values were several orders of magnitude greater than the 2.5 AOB ml−1 detected using a routine liquid most-probable-number assay. Culture-independent techniques described here, in combination with existing chemical indicators, should allow the water industry to obtain more comprehensive data with which to make informed decisions regarding remedial action that may be required either prior to or during an instability event. PMID:16269672

Development of Primer Sets for Loop-Mediated Isothermal Amplification that Enables Rapid and Specific Detection of Streptococcus dysgalactiae, Streptococcus uberis and Streptococcus agalactiae.

PubMed

Wang, Deguo; Liu, Yanhong

2015-05-26

Streptococcus dysgalactiae, Streptococcus uberis and Streptococcus agalactiae are the three main pathogens causing bovine mastitis, with great losses to the dairy industry. Rapid and specific loop-mediated isothermal amplification methods (LAMP) for identification and differentiation of these three pathogens are not available. With the 16S rRNA gene and 16S-23S rRNA intergenic spacers as targets, four sets of LAMP primers were designed for identification and differentiation of S. dysgalactiae, S. uberis and S. agalactiae. The detection limit of all four LAMP primer sets were 0.1 pg DNA template per reaction, the LAMP method with 16S rRNA gene and 16S-23S rRNA intergenic spacers as the targets can differentiate the three pathogens, which is potentially useful in epidemiological studies.

Employment of Near Full-Length Ribosome Gene TA-Cloning and Primer-Blast to Detect Multiple Species in a Natural Complex Microbial Community Using Species-Specific Primers Designed with Their Genome Sequences.

PubMed

Zhang, Huimin; He, Hongkui; Yu, Xiujuan; Xu, Zhaohui; Zhang, Zhizhou

2016-11-01

It remains an unsolved problem to quantify a natural microbial community by rapidly and conveniently measuring multiple species with functional significance. Most widely used high throughput next-generation sequencing methods can only generate information mainly for genus-level taxonomic identification and quantification, and detection of multiple species in a complex microbial community is still heavily dependent on approaches based on near full-length ribosome RNA gene or genome sequence information. In this study, we used near full-length rRNA gene library sequencing plus Primer-Blast to design species-specific primers based on whole microbial genome sequences. The primers were intended to be specific at the species level within relevant microbial communities, i.e., a defined genomics background. The primers were tested with samples collected from the Daqu (also called fermentation starters) and pit mud of a traditional Chinese liquor production plant. Sixteen pairs of primers were found to be suitable for identification of individual species. Among them, seven pairs were chosen to measure the abundance of microbial species through quantitative PCR. The combination of near full-length ribosome RNA gene library sequencing and Primer-Blast may represent a broadly useful protocol to quantify multiple species in complex microbial population samples with species-specific primers.

A prototype stable RNA identification cassette for monitoring plasmids of genetically engineered microorganisms

NASA Technical Reports Server (NTRS)

Hedenstierna, K. O.; Lee, Y. H.; Yang, Y.; Fox, G. E.

1993-01-01

A prototype stable RNA identification cassette for monitoring genetically engineered plasmids carried by strains of Escherichia coli has been developed. The cassette consists of a Vibrio proteolyticus 5S ribosomal RNA (rRNA) gene surrounded by promoters and terminators from the rrnB operon of Escherischia coli. The identifier RNA is expressed and successfully processed so that approximately 30% of the 5S rRNA isolated from either whole cells or 70S ribosomes is of the V. proteolyticus type. Cells carrying the identifier are readily detectable by hybridization. Accurate measurements show that the identification cassette has little effect on fitness compared to a strain containing an analogous plasmid carrying wild type E. coli 5S rRNA, and the V. proteolyticus 5S rRNA gene is not inactivated after prolonged growth. These results demonstrate the feasibility of developing small standardized identification cassettes that can utilize already existing highly sensitive rRNA detection methods. Cassettes of this type could in principle be incorporated into either the engineered regions of recombinant plasmids or their hosts.

Applied Genomics: Data Mining Reveals Species-Specific Malaria Diagnostic Targets More Sensitive than 18S rRNA▿†‡

PubMed Central

Demas, Allison; Oberstaller, Jenna; DeBarry, Jeremy; Lucchi, Naomi W.; Srinivasamoorthy, Ganesh; Sumari, Deborah; Kabanywanyi, Abdunoor M.; Villegas, Leopoldo; Escalante, Ananias A.; Kachur, S. Patrick; Barnwell, John W.; Peterson, David S.; Udhayakumar, Venkatachalam; Kissinger, Jessica C.

2011-01-01

Accurate and rapid diagnosis of malaria infections is crucial for implementing species-appropriate treatment and saving lives. Molecular diagnostic tools are the most accurate and sensitive method of detecting Plasmodium, differentiating between Plasmodium species, and detecting subclinical infections. Despite available whole-genome sequence data for Plasmodium falciparum and P. vivax, the majority of PCR-based methods still rely on the 18S rRNA gene targets. Historically, this gene has served as the best target for diagnostic assays. However, it is limited in its ability to detect mixed infections in multiplex assay platforms without the use of nested PCR. New diagnostic targets are needed. Ideal targets will be species specific, highly sensitive, and amenable to both single-step and multiplex PCRs. We have mined the genomes of P. falciparum and P. vivax to identify species-specific, repetitive sequences that serve as new PCR targets for the detection of malaria. We show that these targets (Pvr47 and Pfr364) exist in 14 to 41 copies and are more sensitive than 18S rRNA when utilized in a single-step PCR. Parasites are routinely detected at levels of 1 to 10 parasites/μl. The reaction can be multiplexed to detect both species in a single reaction. We have examined 7 P. falciparum strains and 91 P. falciparum clinical isolates from Tanzania and 10 P. vivax strains and 96 P. vivax clinical isolates from Venezuela, and we have verified a sensitivity and specificity of ∼100% for both targets compared with a nested 18S rRNA approach. We show that bioinformatics approaches can be successfully applied to identify novel diagnostic targets and improve molecular methods for pathogen detection. These novel targets provide a powerful alternative molecular diagnostic method for the detection of P. falciparum and P. vivax in conventional or multiplex PCR platforms. PMID:21525225

Detection of Verrucomicrobia in a Pasture Soil by PCR-Mediated Amplification of 16S rRNA Genes

PubMed Central

O’Farrell, Katrina A.; Janssen, Peter H.

1999-01-01

Oligonucleotide primers were designed and used to amplify, by PCR, partial 16S rRNA genes of members of the bacterial division Verrucomicrobia in DNA extracted from a pasture soil. By applying most-probable-number theory to the assay, verrucomicrobia appeared to contribute some 0.2% of the soil DNA. Amplified ribosomal DNA restriction analysis of 53 cloned PCR-amplified partial 16S rRNA gene fragments and comparative sequence analysis of 21 nonchimeric partial 16S rRNA genes showed that these primers amplified only 16S rRNA genes of members of the Verrucomicrobia in DNA extracted from the soil. PMID:10473454

Development of species-specific rDNA probes for Giardia by multiple fluorescent in situ hybridization combined with immunocytochemical identification of cyst wall antigens.

PubMed

Erlandsen, Stanley L; Jarroll, Edward; Wallis, Peter; van Keulen, Harry

2005-08-01

In this study, we describe the development of fluorescent oligonucleotide probes to variable regions in the small subunit of 16S rRNA in three distinct Giardia species. Sense and antisense probes (17-22 mer) to variable regions 1, 3, and 8 were labeled with digoxygenin or selected fluorochomes (FluorX, Cy3, or Cy5). Optimal results were obtained with fluorochome-labeled oligonucleotides for detection of rRNA in Giardia cysts. Specificity of fluorescent in situ hybridization (FISH) was shown using RNase digestion and high stringency to diminish the hybridization signal, and oligonucleotide probes for rRNA in Giardia lamblia, Giardia muris, and Giardia ardeae were shown to specifically stain rRNA only within cysts or trophozoites of those species. The fluorescent oligonucleotide specific for rRNA in human isolates of Giardia was positive for ten different strains. A method for simultaneous FISH detection of cysts using fluorescent antibody (genotype marker) and two oligonucleotide probes (species marker) permitted visualization of G. lamblia and G. muris cysts in the same preparation. Testing of an environmental water sample revealed the presence of FISH-positive G. lamblia cysts with a specific rDNA probe for rRNA, while negative cysts were presumed to be of animal or bird origin.

Molecular characterization of 5S ribosomal RNA genes and transcripts in the protozoan parasite Leishmania major.

PubMed

Moreno-Campos, Rodrigo; Florencio-Martínez, Luis E; Nepomuceno-Mejía, Tomás; Rojas-Sánchez, Saúl; Vélez-Ramírez, Daniel E; Padilla-Mejía, Norma E; Figueroa-Angulo, Elisa; Manning-Cela, Rebeca; Martínez-Calvillo, Santiago

2016-12-01

Eukaryotic 5S rRNA, synthesized by RNA polymerase III (Pol III), is an essential component of the large ribosomal subunit. Most organisms contain hundreds of 5S rRNA genes organized into tandem arrays. However, the genome of the protozoan parasite Leishmania major contains only 11 copies of the 5S rRNA gene, which are interspersed and associated with other Pol III-transcribed genes. Here we report that, in general, the number and order of the 5S rRNA genes is conserved between different species of Leishmania. While in most organisms 5S rRNA genes are normally associated with the nucleolus, combined fluorescent in situ hybridization and indirect immunofluorescence experiments showed that 5S rRNA genes are mainly located at the nuclear periphery in L. major. Similarly, the tandemly repeated 5S rRNA genes in Trypanosoma cruzi are dispersed throughout the nucleus. In contrast, 5S rRNA transcripts in L. major were localized within the nucleolus, and scattered throughout the cytoplasm, where mature ribosomes are located. Unlike other rRNA species, stable antisense RNA complementary to 5S rRNA is not detected in L. major.

Detecting 16S rRNA Methyltransferases in Enterobacteriaceae by Use of Arbekacin.

PubMed

McGann, Patrick; Chahine, Sarah; Okafor, Darius; Ong, Ana C; Maybank, Rosslyn; Kwak, Yoon I; Wilson, Kerry; Zapor, Michael; Lesho, Emil; Hinkle, Mary

2016-01-01

16S rRNA methyltransferases confer resistance to most aminoglycosides, but discriminating their activity from that of aminoglycoside-modifying enzymes (AMEs) is challenging using phenotypic methods. We demonstrate that arbekacin, an aminoglycoside refractory to most AMEs, can rapidly detect 16S methyltransferase activity in Enterobacteriaceae with high specificity using the standard disk susceptibility test. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Development of a PCR Assay for the Detection of Spironucleus muris

PubMed Central

Jackson, Glenn A; Livingston, Robert S; Riley, Lela K; Livingston, Beth A; Franklin, Craig L

2013-01-01

Spironucleus muris is a protozoan that can colonize the intestinal tract of many rodent species. Although its effects on animal health and research are debated, S. muris is often included on exclusion lists for rodent facilities. Common diagnostic tests for S. muris are insensitive and typically are performed at postmortem examination. We sought to develop a PCR-based diagnostic test with sufficient sensitivity and specificity for use on fecal samples from live rodents. We designed and optimized a PCR assay that targeted the 16S-like rRNA gene of S. muris. The assay was highly specific, given that samples from mice contaminated with S. muris were PCR positive, whereas samples from mice contaminated with other protozoa were negative. The assay also was highly sensitive, detecting as few as 5 template copies per microliter diluent. All mice positive for S. muris on postmortem exams also were positive by fecal PCR. Moreover, S. muris was detected by PCR in mice negative by postmortem examination but from colonies known to be contaminated as well as in rats and hamsters. To assess protozoal loads in mice of differing ages, the PCR assay was adapted to a quantitative format. Fecal loads of S. muris were highest in 4-wk-old mice and declined with age. The PCR assay developed promises to be a highly specific antemortem diagnostic assay with higher sensitivity than that of existing postmortem tests. PMID:23562099

Validation and Application of a PCR Primer Set to Quantify Fungal Communities in the Soil Environment by Real-Time Quantitative PCR

PubMed Central

Chemidlin Prévost-Bouré, Nicolas; Christen, Richard; Dequiedt, Samuel; Mougel, Christophe; Lelièvre, Mélanie; Jolivet, Claudy; Shahbazkia, Hamid Reza; Guillou, Laure; Arrouays, Dominique; Ranjard, Lionel

2011-01-01

Fungi constitute an important group in soil biological diversity and functioning. However, characterization and knowledge of fungal communities is hampered because few primer sets are available to quantify fungal abundance by real-time quantitative PCR (real-time Q-PCR). The aim in this study was to quantify fungal abundance in soils by incorporating, into a real-time Q-PCR using the SYBRGreen® method, a primer set already used to study the genetic structure of soil fungal communities. To satisfy the real-time Q-PCR requirements to enhance the accuracy and reproducibility of the detection technique, this study focused on the 18S rRNA gene conserved regions. These regions are little affected by length polymorphism and may provide sufficiently small targets, a crucial criterion for enhancing accuracy and reproducibility of the detection technique. An in silico analysis of 33 primer sets targeting the 18S rRNA gene was performed to select the primer set with the best potential for real-time Q-PCR: short amplicon length; good fungal specificity and coverage. The best consensus between specificity, coverage and amplicon length among the 33 sets tested was the primer set FR1 / FF390. This in silico analysis of the specificity of FR1 / FF390 also provided additional information to the previously published analysis on this primer set. The specificity of the primer set FR1 / FF390 for Fungi was validated in vitro by cloning - sequencing the amplicons obtained from a real time Q-PCR assay performed on five independent soil samples. This assay was also used to evaluate the sensitivity and reproducibility of the method. Finally, fungal abundance in samples from 24 soils with contrasting physico-chemical and environmental characteristics was examined and ranked to determine the importance of soil texture, organic carbon content, C∶N ratio and land use in determining fungal abundance in soils. PMID:21931659

Molecular Fingerprint and Dominant Environmental Factors of Nitrite-Dependent Anaerobic Methane-Oxidizing Bacteria in Sediments from the Yellow River Estuary, China.

PubMed

Yan, Pengze; Li, Mingcong; Wei, Guangshan; Li, Han; Gao, Zheng

2015-01-01

Nitrite-dependent anaerobic methane oxidation (n-damo) is performed by "Candidatus Methylomirabilis oxyfera" (M. oxyfera), which connects the carbon and nitrogen global nutrient cycles. In the present study, M. oxyfera-like bacteria sequences were successfully recovered from Yellow River Estuary sediments using specific primers for 16S rRNA and pmoA genes. A M. oxyfera-like sequences analysis based on the 16S rRNA gene revealed greater diversity compared with the pmoA gene; the 16S rRNA gene sequences retrieved from the Yellow River Estuary sediments belong to groups A as well as B and were mainly found in freshwater habitats. Quantitative PCR showed that 16S rRNA gene abundance varied from 9.28±0.11×10(3) to 2.10±0.13×10(5) copies g(-1) (dry weight), and the pmoA gene abundance ranged from 8.63±0.50×10(3) to 1.83±0.18×10(5) copies g(-1) (dry weight). A correlation analysis showed that the total organic carbon (TOC) and ammonium (NH4(+)) as well as the ratio of total phosphorus to total nitrogen (TP/TN) influenced the M. oxyfera-like bacteria distribution in the Yellow River Estuary sediments. These findings will aid in understanding the n-damo bacterial distribution pattern as well as their correlation with surrounding environmental factors in temperate estuarine ecosystems.

Molecular Detection of Helicobacter pylori and its Antimicrobial Resistance in Brazzaville, Congo.

PubMed

Ontsira Ngoyi, Esther Nina; Atipo Ibara, Blaise Irénée; Moyen, Rachelle; Ahoui Apendi, Philestine Clausina; Ibara, Jean Rosaire; Obengui, O; Ossibi Ibara, Roland Bienvenu; Nguimbi, Etienne; Niama, Rock Fabien; Ouamba, Jean Maurille; Yala, Fidèle; Abena, Ange Antoine; Vadivelu, Jamuna; Goh, Khean Lee; Menard, Armelle; Benejat, Lucie; Sifre, Elodie; Lehours, Philippe; Megraud, Francis

2015-08-01

Helicobacter pylori infection is involved in several gastroduodenal diseases which can be cured by antimicrobial treatment. The aim of this study was to determine the prevalence of H. pylori infection and its bacterial resistance to clarithromycin, fluoroquinolones, and tetracycline in Brazzaville, Congo, by using molecular methods. A cross- sectional study was carried out between September 2013 and April 2014. Biopsy specimens were obtained from patients scheduled for an upper gastrointestinal endoscopy and were sent to the French National Reference Center for Campylobacters and Helicobacters where they were tested by molecular methods for detection of H. pylori and clarithromycin resistance by real-time PCR using a fluorescence resonance energy transfer-melting curve analysis (FRET-MCA) protocol, for detection of tetracycline resistance by real-time PCR on 16S rRNA genes (rrnA and rrnB), for detection of point mutations in the quinolone resistance-determining regions (QRDR) of H. pylori gyrA gene, associated with resistance to quinolones, by PCR and sequencing. This study showed a high H. pylori prevalence (89%), low rates of clarithromycin and tetracycline resistance (1.7% and 2.5%, respectively), and a high rate of quinolone resistance (50%). Therefore, the use of standard clarithromycin-based triple therapy is still possible as an empiric first-line treatment as well as prescription of bismuth-based quadruple therapy, which includes tetracycline, but not a levofloxacin-based triple therapy because of the high rate of resistance to fluoroquinolones. © 2015 John Wiley & Sons Ltd.

Development and validation of a citrate synthase directed quantitative PCR marker for soil bacterial communities

DOE Office of Scientific and Technical Information (OSTI.GOV)

Castro Gonzalez, Hector F; Classen, Aimee T; Austin, Emily E

2012-01-01

Molecular innovations in microbial ecology are allowing scientists to correlate microbial community characteristics to a variety of ecosystem functions. However, to date the majority of soil microbial ecology studies target phylogenetic rRNA markers, while a smaller number target functional markers linked to soil processes. We validated a new primer set targeting citrate synthase (gtlA), a central enzyme in the citric acid cycle linked to aerobic respiration. Primers for a 225 bp fragment suitable for qPCR were tested for specificity and assay performance verified on multiple soils. Clone libraries of the PCR-amplified gtlA gene exhibited high diversity and recovered most majormore » groups identified in a previous 16S rRNA gene study. Comparisons among bacterial communities based on gtlA sequencing using UniFrac revealed differences among the experimental soils studied. Conditions for gtlA qPCR were optimized and calibration curves were highly linear (R2 > 0.99) over six orders of magnitude (4.56 10^5 to 4.56 10^11 copies), with high amplification efficiencies (>1.7). We examined the performance of the gtlA qPCR across a variety of soils and ecosystems, spanning forests, old fields and agricultural areas. We were able to amplify gtlA genes in all tested soils, and detected differences in gtlA abundance within and among environments. These results indicate that a fully developed gtlA-targeted qPCR approach may have potential to link microbial community characteristics with changes in soil respiration.« less

Bacterial discrimination by means of a universal array approach mediated by LDR (ligase detection reaction)

PubMed Central

Busti, Elena; Bordoni, Roberta; Castiglioni, Bianca; Monciardini, Paolo; Sosio, Margherita; Donadio, Stefano; Consolandi, Clarissa; Rossi Bernardi, Luigi; Battaglia, Cristina; De Bellis, Gianluca

2002-01-01

Background PCR amplification of bacterial 16S rRNA genes provides the most comprehensive and flexible means of sampling bacterial communities. Sequence analysis of these cloned fragments can provide a qualitative and quantitative insight of the microbial population under scrutiny although this approach is not suited to large-scale screenings. Other methods, such as denaturing gradient gel electrophoresis, heteroduplex or terminal restriction fragment analysis are rapid and therefore amenable to field-scale experiments. A very recent addition to these analytical tools is represented by microarray technology. Results Here we present our results using a Universal DNA Microarray approach as an analytical tool for bacterial discrimination. The proposed procedure is based on the properties of the DNA ligation reaction and requires the design of two probes specific for each target sequence. One oligo carries a fluorescent label and the other a unique sequence (cZipCode or complementary ZipCode) which identifies a ligation product. Ligated fragments, obtained in presence of a proper template (a PCR amplified fragment of the 16s rRNA gene) contain either the fluorescent label or the unique sequence and therefore are addressed to the location on the microarray where the ZipCode sequence has been spotted. Such an array is therefore "Universal" being unrelated to a specific molecular analysis. Here we present the design of probes specific for some groups of bacteria and their application to bacterial diagnostics. Conclusions The combined use of selective probes, ligation reaction and the Universal Array approach yielded an analytical procedure with a good power of discrimination among bacteria. PMID:12243651

Dynamic of active microorganisms inhabiting a bioleaching industrial heap of low‐grade copper sulfide ore monitored by real‐time PCR and oligonucleotide prokaryotic acidophile microarray

PubMed Central

Remonsellez, Francisco; Galleguillos, Felipe; Moreno‐Paz, Mercedes; Parro, Víctor; Acosta, Mauricio; Demergasso, Cecilia

2009-01-01

Summary The bioleaching of metal sulfide has developed into a very important industrial process and understanding the microbial dynamic is key to advancing commercial bioleaching operations. Here we report the first quantitative description of the dynamic of active communities in an industrial bioleaching heap. Acidithiobacillus ferrooxidans was the most abundant during the first part of the leaching cycle, while the abundance of Leptospirillum ferriphilum and Ferroplasma acidiphilum increased with age of the heap. Acidithiobacillus thiooxidans kept constant throughout the leaching cycle, and Firmicutes group showed a low and a patchy distribution in the heap. The Acidiphilium‐like bacteria reached their highest abundance corresponding to the amount of autotrophs. The active microorganisms in the leaching system were determined using two RNA‐based sensitive techniques. In most cases, the 16S rRNA copy numbers of At. ferrooxidans, L. ferriphilum, At. thiooxidans and F. acidiphilum, was concomitant with the DNA copy numbers, whereas Acidiphilium‐like bacteria and some Firmicutes members did not show a clear correlation between 16S rRNA accumulation and DNA copy numbers. However, the prokaryotic acidophile microarray (PAM) analysis showed active members of Alphaproteobacteria in all samples and of Sulfobacillus genus in older ones. Also, new active groups such as Actinobacteria and Acidobacterium genus were detected by PAM. The results suggest that changes during the leaching cycle in chemical and physical conditions, such as pH and Fe3+/Fe2+ ion rate, are primary factors shaping the microbial dynamic in the heap. PMID:21255296

Evaluation of qPCR-Based Assays for Leprosy Diagnosis Directly in Clinical Specimens

PubMed Central

Sarno, Euzenir Nunes; Moraes, Milton Ozório

2011-01-01

The increased reliability and efficiency of the quantitative polymerase chain reaction (qPCR) makes it a promising tool for performing large-scale screening for infectious disease among high-risk individuals. To date, no study has evaluated the specificity and sensitivity of different qPCR assays for leprosy diagnosis using a range of clinical samples that could bias molecular results such as difficult-to-diagnose cases. In this study, qPCR assays amplifying different M. leprae gene targets, sodA, 16S rRNA, RLEP and Ag 85B were compared for leprosy differential diagnosis. qPCR assays were performed on frozen skin biopsy samples from a total of 62 patients: 21 untreated multibacillary (MB), 26 untreated paucibacillary (PB) leprosy patients, as well as 10 patients suffering from other dermatological diseases and 5 healthy donors. To develop standardized protocols and to overcome the bias resulted from using chromosome count cutoffs arbitrarily defined for different assays, decision tree classifiers were used to estimate optimum cutoffs and to evaluate the assays. As a result, we found a decreasing sensitivity for Ag 85B (66.1%), 16S rRNA (62.9%), and sodA (59.7%) optimized assay classifiers, but with similar maximum specificity for leprosy diagnosis. Conversely, the RLEP assay showed to be the most sensitive (87.1%). Moreover, RLEP assay was positive for 3 samples of patients originally not diagnosed as having leprosy, but these patients developed leprosy 5–10 years after the collection of the biopsy. In addition, 4 other samples of patients clinically classified as non-leprosy presented detectable chromosome counts in their samples by the RLEP assay suggesting that those patients either had leprosy that was misdiagnosed or a subclinical state of leprosy. Overall, these results are encouraging and suggest that RLEP assay could be useful as a sensitive diagnostic test to detect M. leprae infection before major clinical manifestations. PMID:22022631

Development and evaluation of new primers for PCR-based identification of Prevotella intermedia.

PubMed

Zhou, Yanbin; Liu, Dali; Wang, Yiwei; Zhu, Cailian; Liang, Jingping; Shu, Rong

2014-08-01

The aim of this study was to develop new Prevotella intermedia-specific PCR primers based on the 16S rRNA. The new primer set, Pi-192 and Pi-468, increased the accuracy of PCR-based P. intermedia identification and could be useful in the detection of P. intermedia as well as epidemiological studies on periodontal disease. Copyright © 2014 Elsevier Ltd. All rights reserved.

PCR detection of uncultured rumen bacteria.

PubMed

Rosero, Jaime A; Strosová, Lenka; Mrázek, Jakub; Fliegerová, Kateřina; Kopečný, Jan

2012-07-01

16S rRNA sequences of ruminal uncultured bacterial clones from public databases were phylogenetically examined. The sequences were found to form two unique clusters not affiliated with any known bacterial species: cluster of unidentified sequences of free floating rumen fluid uncultured bacteria (FUB) and cluster of unidentified sequences of bacteria associated with rumen epithelium (AUB). A set of PCR primers targeting 16S rRNA of ruminal free uncultured bacteria and rumen epithelium adhering uncultured bacteria was designed based on these sequences. FUB primers were used for relative quantification of uncultured bacteria in ovine rumen samples. The effort to increase the population size of FUB group has been successful in sulfate reducing broth and culture media supplied with cellulose.

Quantitative Tetraplex Real-Time Polymerase Chain Reaction Assay with TaqMan Probes Discriminates Cattle, Buffalo, and Porcine Materials in Food Chain.

PubMed

Hossain, M A Motalib; Ali, Md Eaqub; Sultana, Sharmin; Asing; Bonny, Sharmin Quazi; Kader, Md Abdul; Rahman, M Aminur

2017-05-17

Cattle, buffalo, and porcine materials are widely adulterated, and their quantification might safeguard health, religious, economic, and social sanctity. Recently, conventional polymerase chain reaction (PCR) and PCR-restriction fragment length polymorphism (RFLP) assays have been documented but they are just suitable for identification, cannot quantify adulterations. We described here a quantitative tetraplex real-time PCR assay with TaqMan Probes to quantify contributions from cattle, buffalo, and porcine materials simultaneously. Amplicon-sizes were very short (106-, 90-, and 146-bp for cattle, buffalo, and porcine) because longer targets could be broken down, bringing serious ambiguity in molecular diagnostics. False negative detection was eliminated through an endogenous control (141-bp site of eukaryotic 18S rRNA). Analysis of 27 frankfurters and 27 meatballs reflected 84-115% target recovery at 0.1-10% adulterations. Finally, a test of 36 commercial products revealed 71% beef frankfurters, 100% meatballs, and 85% burgers contained buffalo adulteration, but no porcine was found in beef products.

Identification of New Single Nucleotide Polymorphism-Based Markers for Inter- and Intraspecies Discrimination of Obligate Bacterial Parasites (Pasteuria spp.) of Invertebrates ▿ †

PubMed Central

Mauchline, Tim H.; Knox, Rachel; Mohan, Sharad; Powers, Stephen J.; Kerry, Brian R.; Davies, Keith G.; Hirsch, Penny R.

2011-01-01

Protein-encoding and 16S rRNA genes of Pasteuria penetrans populations from a wide range of geographic locations were examined. Most interpopulation single nucleotide polymorphisms (SNPs) were detected in the 16S rRNA gene. However, in order to fully resolve all populations, these were supplemented with SNPs from protein-encoding genes in a multilocus SNP typing approach. Examination of individual 16S rRNA gene sequences revealed the occurrence of “cryptic” SNPs which were not present in the consensus sequences of any P. penetrans population. Additionally, hierarchical cluster analysis separated P. penetrans 16S rRNA gene clones into four groups, and one of which contained sequences from the most highly passaged population, demonstrating that it is possible to manipulate the population structure of this fastidious bacterium. The other groups were made from representatives of the other populations in various proportions. Comparison of sequences among three Pasteuria species, namely, P. penetrans, P. hartismeri, and P. ramosa, showed that the protein-encoding genes provided greater discrimination than the 16S rRNA gene. From these findings, we have developed a toolbox for the discrimination of Pasteuria at both the inter- and intraspecies levels. We also provide a model to monitor genetic variation in other obligate hyperparasites and difficult-to-culture microorganisms. PMID:21803895

Identification of new single nucleotide polymorphism-based markers for inter- and intraspecies discrimination of obligate bacterial parasites (Pasteuria spp.) of invertebrates.

PubMed

Mauchline, Tim H; Knox, Rachel; Mohan, Sharad; Powers, Stephen J; Kerry, Brian R; Davies, Keith G; Hirsch, Penny R

2011-09-01

Protein-encoding and 16S rRNA genes of Pasteuria penetrans populations from a wide range of geographic locations were examined. Most interpopulation single nucleotide polymorphisms (SNPs) were detected in the 16S rRNA gene. However, in order to fully resolve all populations, these were supplemented with SNPs from protein-encoding genes in a multilocus SNP typing approach. Examination of individual 16S rRNA gene sequences revealed the occurrence of "cryptic" SNPs which were not present in the consensus sequences of any P. penetrans population. Additionally, hierarchical cluster analysis separated P. penetrans 16S rRNA gene clones into four groups, and one of which contained sequences from the most highly passaged population, demonstrating that it is possible to manipulate the population structure of this fastidious bacterium. The other groups were made from representatives of the other populations in various proportions. Comparison of sequences among three Pasteuria species, namely, P. penetrans, P. hartismeri, and P. ramosa, showed that the protein-encoding genes provided greater discrimination than the 16S rRNA gene. From these findings, we have developed a toolbox for the discrimination of Pasteuria at both the inter- and intraspecies levels. We also provide a model to monitor genetic variation in other obligate hyperparasites and difficult-to-culture microorganisms.

Distance-based microfluidic quantitative detection methods for point-of-care testing.

PubMed

Tian, Tian; Li, Jiuxing; Song, Yanling; Zhou, Leiji; Zhu, Zhi; Yang, Chaoyong James

2016-04-07

Equipment-free devices with quantitative readout are of great significance to point-of-care testing (POCT), which provides real-time readout to users and is especially important in low-resource settings. Among various equipment-free approaches, distance-based visual quantitative detection methods rely on reading the visual signal length for corresponding target concentrations, thus eliminating the need for sophisticated instruments. The distance-based methods are low-cost, user-friendly and can be integrated into portable analytical devices. Moreover, such methods enable quantitative detection of various targets by the naked eye. In this review, we first introduce the concept and history of distance-based visual quantitative detection methods. Then, we summarize the main methods for translation of molecular signals to distance-based readout and discuss different microfluidic platforms (glass, PDMS, paper and thread) in terms of applications in biomedical diagnostics, food safety monitoring, and environmental analysis. Finally, the potential and future perspectives are discussed.

Pig Manure Contamination Marker Selection Based on the Influence of Biological Treatment on the Dominant Fecal Microbial Groups▿

PubMed Central

Marti, Romain; Dabert, Patrick; Pourcher, Anne-Marie

2009-01-01

The objective of this study was to identify a microbial marker for pig manure contamination. We quantified the persistence of four dominant bacterial groups from the pig intestinal tract throughout manure handling at 10 livestock operations (including aerobic digestion) by using molecular typing. The partial 16S rRNA genes of Bacteroides-Prevotella, Eubacterium-Clostridiaceae, Bacillus-Streptococcus-Lactobacillus (BSL), and Bifidobacterium group isolates were amplified and analyzed by capillary electrophoresis single-strand conformation polymorphism. The most dominant bacterial populations were identified by cloning and sequencing their 16S rRNA genes. The results showed that Bifidobacterium spp. and, to a lesser extent, members of the BSL group, were less affected by the aerobic treatment than either Eubacterium-Clostridiaceae or Bacteroides-Prevotella. Two Bifidobacterium species found in raw manure were still present in manure during land application, suggesting that they can survive outside the pig intestinal tract and also survive aerobic treatment. The 16S-23S rRNA internal transcribed spacer of one species, Bifidobacterium thermacidophilum subsp. porcinum, was sequenced, and a specific pair of primers was designed for its detection in the environment. With this nested PCR assay, this potential marker was not detected in samples from 30 bovine, 30 poultry, and 28 human fecal samples or in 15 urban wastewater effluents. As it was detected in runoff waters after spreading of pig manure, we propose this marker as a suitable microbial indicator of pig manure contamination. PMID:19525269

Pig manure contamination marker selection based on the influence of biological treatment on the dominant fecal microbial groups.

PubMed

Marti, Romain; Dabert, Patrick; Pourcher, Anne-Marie

2009-08-01

The objective of this study was to identify a microbial marker for pig manure contamination. We quantified the persistence of four dominant bacterial groups from the pig intestinal tract throughout manure handling at 10 livestock operations (including aerobic digestion) by using molecular typing. The partial 16S rRNA genes of Bacteroides-Prevotella, Eubacterium-Clostridiaceae, Bacillus-Streptococcus-Lactobacillus (BSL), and Bifidobacterium group isolates were amplified and analyzed by capillary electrophoresis single-strand conformation polymorphism. The most dominant bacterial populations were identified by cloning and sequencing their 16S rRNA genes. The results showed that Bifidobacterium spp. and, to a lesser extent, members of the BSL group, were less affected by the aerobic treatment than either Eubacterium-Clostridiaceae or Bacteroides-Prevotella. Two Bifidobacterium species found in raw manure were still present in manure during land application, suggesting that they can survive outside the pig intestinal tract and also survive aerobic treatment. The 16S-23S rRNA internal transcribed spacer of one species, Bifidobacterium thermacidophilum subsp. porcinum, was sequenced, and a specific pair of primers was designed for its detection in the environment. With this nested PCR assay, this potential marker was not detected in samples from 30 bovine, 30 poultry, and 28 human fecal samples or in 15 urban wastewater effluents. As it was detected in runoff waters after spreading of pig manure, we propose this marker as a suitable microbial indicator of pig manure contamination.

Expedited quantification of mutant ribosomal RNA by binary deoxyribozyme (BiDz) sensors.

PubMed

Gerasimova, Yulia V; Yakovchuk, Petro; Dedkova, Larisa M; Hecht, Sidney M; Kolpashchikov, Dmitry M

2015-10-01

Mutations in ribosomal RNA (rRNA) have traditionally been detected by the primer extension assay, which is a tedious and multistage procedure. Here, we describe a simple and straightforward fluorescence assay based on binary deoxyribozyme (BiDz) sensors. The assay uses two short DNA oligonucleotides that hybridize specifically to adjacent fragments of rRNA, one of which contains a mutation site. This hybridization results in the formation of a deoxyribozyme catalytic core that produces the fluorescent signal and amplifies it due to multiple rounds of catalytic action. This assay enables us to expedite semi-quantification of mutant rRNA content in cell cultures starting from whole cells, which provides information useful for optimization of culture preparation prior to ribosome isolation. The method requires less than a microliter of a standard Escherichia coli cell culture and decreases analysis time from several days (for primer extension assay) to 1.5 h with hands-on time of ∼10 min. It is sensitive to single-nucleotide mutations. The new assay simplifies the preliminary analysis of RNA samples and cells in molecular biology and cloning experiments and is promising in other applications where fast detection/quantification of specific RNA is required. © 2015 Gerasimova et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.

Adenovirus and mycoplasma infection in an ornate box turtle (Terrapene ornata ornata) in Hungary.

PubMed

Farkas, Szilvia L; Gál, János

2009-07-02

A female, adult ornate box turtle (Terrapene ornata ornata) with fatty liver was submitted for virologic examination in Hungary. Signs of an adenovirus infection including degeneration of the liver cells, enlarged nuclei and intranuclear inclusion bodies were detected by light microscopic examination. The presence of an adenovirus was later confirmed by obtaining partial sequence data from the adenoviral DNA-dependent DNA-polymerase. Phylogenetic analyses revealed that this novel chelonian adenovirus was distinct from previously described reptilian adenoviruses, not belonging to any of the recognized genera of the family Adenoviridae. As a part of the routine diagnostic procedure for chelonians the detection of herpes-, rana- and iridoviruses together with Mycoplasma spp. was attempted. Amplicons were generated by a general mycoplasma polymerase chain reaction (PCR) targeting the 16S/23S ribosomal RNA (rRNA) intergenic spacer region, as well as, a specific Mycoplasma agassizii PCR targeting the 16S rRNA gene. Based on the analyses of partial sequences of the 16S rRNA gene, the Mycoplasma sp. of the ornate box turtle seemed to be identical with the recently described eastern box turtle (Terrapene carolina carolina) Mycoplasma sp. This is the first report of a novel chelonian adenovirus and a mycoplasma infection in an ornate box turtle (T. ornata ornata) in Europe.

DECIPHER, a Search-Based Approach to Chimera Identification for 16S rRNA Sequences

PubMed Central

Wright, Erik S.; Yilmaz, L. Safak

2012-01-01

DECIPHER is a new method for finding 16S rRNA chimeric sequences by the use of a search-based approach. The method is based upon detecting short fragments that are uncommon in the phylogenetic group where a query sequence is classified but frequently found in another phylogenetic group. The algorithm was calibrated for full sequences (fs_DECIPHER) and short sequences (ss_DECIPHER) and benchmarked against WigeoN (Pintail), ChimeraSlayer, and Uchime using artificially generated chimeras. Overall, ss_DECIPHER and Uchime provided the highest chimera detection for sequences 100 to 600 nucleotides long (79% and 81%, respectively), but Uchime's performance deteriorated for longer sequences, while ss_DECIPHER maintained a high detection rate (89%). Both methods had low false-positive rates (1.3% and 1.6%). The more conservative fs_DECIPHER, benchmarked only for sequences longer than 600 nucleotides, had an overall detection rate lower than that of ss_DECIPHER (75%) but higher than those of the other programs. In addition, fs_DECIPHER had the lowest false-positive rate among all the benchmarked programs (<0.20%). DECIPHER was outperformed only by ChimeraSlayer and Uchime when chimeras were formed from closely related parents (less than 10% divergence). Given the differences in the programs, it was possible to detect over 89% of all chimeras with just the combination of ss_DECIPHER and Uchime. Using fs_DECIPHER, we detected between 1% and 2% additional chimeras in the RDP, SILVA, and Greengenes databases from which chimeras had already been removed with Pintail or Bellerophon. DECIPHER was implemented in the R programming language and is directly accessible through a webpage or by downloading the program as an R package (http://DECIPHER.cee.wisc.edu). PMID:22101057

High throughput pyrosequencing technology for molecular differential detection of Babesia vogeli, Hepatozoon canis, Ehrlichia canis and Anaplasma platys in canine blood samples.

PubMed

Kaewkong, Worasak; Intapan, Pewpan M; Sanpool, Oranuch; Janwan, Penchom; Thanchomnang, Tongjit; Kongklieng, Amornmas; Tantrawatpan, Chairat; Boonmars, Thidarut; Lulitanond, Viraphong; Taweethavonsawat, Piyanan; Chungpivat, Sudchit; Maleewong, Wanchai

2014-06-01

Canine babesiosis, hepatozoonosis, ehrlichiosis, and anaplasmosis are tick-borne diseases caused by different hemopathogens. These diseases are causes of morbidity and mortality in dogs. The classic method for parasite detection and differentiation is based on microscopic observation of blood smears. The limitations of the microscopic method are that its performance requires a specially qualified person with professional competence, and it is ineffective in differentiating closely related species. This study applied PCR amplification with high throughput pyrosequencing for molecular differential detection of the following 4 hemoparasites common to tropical areas in dog blood samples: Babesia vogeli, Hepatozoon canis, Ehrlichia canis, and Anaplasma platys. PCR was initially used to amplify specific target regions of the ribosomal RNA genes of each parasite using 2 primer pairs that included 18S rRNA for protozoa (B. vogeli and H. canis) and 16S rRNA for rickettsia (E. canis and A. platys). Babesia vogeli and H. canis were discriminated using 9 nucleotide positions out of 30 base pairs, whereas E. canis and A. platys were differentiated using 15 nucleotide positions out of 34 base pairs that were determined from regions adjacent to 3' ends of the sequencing primers. This method provides a challenging alternative for a rapid diagnosis and surveillance of these tick-borne diseases in canines. Copyright © 2014 Elsevier GmbH. All rights reserved.

Selection of the internal control gene for real-time quantitative rt-PCR assays in temperature treated Leptospira.

PubMed

Carrillo-Casas, Erika Margarita; Hernández-Castro, Rigoberto; Suárez-Güemes, Francisco; de la Peña-Moctezuma, Alejandro

2008-06-01

Analysis of gene expression requires sensitive, precise, and reproducible measurements for specific mRNA sequences. To avoid bias, real-time RT-PCR is referred to one or several internal control genes. Here, we sought to identify a gene to be used as normalizer by analyzing three functional distinct housekeeping genes (lipL41, flaB, and 16S rRNA) for their expression level and stability in temperature treated Leptospira cultures. Leptospira interrogans serovar Hardjo subtype Hardjoprajitno was cultured at 30 degrees C for 7 days until a density of 10(6) cells/ml was reached and then shifted to physiological temperatures (37 degrees C and 42 degrees C) and to environmental temperatures (25 degrees C and 30 degrees C) during a 24 h period. cDNA was amplified by quantitative PCR using SYBR Green I technology and gene-specific primers for lipL41, flaB, and 16S rRNA. Expression stability (M) was assessed by geNorm and Normfinder v.18. 16S rRNA registered an average expression stability of M = 1.1816, followed by flaB (M = 1.682) and lipL41 (M = 1.763). 16S rRNA was identified as the most stable gene and can be used as a normalizer, as it showed greater expression stability than lipL41 and flaB in the four temperature treatments. Hence, comparisons of gene expression will have a higher sensitivity and specificity.

Detection and quantification of major toxigenic Microcystis genotypes in Moo-Tan reservoir and associated water treatment plant.

PubMed

Yen, Hung-Kai; Lin, Tsair-Fuh; Tseng, I-Cheng

2012-02-01

Two molecular methods, denaturing gradient gel electrophoresis (DGGE) and quantitative real-time polymerase chain reaction (qPCR) with the Universal ProbeLibrary (UPL) probe, were developed and used for the characterization and quantification of several microcystin producers in Moo-Tan Reservoir (MTR), Taiwan and its associated water treatment plant (Shih-Men Water Treatment Plant, SMWTP). Internal transcribed spacer (ITS) sequence, a highly diversified region between the 16S rRNA and 23S rRNA genes, was used to further identify the isolated strains from MTR and also used in DGGE for the detection of the specific DNA fragments and biomarkers for 11 strains observed in MTR. These ITS-DGGE biomarkers were successfully applied to monitor the community changes of potential toxigenic Microcystis sp. over a period of five years. Two highly specific primers were combined with UPL probes to measure microcystins synthesis gene (mcyB) and phycocyanin intergenic spacer region (cpcB) concentrations in water samples. The copy concentrations of UPL-mcyB and UPL-cpcB correlated well with MC-RR concentrations/water temperature and Microcystis sp. cell numbers in the water samples, respectively. For SMWTP, toxin concentrations were low, but the DGGE bands clearly demonstrated the presence of potential microcystin producers in both water treatment plants and finished water samples. It was demonstrated that toxigenic Microcystis sp. may penetrate through the treatment processes and pose a potential risk to human health in the drinking water systems.

Acetoclastic methane formation from Eucalyptus detritus in pristine hydrocarbon-rich river sediments by Methanosarcinales.

PubMed

Beckmann, Sabrina; Manefield, Mike

2014-12-01

Pristine hydrocarbon-rich river sediments in the Greater Blue Mountains World Heritage Area (Australia) release substantial amounts of methane. The present study aimed to unravel for the first time the active methanogens mediating methane formation and exploiting the bacterial diversity potentially involved in the trophic network. Quantitative PCR of 16S rRNA gene and functional genes as well as 454 pyrosequencing were used to address the unknown microbial diversity and abundance. Methane-releasing sediment cores derived from three different river sites of the Tootie River. Highest methane production rates of 10.8 ± 0.5 μg g(-1)(wet weight) day(-1) were detected in 40 cm sediment depth being in congruence with the detection of the highest abundances of the archaeal 16S rRNA gene and the methyl-coenzyme M reductase (mcrA) genes. Stable carbon and hydrogen isotopic signatures of the produced methane indicated an acetoclastic origin. Long-term enrichment cultures amended with either acetate or H2/CO2 revealed acetoclastic methanogenesis as key methane-formation process mediated by members of the order Methanosarcinales. Conditions prevailing in the river sediments might be suitable for hydrocarbon-degrading bacteria observed in the river sediments that were previously unclassified or closely related to the Bacteroidetes/Chlorobi group, the Firmicutes and the Chloroflexi group fuelling acetoclastic methanogensis in pristine river sediments. © 2014 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved.

Droplet microfluidics for amplification-free genetic detection of single cells.

PubMed

Rane, Tushar D; Zec, Helena C; Puleo, Chris; Lee, Abraham P; Wang, Tza-Huei

2012-09-21

In this article we present a novel droplet microfluidic chip enabling amplification-free detection of single pathogenic cells. The device streamlines multiple functionalities to carry out sample digitization, cell lysis, probe-target hybridization for subsequent fluorescent detection. A peptide nucleic acid fluorescence resonance energy transfer probe (PNA beacon) is used to detect 16S rRNA present in pathogenic cells. Initially the sensitivity and quantification abilities of the platform are tested using a synthetic target mimicking the actual expression level of 16S rRNA in single cells. The capability of the device to perform "sample-to-answer" pathogen detection of single cells is demonstrated using E. coli as a model pathogen.

Development and validation of a real-time quantitative PCR assay to detect Xanthomonas axonopodis pv. allii from onion seed.

PubMed

Robène, Isabelle; Perret, Marion; Jouen, Emmanuel; Escalon, Aline; Maillot, Marie-Véronique; Chabirand, Aude; Moreau, Aurélie; Laurent, Annie; Chiroleu, Frédéric; Pruvost, Olivier

2015-07-01

Bacterial blight of onion is an emerging disease threatening world onion production. The causal agent Xanthomonas axonopodis pv. allii is seed transmitted and a reliable and sensitive tool is needed to monitor seed exchanges. A triplex quantitative real-time PCR assay was developed targeting two X. axonopodis pv. allii-specific markers and an internal control chosen in 5.8S rRNA gene from Alliaceae. Amplification of at least one marker indicates the presence of the bacterium in seed extracts. This real-time PCR assay detected all the 79 X. axonopodis pv. allii strains tested and excluded 85.2% of the 135 non-target strains and particularly all 39 saprophytic and pathogenic bacteria associated with onion. Cross-reactions were mainly obtained for strains assigned to nine phylogenetically related X. axonopodis pathovars. The cycle cut-off was estimated statistically at 36.3 considering a risk of false positive of 1%. The limit of detection obtained in at least 95% of the time (LOD 95%) was 5×10(3) CFU/g (colony forming unit/g). The sensitivity threshold was found to be 1 infected seed in 32,790 seeds. This real-time PCR assay should be useful for preventing the long-distance spread of X. axonopodis pv. allii via contaminated seed lots and determining the epidemiology of the bacterium. Copyright © 2015 Elsevier B.V. All rights reserved.

Detection of Bacillus spores using PCR and FTA filters.

PubMed

Lampel, Keith A; Dyer, Deanne; Kornegay, Leroy; Orlandi, Palmer A

2004-05-01

Emphasis has been placed on developing and implementing rapid detection systems for microbial pathogens. We have explored the utility of expanding FTA filter technology for the preparation of template DNA for PCR from bacterial spores. Isolated spores from several Bacillus spp., B. subtilis, B. cereus, and B. megaterium, were applied to FTA filters, and specific DNA products were amplified by PCR. Spore preparations were examined microscopically to ensure that the presence of vegetative cells, if any, did not yield misleading results. PCR primers SRM86 and SRM87 targeted a conserved region of bacterial rRNA genes, whereas primers Bsub5F and Bsub3R amplified a product from a conserved sequence of the B. subtilis rRNA gene. With the use of the latter set of primers for nested PCR, the sensitivity of the PCR-based assay was increased. Overall, 53 spores could be detected after the first round of PCR, and the sensitivity was increased to five spores by nested PCR. FTA filters are an excellent platform to remove PCR inhibitors and have universal applications for environmental, clinical, and food samples.

Molecular characterization and specific detection of Anaplasma species (AP-sd) in sika deer and its first detection in wild brown bears and rodents in Hokkaido, Japan.

PubMed

Moustafa, Mohamed Abdallah Mohamed; Lee, Kyunglee; Taylor, Kyle; Nakao, Ryo; Sashika, Mariko; Shimozuru, Michito; Tsubota, Toshio

2015-12-01

A previously undescribed Anaplasma species (herein referred to as AP-sd) has been detected in sika deer, cattle and ticks in Japan. Despite being highly similar to some strains of A. phagocytophilum, AP-sd has never been detected in humans. Its ambiguous epidemiology and the lack of tools for its specific detection make it difficult to understand and interpret the prevalence of this Anaplasma species. We developed a method for specific detection, and examined AP-sd prevalence in Hokkaido wildlife. Our study included 250 sika deer (Cervus nippon yesoensis), 13 brown bears (Ursus arctos yesoensis) and 252 rodents including 138 (Apodemus speciosus), 45 (Apodemus argenteus), 42 (Myodes rufocanus) and 27 (Myodes rutilus) were collected from Hokkaido island, northern Japan, collected during 2010 to 2015. A 770 bp and 382 bp segment of the 16S rRNA and gltA genes, respectively, were amplified by nested PCR. Results were confirmed by cloning and sequencing of the positive PCR products. A reverse line blot hybridization (RLB) based on the 16S rRNA gene was then developed for the specific detection of AP-sd. The prevalence of AP-sd by nested PCR in sika deer was 51% (128/250). We detected this Anaplasma sp. for the first time in wild brown bears and rodents with a prevalence of 15% (2/13) and 2.4% (6/252), respectively. The sequencing results of the 16S rRNA and gltA gene amplicons were divergent from the selected A. phagocytophilum sequences in GenBank. Using a newly designed AP-sd specific probe for RLB has enabled us to specifically detect this Anaplasma species. Besides sika deer and cattle, wild brown bears and rodents were identified as potential reservoir hosts for AP-sd. This study provided a high throughput molecular method that specifically detects AP-sd, and which can be used to investigate its ecology and its potential as a threat to humans in Japan. Copyright © 2015 Elsevier B.V. All rights reserved.

Quantitative detection of bovine and porcine gelatin difference using surface plasmon resonance based biosensor

NASA Astrophysics Data System (ADS)

Wardani, Devy P.; Arifin, Muhammad; Suharyadi, Edi; Abraha, Kamsul

2015-05-01

Gelatin is a biopolymer derived from collagen that is widely used in food and pharmaceutical products. Due to some religion restrictions and health issues regarding the gelatin consumption which is extracted from certain species, it is necessary to establish a robust, reliable, sensitive and simple quantitative method to detect gelatin from different parent collagen species. To the best of our knowledge, there has not been a gelatin differentiation method based on optical sensor that could detect gelatin from different species quantitatively. Surface plasmon resonance (SPR) based biosensor is known to be a sensitive, simple and label free optical method for detecting biomaterials that is able to do quantitative detection. Therefore, we have utilized SPR-based biosensor to detect the differentiation between bovine and porcine gelatin in various concentration, from 0% to 10% (w/w). Here, we report the ability of SPR-based biosensor to detect difference between both gelatins, its sensitivity toward the gelatin concentration change, its reliability and limit of detection (LOD) and limit of quantification (LOQ) of the sensor. The sensor's LOD and LOQ towards bovine gelatin concentration are 0.38% and 1.26% (w/w), while towards porcine gelatin concentration are 0.66% and 2.20% (w/w), respectively. The results show that SPR-based biosensor is a promising tool for detecting gelatin from different raw materials quantitatively.

Potential applications of next generation DNA sequencing of 16S rRNA gene amplicons in microbial water quality monitoring

PubMed Central

Vierheilig, J.; Savio, D.; Ley, R. E.; Mach, R. L.; Farnleitner, A. H.

2016-01-01

The applicability of next generation DNA sequencing (NGS) methods for water quality assessment has so far not been broadly investigated. This study set out to evaluate the potential of an NGS-based approach in a complex catchment with importance for drinking water abstraction. In this multicompartment investigation, total bacterial communities in water, faeces, soil, and sediment samples were investigated by 454 pyrosequencing of bacterial 16S rRNA gene amplicons to assess the capabilities of this NGS method for (i) the development and evaluation of environmental molecular diagnostics, (ii) direct screening of the bulk bacterial communities, and (iii) the detection of faecal pollution in water. Results indicate that NGS methods can highlight potential target populations for diagnostics and will prove useful for the evaluation of existing and the development of novel DNA-based detection methods in the field of water microbiology. The used approach allowed unveiling of dominant bacterial populations but failed to detect populations with low abundances such as faecal indicators in surface waters. In combination with metadata, NGS data will also allow the identification of drivers of bacterial community composition during water treatment and distribution, highlighting the power of this approach for monitoring of bacterial regrowth and contamination in technical systems. PMID:26606090

Identification of the bacterial endosymbionts of the marine ciliate Euplotes magnicirratus (Ciliophora, Hypotrichia) and proposal of 'Candidatus Devosia euplotis'.

PubMed

Vannini, Claudia; Rosati, Giovanna; Verni, Franco; Petroni, Giulio

2004-07-01

This paper reports the identification of bacterial endosymbionts that inhabit the cytoplasm of the marine ciliated protozoon Euplotes magnicirratus. Ultrastructural and full-cycle rRNA approaches were used to reveal the identity of these bacteria. Based on analysis of 16S rRNA gene sequences, evolutionary trees were constructed; these placed the endosymbiont in the genus Devosia in the alpha-Proteobacteria. The validity of this finding was also shown by fluorescence in situ hybridization with a Devosia-specific oligonucleotide probe. Differences at the 16S rRNA gene level (which allowed the construction of a species-specific oligonucleotide probe) and the peculiar habitat indicate that the endosymbiont represents a novel species. As its cultivation has not been successful to date, the provisional name 'Candidatus Devosia euplotis' is proposed. The species- and group-specific probes designed in this study could represent convenient tools for the detection of 'Candidatus Devosia euplotis' and Devosia-like bacteria in the environment.

Electrokinetic stringency control in self-assembled monolayer-based biosensors for multiplex urinary tract infection diagnosis.

PubMed

Liu, Tingting; Sin, Mandy L Y; Pyne, Jeff D; Gau, Vincent; Liao, Joseph C; Wong, Pak Kin

2014-01-01

Rapid detection of bacterial pathogens is critical toward judicious management of infectious diseases. Herein, we demonstrate an in situ electrokinetic stringency control approach for a self-assembled monolayer-based electrochemical biosensor toward urinary tract infection diagnosis. The in situ electrokinetic stringency control technique generates Joule heating induced temperature rise and electrothermal fluid motion directly on the sensor to improve its performance for detecting bacterial 16S rRNA, a phylogenetic biomarker. The dependence of the hybridization efficiency reveals that in situ electrokinetic stringency control is capable of discriminating single-base mismatches. With electrokinetic stringency control, the background noise due to the matrix effects of clinical urine samples can be reduced by 60%. The applicability of the system is demonstrated by multiplex detection of three uropathogenic clinical isolates with similar 16S rRNA sequences. The results demonstrate that electrokinetic stringency control can significantly improve the signal-to-noise ratio of the biosensor for multiplex urinary tract infection diagnosis. Urinary tract infections remain a significant cause of mortality and morbidity as secondary conditions often related to chronic diseases or to immunosuppression. Rapid and sensitive identification of the causative organisms is critical in the appropriate management of this condition. These investigators demonstrate an in situ electrokinetic stringency control approach for a self-assembled monolayer-based electrochemical biosensor toward urinary tract infection diagnosis, establishing that such an approach significantly improves the biosensor's signal-to-noise ratio. © 2013.

Selective progressive response of soil microbial community to wild oat roots.

PubMed

DeAngelis, Kristen M; Brodie, Eoin L; DeSantis, Todd Z; Andersen, Gary L; Lindow, Steven E; Firestone, Mary K

2009-02-01

Roots moving through soil induce physical and chemical changes that differentiate rhizosphere from bulk soil, and the effects of these changes on soil microorganisms have long been a topic of interest. The use of a high-density 16S rRNA microarray (PhyloChip) for bacterial and archaeal community analysis has allowed definition of the populations that respond to the root within the complex grassland soil community; this research accompanies compositional changes reported earlier, including increases in chitinase- and protease-specific activity, cell numbers and quorum sensing signal. PhyloChip results showed a significant change compared with bulk soil in relative abundance for 7% of the total rhizosphere microbial community (147 of 1917 taxa); the 7% response value was confirmed by16S rRNA terminal restriction fragment length polymorphism analysis. This PhyloChip-defined dynamic subset was comprised of taxa in 17 of the 44 phyla detected in all soil samples. Expected rhizosphere-competent phyla, such as Proteobacteria and Firmicutes, were well represented, as were less-well-documented rhizosphere colonizers including Actinobacteria, Verrucomicrobia and Nitrospira. Richness of Bacteroidetes and Actinobacteria decreased in soil near the root tip compared with bulk soil, but then increased in older root zones. Quantitative PCR revealed rhizosphere abundance of beta-Proteobacteria and Actinobacteria at about 10(8) copies of 16S rRNA genes per g soil, with Nitrospira having about 10(5) copies per g soil. This report demonstrates that changes in a relatively small subset of the soil microbial community are sufficient to produce substantial changes in functions observed earlier in progressively more mature rhizosphere zones.

Molecular detection of tick-borne protozoan parasites in a population of domestic cats in midwestern Brazil.

PubMed

Braga, Ísis Assis; de Souza Ramos, Dirceu Guilherme; Marcili, Arlei; Melo, Andréia Lima Tomé; Taques, Isis Indaiara Gonçalves Granjeiro; Amude, Alexandre Mendes; Chitarra, Cristiane Silva; Nakazato, Luciano; Dutra, Valéria; de Campos Pacheco, Richard; Aguiar, Daniel Moura

2016-07-01

Some tick-borne pathogens that infect domestic cats have been considered emergent in veterinary medicine. Occurrences of Hepatozoon spp., Babesia spp. and Cytauxzoon spp. have been described in several regions of Brazil. This paper offers a comprehensive analysis of the 18S rRNA gene of a Hepatozoon sp. strain detected in domestic cats in the metropolitan area of Cuiabá, in Midwestern Brazil. Based on a molecular analysis, we detected the presence of Hepatozoon species circulating among cats in this region. The aforementioned strain is closely related to other isolates of H. felis detected in wild felids. Moreover, a phylogenetic analysis indicates that this genotype is grouped into a clade of 18S rRNA sequences previously described for the genus Hepatozoon in wild felids around the world. Hepatozoon felis strains detected in cats from Spain and Israel showed, respectively, 98% and 97% identity to our sequence and are clustered on a separate branch of the phylogenetic tree. This finding suggests a high diversity of Hepatozoon genotypes occurring in cats in Europe and South America. None of the analyzed cats were positive for Babesia spp. or Cytauxzoon spp. by PCR analysis. Copyright © 2016 Elsevier GmbH. All rights reserved.

Increased 5S rRNA oxidation in Alzheimer's disease.

PubMed

Ding, Qunxing; Zhu, Haiyan; Zhang, Bing; Soriano, Augusto; Burns, Roxanne; Markesbery, William R

2012-01-01

It is widely accepted that oxidative stress is involved in neurodegenerative disorders such as Alzheimer's disease (AD). Ribosomal RNA (rRNA) is one of the most abundant molecules in most cells and is affected by oxidative stress in the human brain. Previous data have indicated that total rRNA levels were decreased in the brains of subjects with AD and mild cognitive impairment concomitant with an increase in rRNA oxidation. In addition, level of 5S rRNA, one of the essential components of the ribosome complex, was significantly lower in the inferior parietal lobule (IP) brain area of subjects with AD compared with control subjects. To further evaluate the alteration of 5S rRNA in neurodegenerative human brains, multiple brain regions from both AD and age-matched control subjects were used in this study, including IP, superior and middle temporal gyro, temporal pole, and cerebellum. Different molecular pools including 5S rRNA integrated into ribosome complexes, free 5S rRNA, cytoplasmic 5S rRNA, and nuclear 5S rRNA were studied. Free 5S rRNA levels were significantly decreased in the temporal pole region of AD subjects and the oxidation of ribosome-integrated and free 5S rRNA was significantly increased in multiple brain regions in AD subjects compared with controls. Moreover, a greater amount of oxidized 5S rRNA was detected in the cytoplasm and nucleus of AD subjects compared with controls. These results suggest that the increased oxidation of 5S rRNA, especially the oxidation of free 5S rRNA, may be involved in the neurodegeneration observed in AD.

Occurrence of Acquired 16S rRNA Methyltransferase-Mediated Aminoglycoside Resistance in Clinical Isolates of Enterobacteriaceae within a Tertiary Referral Hospital of Northeast India

PubMed Central

Wangkheimayum, Jayalaxmi; Paul, Deepjyoti; Dhar, Debadatta; Nepram, Rajlakshmi; Chetri, Shiela; Bhowmik, Deepshikha; Chakravarty, Atanu

2017-01-01

ABSTRACT The methylation of a ribosomal target leads to a high level of resistance to all clinically relevant aminoglycoside antibiotics, so early detection of these resistance determinants will help to reduce the incidence of treatment failures as well as lessen the dissemination rate. Here, we characterized different 16S rRNA methyltransferases responsible for aminoglycoside resistance and their epidemiological background in clinical isolates of Enterobacteriaceae in a tertiary referral hospital in India. All aminoglycoside-resistant isolates were screened for different 16S rRNA methyltransferases by PCR assay, and incompatibility typing of the conjugable plasmid harboring resistance genes was performed by PCR-based replicon typing. An assay for the stability and elimination of these resistance plasmids was performed. The coexistence of extended-spectrum β-lactamases and metallo-β-lactamases was also detected, and the heterogeneity of these isolates was determined by enterobacterial repetitive intergenic consensus PCR. The PCR assay revealed the presence of armA, rmtA, rmtB, rmtC, and rmtD in single and multiple combinations, and these were carried by a diverse group of Inc plasmids. Plasmids harboring these resistance determinants were highly stable and maintained until the 55th serial passage, but SDS treatment could easily eliminate the plasmids harboring the resistance determinants. The coexistence of blaTEM, blaPER, blaGES, and blaSHV, as well as blaVIM and blaNDM, within these isolates was also detected. Strains with different clonal patterns of aminoglycoside resistance were found to spread in this hospital setting. We observed that the 16S rRNA methyltransferase genes were encoded within different Inc plasmid types, suggesting diverse origins and sources of acquisition. Therefore, the present study is of epidemiological importance and can have a role in infection control policy in hospital settings. PMID:28320725

Estimates of Soil Bacterial Ribosome Content and Diversity Are Significantly Affected by the Nucleic Acid Extraction Method Employed

PubMed Central

Wüst, Pia K.; Nacke, Heiko; Kaiser, Kristin; Marhan, Sven; Sikorski, Johannes; Kandeler, Ellen; Daniel, Rolf

2016-01-01

Modern sequencing technologies allow high-resolution analyses of total and potentially active soil microbial communities based on their DNA and RNA, respectively. In the present study, quantitative PCR and 454 pyrosequencing were used to evaluate the effects of different extraction methods on the abundance and diversity of 16S rRNA genes and transcripts recovered from three different types of soils (leptosol, stagnosol, and gleysol). The quality and yield of nucleic acids varied considerably with respect to both the applied extraction method and the analyzed type of soil. The bacterial ribosome content (calculated as the ratio of 16S rRNA transcripts to 16S rRNA genes) can serve as an indicator of the potential activity of bacterial cells and differed by 2 orders of magnitude between nucleic acid extracts obtained by the various extraction methods. Depending on the extraction method, the relative abundances of dominant soil taxa, in particular Actinobacteria and Proteobacteria, varied by a factor of up to 10. Through this systematic approach, the present study allows guidelines to be deduced for the selection of the appropriate extraction protocol according to the specific soil properties, the nucleic acid of interest, and the target organisms. PMID:26896137

Use of a Hierarchical Oligonucleotide Primer Extension Approach for Multiplexed Relative Abundance Analysis of Methanogens in Anaerobic Digestion Systems

PubMed Central

Chuang, Hui-Ping; Hsu, Mao-Hsuan; Chen, Wei-Yu

2013-01-01

In this study, we established a rapid multiplex method to detect the relative abundances of amplified 16S rRNA genes from known cultivatable methanogens at hierarchical specificities in anaerobic digestion systems treating industrial wastewater and sewage sludge. The method was based on the hierarchical oligonucleotide primer extension (HOPE) technique and combined with a set of 27 primers designed to target the total archaeal populations and methanogens from 22 genera within 4 taxonomic orders. After optimization for their specificities and detection sensitivity under the conditions of multiple single-nucleotide primer extension reactions, the HOPE approach was applied to analyze the methanogens in 19 consortium samples from 7 anaerobic treatment systems (i.e., 513 reactions). Among the samples, the methanogen populations detected with order-level primers accounted for >77.2% of the PCR-amplified 16S rRNA genes detected using an Archaea-specific primer. The archaeal communities typically consisted of 2 to 7 known methanogen genera within the Methanobacteriales, Methanomicrobiales, and Methanosarcinales and displayed population dynamic and spatial distributions in anaerobic reactor operations. Principal component analysis of the HOPE data further showed that the methanogen communities could be clustered into 3 distinctive groups, in accordance with the distribution of the Methanosaeta, Methanolinea, and Methanomethylovorans, respectively. This finding suggested that in addition to acetotrophic and hydrogenotrophic methanogens, the methylotrophic methanogens might play a key role in the anaerobic treatment of industrial wastewater. Overall, the results demonstrated that the HOPE approach is a specific, rapid, and multiplexing platform to determine the relative abundances of targeted methanogens in PCR-amplified 16S rRNA gene products. PMID:24077716

Genetic diversity among Babesia rossi detected in naturally infected dogs in Abeokuta, Nigeria, based on 18S rRNA gene sequences.

PubMed

Takeet, Michael I; Oyewusi, Adeoye J; Abakpa, Simon A V; Daramola, Olukayode O; Peters, Sunday O

2017-03-01

Adequate knowledge of the genetic diversity among Babesia species infecting dogs is necessary for a better understanding of the epidemiology and control of canine babesiosis. Hence, this study determined the genetic diversity among the Babesia rossi detected in dogs presented for routine examination in Veterinary Hospitals in Abeokuta, Nigeria. Blood were randomly collected from 209 dogs. Field-stained thin smears were made and DNA extracted from the blood. Partial region of the 18S small subunit ribosomal RNA (rRNA) gene was amplified, sequenced and analysed. Babesia species was detected in 16 (7.7%) of the dogs by microscopy. Electrophoresed PCR products from 39 (18.66%) dogs revealed band size of 450 bp and 2 (0.95%) dogs had band size of 430 bp. The sequences obtained from 450 bp amplicon displayed homology of 99.74% (387/388) with partial sequences of 18S rRNA gene of Babesia rossi in the GeneBank. Of the two sequences that had 430 bp amplicon, one was identified as T. annulata and second as T. ovis. A significantly (p<0.05) higher prevalence of B. rossi was detected by PCR compared to microscopy. The mean PCV of Babesia infected dogs was significantly (p<0.05) lower than non-infected dogs. Phylogenetic analysis revealed minimal diversity among B. rossi with the exception of one sequence that was greatly divergent from the others. This study suggests that more than one genotype of B. rossi may be in circulation among the dog population in the study area and this may have potential implication on clinical outcome of canine babesiosis.

PCR-free quantitative detection of genetically modified organism from raw materials – A novel electrochemiluminescence-based bio-barcode method

PubMed Central

Zhu, Debin; Tang, Yabing; Xing, Da; Chen, Wei R.

2018-01-01

Bio-barcode assay based on oligonucleotide-modified gold nanoparticles (Au-NPs) provides a PCR-free method for quantitative detection of nucleic acid targets. However, the current bio-barcode assay requires lengthy experimental procedures including the preparation and release of barcode DNA probes from the target-nanoparticle complex, and immobilization and hybridization of the probes for quantification. Herein, we report a novel PCR-free electrochemiluminescence (ECL)-based bio-barcode assay for the quantitative detection of genetically modified organism (GMO) from raw materials. It consists of tris-(2’2’-bipyridyl) ruthenium (TBR)-labele barcode DNA, nucleic acid hybridization using Au-NPs and biotin-labeled probes, and selective capture of the hybridization complex by streptavidin-coated paramagnetic beads. The detection of target DNA is realized by direct measurement of ECL emission of TBR. It can quantitatively detect target nucleic acids with high speed and sensitivity. This method can be used to quantitatively detect GMO fragments from real GMO products. PMID:18386909

PCR-free quantitative detection of genetically modified organism from raw materials. An electrochemiluminescence-based bio bar code method.

PubMed

Zhu, Debin; Tang, Yabing; Xing, Da; Chen, Wei R

2008-05-15

A bio bar code assay based on oligonucleotide-modified gold nanoparticles (Au-NPs) provides a PCR-free method for quantitative detection of nucleic acid targets. However, the current bio bar code assay requires lengthy experimental procedures including the preparation and release of bar code DNA probes from the target-nanoparticle complex and immobilization and hybridization of the probes for quantification. Herein, we report a novel PCR-free electrochemiluminescence (ECL)-based bio bar code assay for the quantitative detection of genetically modified organism (GMO) from raw materials. It consists of tris-(2,2'-bipyridyl) ruthenium (TBR)-labeled bar code DNA, nucleic acid hybridization using Au-NPs and biotin-labeled probes, and selective capture of the hybridization complex by streptavidin-coated paramagnetic beads. The detection of target DNA is realized by direct measurement of ECL emission of TBR. It can quantitatively detect target nucleic acids with high speed and sensitivity. This method can be used to quantitatively detect GMO fragments from real GMO products.

Identification of thermophilic bacterial strains producing thermotolerant hydrolytic enzymes from manure compost.

PubMed

Charbonneau, David M; Meddeb-Mouelhi, Fatma; Boissinot, Maurice; Sirois, Marc; Beauregard, Marc

2012-03-01

Ten thermophilic bacterial strains were isolated from manure compost. Phylogenetic analysis based on 16S rRNA genes and biochemical characterization allowed identification of four different species belonging to four genera: Geobacillus thermodenitrificans, Bacillus smithii, Ureibacillus suwonensis and Aneurinibacillus thermoaerophilus. PCR-RFLP profiles of the 16S-ITS-23S rRNA region allowed us to distinguish two subgroups among the G. thermodenitrificans isolates. Isolates were screened for thermotolerant hydrolytic activities (60-65°C). Thermotolerant lipolytic activities were detected for G. thermodenitrificans, A. thermoaerophilus and B. smithii. Thermotolerant protease, α-amylase and xylanase activities were also observed in the G. thermodenitrificans group. These species represent a source of potential novel thermostable enzymes for industrial applications.

Quantification of Microbial Communities in Subsurface Marine Sediments of the Black Sea and off Namibia.

PubMed

Schippers, Axel; Kock, Dagmar; Höft, Carmen; Köweker, Gerrit; Siegert, Michael

2012-01-01

Organic-rich subsurface marine sediments were taken by gravity coring up to a depth of 10 m below seafloor at six stations from the anoxic Black Sea and the Benguela upwelling system off Namibia during the research cruises Meteor 72-5 and 76-1, respectively. The quantitative microbial community composition at various sediment depths was analyzed using total cell counting, catalyzed reporter deposition - fluorescence in situ hybridization (CARD-FISH) and quantitative real-time PCR (Q-PCR). Total cell counts decreased with depths from 10(9) to 10(10) cells/mL at the sediment surface to 10(7)-10(9) cells/mL below one meter depth. Based on CARD-FISH and Q-PCR analyses overall similar proportions of Bacteria and Archaea were found. The down-core distribution of prokaryotic and eukaryotic small subunit ribosomal RNA genes (16S and 18S rRNA) as well as functional genes involved in different biogeochemical processes was quantified using Q-PCR. Crenarchaeota and the bacterial candidate division JS-1 as well as the classes Anaerolineae and Caldilineae of the phylum Chloroflexi were highly abundant. Less abundant but detectable in most of the samples were Eukarya as well as the metal and sulfate-reducing Geobacteraceae (only in the Benguela upwelling influenced sediments). The functional genes cbbL, encoding for the large subunit of RuBisCO, the genes dsrA and aprA, indicative of sulfate-reducers as well as the mcrA gene of methanogens were detected in the Benguela upwelling and Black Sea sediments. Overall, the high organic carbon content of the sediments goes along with high cell counts and high gene copy numbers, as well as an equal abundance of Bacteria and Archaea.

Development of multiplex polymerase chain reaction for detection of Ehrlichia canis, Babesia spp and Hepatozoon canis in canine blood.

PubMed

Kledmanee, Kan; Suwanpakdee, Sarin; Krajangwong, Sakranmanee; Chatsiriwech, Jarin; Suksai, Parut; Suwannachat, Pongpun; Sariya, Ladawan; Buddhirongawatr, Ruangrat; Charoonrut, Phingphol; Chaichoun, Kridsada

2009-01-01

A multiplex polymerase chain reaction (PCR) has been developed for simultaneous detection of canine blood parasites, Ehrlichia canis, Babesia spp and Hepatozoon canis, from blood samples in a single reaction. The multiplex PCR primers were specific to E. canis VirB9, Babesia spp 16S rRNA and H. canis 16S rRNA genes. Specificity of the amplicons was confirmed by DNA sequencing. The assay was evaluated using normal canine and infected blood samples, which were detected by microscopic examination. This multiplex PCR offers scope for simultaneous detection of three important canine blood parasites and should be valuable in monitoring parasite infections in dogs and ticks.

Development of a single-tube loop-mediated isothermal amplification assay for detection of four pathogens of bacterial meningitis.

PubMed

Huy, Nguyen Tien; Hang, Le Thi Thuy; Boamah, Daniel; Lan, Nguyen Thi Phuong; Van Thanh, Phan; Watanabe, Kiwao; Huong, Vu Thi Thu; Kikuchi, Mihoko; Ariyoshi, Koya; Morita, Kouichi; Hirayama, Kenji

2012-12-01

Several loop-mediated isothermal amplification (LAMP) assays have been developed to detect common causative pathogens of bacterial meningitis (BM). However, no LAMP assay is reported to detect Streptococcus agalactiae and Streptococcus suis, which are also among common pathogens of BM. Moreover, it is laborious and expensive by performing multiple reactions for each sample to detect bacterial pathogen. Thus, we aimed to design and develop a single-tube LAMP assay capable of detecting multiple bacterial species, based on the nucleotide sequences of the 16S rRNA genes of the bacteria. The nucleotide sequences of the 16S rRNA genes of main pathogens involved in BM were aligned to identify conserved regions, which were further used to design broad range specific LAMP assay primers. We successfully designed a set of broad range specific LAMP assay primers for simultaneous detection of four species including Staphylococcus aureus, Streptococcus pneumoniae, S. suis and S. agalactiae. The broad range LAMP assay was highly specific without cross-reactivity with other bacteria including Haemophilus influenzae, Neisseria meningitidis and Escherichia coli. The sensitivity of our LAMP assay was 100-1000 times higher compared with the conventional PCR assay. The bacterial species could be identified after digestion of the LAMP products with restriction endonuclease DdeI and HaeIII. © 2012 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved.

A probe-based quantitative PCR assay for detecting Tetracapsuloides bryosalmonae in fish tissue and environmental DNA water samples

USGS Publications Warehouse

Hutchins, Patrick; Sepulveda, Adam; Martin, Renee; Hopper, Lacey

2017-01-01

A probe-based quantitative real-time PCR assay was developed to detect Tetracapsuloides bryosalmonae, which causes proliferative kidney disease in salmonid fish, in kidney tissue and environmental DNA (eDNA) water samples. The limits of detection and quantification were 7 and 100 DNA copies for calibration standards and T. bryosalmonae was reliably detected down to 100 copies in tissue and eDNA samples. The assay presented here is a highly sensitive and quantitative tool for detecting T. bryosalmonae with potential applications for tissue diagnostics and environmental detection.

16S rRNA beacons for bacterial monitoring during human space missions.

PubMed

Larios-Sanz, Maia; Kourentzi, Katerina D; Warmflash, David; Jones, Jeffrey; Pierson, Duane L; Willson, Richard C; Fox, George E

2007-04-01

Microorganisms are unavoidable in space environments and their presence has, at times, been a source of problems. Concerns about disease during human space missions are particularly important considering the significant changes the immune system incurs during spaceflight and the history of microbial contamination aboard the Mir space station. Additionally, these contaminants may have adverse effects on instrumentation and life-support systems. A sensitive, highly specific system to detect, characterize, and monitor these microbial populations is essential. Herein we describe a monitoring approach that uses 16S rRNA targeted molecular beacons to successfully detect several specific bacterial groupings. This methodology will greatly simplify in-flight monitoring by minimizing sample handling and processing. We also address and provide solutions to target accessibility problems encountered in hybridizations that target 16S rRNA.

A Rapid Protocol of Crude RNA/DNA Extraction for RT-qPCR Detection and Quantification of 'Candidatus Phytoplasma prunorum'

PubMed Central

Minguzzi, Stefano; Terlizzi, Federica; Lanzoni, Chiara; Poggi Pollini, Carlo; Ratti, Claudio

2016-01-01

Many efforts have been made to develop a rapid and sensitive method for phytoplasma and virus detection. Taking our cue from previous works, different rapid sample preparation methods have been tested and applied to Candidatus Phytoplasma prunorum (‘Ca. P. prunorum’) detection by RT-qPCR. A duplex RT-qPCR has been optimized using the crude sap as a template to simultaneously amplify a fragment of 16S rRNA of the pathogen and 18S rRNA of the host plant. The specific plant 18S rRNA internal control allows comparison and relative quantification of samples. A comparison between DNA and RNA contribution to qPCR detection is provided, showing higher contribution of the latter. The method presented here has been validated on more than a hundred samples of apricot, plum and peach trees. Since 2013, this method has been successfully applied to monitor ‘Ca. P. prunorum’ infections in field and nursery. A triplex RT-qPCR assay has also been optimized to simultaneously detect ‘Ca. P. prunorum’ and Plum pox virus (PPV) in Prunus. PMID:26742106

Using secondary structure to identify ribosomal numts: cautionary examples from the human genome.

PubMed

Olson, Link E; Yoder, Anne D

2002-01-01

The identification of inadvertently sequenced mitochondrial pseudogenes (numts) is critical to any study employing mitochondrial DNA sequence data. Failure to discriminate numts correctly can confound phylogenetic reconstruction and studies of molecular evolution. This is especially problematic for ribosomal mtDNA genes. Unlike protein-coding loci, whose pseudogenes tend to accumulate diagnostic frameshift or premature stop mutations, functional ribosomal genes are not constrained to maintain a reading frame and can accumulate insertion-deletion events of varying length, particularly in nonpairing regions. Several authors have advocated using structural features of the transcribed rRNA molecule to differentiate functional mitochondrial rRNA genes from their nuclear paralogs. We explored this approach using the mitochondrial 12S rRNA gene and three known 12S numts from the human genome in the context of anthropoid phylogeny and the inferred secondary structure of primate 12S rRNA. Contrary to expectation, each of the three human numts exhibits striking concordance with secondary structure models, with little, if any, indication of their pseudogene status, and would likely escape detection based on structural criteria alone. Furthermore, we show that the unwitting inclusion of a particularly ancient (18-25 Myr old) and surprisingly cryptic human numt in a phylogenetic analysis would yield a well-supported but dramatically incorrect conclusion regarding anthropoid relationships. Though we endorse the use of secondary structure models for inferring positional homology wholeheartedly, we caution against reliance on structural criteria for the discrimination of rRNA numts, given the potential fallibility of this approach.

Development of loop-mediated isothermal amplification with Plasmodium falciparum unique genes for molecular diagnosis of human malaria.

PubMed

Zhang, Yijing; Yao, Yi; Du, Weixing; Wu, Kai; Xu, Wenyue; Lin, Min; Tan, Huabing; Li, Jian

2017-07-01

In order to achieve better outcomes for treatment and in the prophylaxis of malaria, it is imperative to develop a sensitive, specific, and accurate assay for early diagnosis of Plasmodium falciparum infection, which is the major cause of malaria. In this study, we aimed to develop a loop-mediated isothermal amplification (LAMP) assay with P. falciparum unique genes for sensitive, specific, and accurate detection of P. falciparum infection. The unique genes of P. falciparum were randomly selected from PlasmoDB. The LAMP primers of the unique genes were designed using PrimerExplorer V4. LAMP assays with primers from unique genes of P. falciparum and conserved 18S rRNA gene were developed and their sensitivity was assessed. The specificity of the most sensitive LAMP assay was further examined using genomic DNA from Plasmodium vivax, Plasmodium yoelii and Toxoplasma gondii. Finally, the unique gene-based LAMP assay was validated using clinical samples of P. falciparum infection cases. A total of 31 sets of top-scored LAMP primers from nine unique genes were selected from the pools of designed primers. The LAMP assay with PF3D7_1253300-5 was the most sensitive with the detection limit 5 parasites/μl, and it displayed negative LAMP assay with the genomic DNA samples of P. vivax, P. yoelii, and T. gondii. The LAMP assay with PF3D7_0112300 (18S rRNA) was less sensitive with the detection limit 50 parasites/μl, and it displayed negative LAMP assay with the genomic DNA samples of P. yoelii and T. gondii, but displayed positive LAMP detection with P. vivax. The positive detection rate of the LAMP assay with PF3D7_1253300-5 was 90% (27/30), higher than that (80%, 24/30) of the positive rate of PF3D7_0112300 (18S rRNA) in examining clinical samples of P. falciparum infection cases. The LAMP assay with the primer set PF3D7_1253300-5 was more sensitive, specific, and accurate than those with PF3D7_0112300 (18S rRNA) in examining P. falciparum infection, and therefore it is a promising tool for diagnosis of P. falciparum infection.

Identification of vaginal fluid, saliva, and feces using microbial signatures in a Han Chinese population.

PubMed

Zou, Kai-Nan; Ren, Li-Jie; Ping, Yuan; Ma, Ke; Li, Hui; Cao, Yu; Zhou, Huai-Gu; Wei, Yi-Liang

2016-10-01

In recent years, forensic scientists have focused on the discrimination of body fluids using microbial signatures. In this study, we performed PCR-based detection of microbial signatures of vaginal fluid, saliva, and feces in a Han Chinese population. We investigated the 16S rRNA genes of Lactobacillus crispatus, Lactobacillus gasseri, Lactobacillus jensenii, Lactobacillus iners, and Atopobium vaginae in vaginal fluid, the 16S rRNA and the glucosyltransferase enzyme genes of Streptococcus salivarius and Streptococcus mutans in saliva, and the 16S rRNA genes of Enterococcus species, the RNA polymerase β-subunit gene of Bacteroides uniformis and Bacteroides vulgatus, and the α-1-6 mannanase gene of Bacteroides thetaiotaomicron in feces. As a result, the detection proportions of L. crispatus, L. gasseri, L. jensenii, L. iners, and A. vaginae were 15/16, 5/16, 8/16, 14/16, and 3/16 in 16 vaginal fluid donors, respectively. L. crispatus and L. jensenii were specifically detected in vaginal fluid; L. gasseri, L. iners, and A. vaginae were also detected in non-vaginal fluid. S. salivarius and S. mutans were not specifically detected in saliva. The detection proportions of Enterococcus species, B. uniformis, B. vulgatus, and B. thetaiotaomicron in 16 feces samples were 16/16, 12/16, 15/16, and 11/16, respectively. B. uniformis and B. thetaiotaomicron were specifically detected in feces. In addition, DNA samples prepared for the identification of body fluid can also be used for individual identification by short tandem repeat typing. The mean detection sensitivities of L. crispatus and L. jensenii were 0.362 and 0.249 pg/uL, respectively. In conclusion, L. crispatus, L. jensenii, B. uniformis, and B. thetaiotaomicron can be used as effective markers for forensic identification of vaginal fluid and feces. Copyright © 2016 Elsevier Ltd and Faculty of Forensic and Legal Medicine. All rights reserved.

Saccharomyces cerevisiae RNA Polymerase I Terminates Transcription at the Reb1 Terminator In Vivo

PubMed Central

Reeder, Ronald H.; Guevara, Palmira; Roan, Judith G.

1999-01-01

We have mapped transcription termination sites for RNA polymerase I in the yeast Saccharomyces cerevisiae. S1 nuclease mapping shows that the primary terminator is the Reb1p terminator located at +93 downstream of the 3′ end of 25S rRNA. Reverse transcription coupled with quantitative PCR shows that approximately 90% of all transcripts terminate at this site. Transcripts which read through the +93 site quantitatively terminate at a fail-safe terminator located further downstream at +250. Inactivation of Rnt1p (an RNase III involved in processing the 3′ end of 25S rRNA) greatly stabilizes transcripts extending to both sites and increases readthrough at the +93 site. In vivo assay of mutants of the Reb1p terminator shows that this site operates in vivo by the same mechanism as has previously been delineated through in vitro studies. PMID:10523625

Application of clone library analysis and real-time PCR for comparison of microbial communities in a low-grade copper sulfide ore bioheap leachate.

PubMed

Bowei, Chen; Xingyu, Liu; Wenyan, Liu; Jiankang, Wen

2009-11-01

The microbial communities of leachate from a bioleaching heap located in China were analyzed using the 16S rRNA gene clone library and real-time quantitative PCR. Both methods showed that Leptospirillum spp. were the dominant bacteria, and Ferroplasma acidiphilum were the only archaea detected in the leachate. Clone library results indicated that nine operational taxonomic units (OTUs) were obtained, which fell into four divisions, the Nitrospirae (74%), the gamma-Proteobacteria (14%), the Actinobacteria (6%) and the Euryarchaeota (6%). The results obtained by real-time PCR in some ways were the same as clone library analysis. Furthermore, Sulfobacillus spp., detected only by real-time PCR, suggests that real-time PCR was a reliable technology to study the microbial communities in bioleaching environments. It is a useful tool to assist clone library analysis, to further understand microbial consortia and to have comprehensive and exact microbiological information about bioleaching environments. Finally, the interactions among the microorganisms detected in the leachate were summarized according to the characteristics of these species.

Growth properties associated with A-U replacement of specific G-C base pairs in 16S rRNA from Escherichia coli.

PubMed Central

Triman, K L

1995-01-01

Mutations that disrupt each of seven specific G-C base pairs in 16S rRNA from Escherichia coli confer loss of expression of a plasmid-encoded 16S rRNA selectable marker (spectinomycin resistance). However, A-U replacement of G-C base pairs at nucleotides 359/52 or 1292/1245 in 16S rRNA permits normal expression of the marker. By contrast, A-U replacements at 146/176, 153/168, 350/339, or 1293/1244 are associated with loss of expression of the marker. These genetic studies are designed to determine the importance of specific base pairs by assessment of the structural and functional impairments of 16S rRNA molecules resulting from expression of base pair substitutions at these positions. PMID:7543481

Cultivable Anaerobic Microbiota of Severe Early Childhood Caries▿¶

PubMed Central

Tanner, A. C. R.; Mathney, J. M. J.; Kent, R. L.; Chalmers, N. I.; Hughes, C. V.; Loo, C. Y.; Pradhan, N.; Kanasi, E.; Hwang, J.; Dahlan, M. A.; Papadopolou, E.; Dewhirst, F. E.

2011-01-01

Severe early childhood caries (ECC), while strongly associated with Streptococcus mutans using selective detection (culture, PCR), has also been associated with a widely diverse microbiota using molecular cloning approaches. The aim of this study was to evaluate the microbiota of severe ECC using anaerobic culture. The microbial composition of dental plaque from 42 severe ECC children was compared with that of 40 caries-free children. Bacterial samples were cultured anaerobically on blood and acid (pH 5) agars. Isolates were purified, and partial sequences for the 16S rRNA gene were obtained from 5,608 isolates. Sequence-based analysis of the 16S rRNA isolate libraries from blood and acid agars of severe ECC and caries-free children had >90% population coverage, with greater diversity occurring in the blood isolate library. Isolate sequences were compared with taxon sequences in the Human Oral Microbiome Database (HOMD), and 198 HOMD taxa were identified, including 45 previously uncultivated taxa, 29 extended HOMD taxa, and 45 potential novel groups. The major species associated with severe ECC included Streptococcus mutans, Scardovia wiggsiae, Veillonella parvula, Streptococcus cristatus, and Actinomyces gerensceriae. S. wiggsiae was significantly associated with severe ECC children in the presence and absence of S. mutans detection. We conclude that anaerobic culture detected as wide a diversity of species in ECC as that observed using cloning approaches. Culture coupled with 16S rRNA identification identified over 74 isolates for human oral taxa without previously cultivated representatives. The major caries-associated species were S. mutans and S. wiggsiae, the latter of which is a candidate as a newly recognized caries pathogen. PMID:21289150

Evaluation of a Real-Time PCR Test for the Detection and Discrimination of Theileria Species in the African Buffalo (Syncerus caffer)

PubMed Central

Chaisi, Mamohale E.; Janssens, Michiel E.; Vermeiren, Lieve; Oosthuizen, Marinda C.; Collins, Nicola E.; Geysen, Dirk

2013-01-01

A quantitative real-time PCR (qPCR) assay based on the cox III gene was evaluated for the simultaneous detection and discrimination of Theileria species in buffalo and cattle blood samples from South Africa and Mozambique using melting curve analysis. The results obtained were compared to those of the reverse line blot (RLB) hybridization assay for the simultaneous detection and differentiation of Theileria spp. in mixed infections, and to the 18S rRNA qPCR assay results for the specific detection of Theileria parva. Theileria parva, Theileria sp. (buffalo), Theileria taurotragi, Theileria buffeli and Theileria mutans were detected by the cox III assay. Theileria velifera was not detected from any of the samples analysed. Seventeen percent of the samples had non-species specific melting peaks and 4.5% of the samples were negative or below the detection limit of the assay. The cox III assay identified more T. parva and Theileria sp. (buffalo) positive samples than the RLB assay, and also detected more T. parva infections than the 18S assay. However, only a small number of samples were positive for the benign Theileria spp. To our knowledge T. taurotragi has never been identified from the African buffalo, its identification in some samples by the qPCR assay was unexpected. Because of these discrepancies in the results, cox III qPCR products were cloned and sequenced. Sequence analysis indicated extensive inter- and intra-species variations in the probe target regions of the cox III gene sequences of the benign Theileria spp. and therefore explains their low detection. The cox III assay is specific for the detection of T. parva infections in cattle and buffalo. Sequence data generated from this study can be used for the development of a more inclusive assay for detection and differentiation of all variants of the mildly pathogenic and benign Theileria spp. of buffalo and cattle. PMID:24146782

Evaluation of a real-time PCR test for the detection and discrimination of theileria species in the African buffalo (Syncerus caffer).

PubMed

Chaisi, Mamohale E; Janssens, Michiel E; Vermeiren, Lieve; Oosthuizen, Marinda C; Collins, Nicola E; Geysen, Dirk

2013-01-01

A quantitative real-time PCR (qPCR) assay based on the cox III gene was evaluated for the simultaneous detection and discrimination of Theileria species in buffalo and cattle blood samples from South Africa and Mozambique using melting curve analysis. The results obtained were compared to those of the reverse line blot (RLB) hybridization assay for the simultaneous detection and differentiation of Theileria spp. in mixed infections, and to the 18S rRNA qPCR assay results for the specific detection of Theileria parva. Theileria parva, Theileria sp. (buffalo), Theileria taurotragi, Theileria buffeli and Theileria mutans were detected by the cox III assay. Theileria velifera was not detected from any of the samples analysed. Seventeen percent of the samples had non-species specific melting peaks and 4.5% of the samples were negative or below the detection limit of the assay. The cox III assay identified more T. parva and Theileria sp. (buffalo) positive samples than the RLB assay, and also detected more T. parva infections than the 18S assay. However, only a small number of samples were positive for the benign Theileria spp. To our knowledge T. taurotragi has never been identified from the African buffalo, its identification in some samples by the qPCR assay was unexpected. Because of these discrepancies in the results, cox III qPCR products were cloned and sequenced. Sequence analysis indicated extensive inter- and intra-species variations in the probe target regions of the cox III gene sequences of the benign Theileria spp. and therefore explains their low detection. The cox III assay is specific for the detection of T. parva infections in cattle and buffalo. Sequence data generated from this study can be used for the development of a more inclusive assay for detection and differentiation of all variants of the mildly pathogenic and benign Theileria spp. of buffalo and cattle.

Differential resistance of drinking water bacterial populations to monochloramine disinfection.

PubMed

Chiao, Tzu-Hsin; Clancy, Tara M; Pinto, Ameet; Xi, Chuanwu; Raskin, Lutgarde

2014-04-01

The impact of monochloramine disinfection on the complex bacterial community structure in drinking water systems was investigated using culture-dependent and culture-independent methods. Changes in viable bacterial diversity were monitored using culture-independent methods that distinguish between live and dead cells based on membrane integrity, providing a highly conservative measure of viability. Samples were collected from lab-scale and full-scale drinking water filters exposed to monochloramine for a range of contact times. Culture-independent detection of live cells was based on propidium monoazide (PMA) treatment to selectively remove DNA from membrane-compromised cells. Quantitative PCR (qPCR) and pyrosequencing of 16S rRNA genes was used to quantify the DNA of live bacteria and characterize the bacterial communities, respectively. The inactivation rate determined by the culture-independent PMA-qPCR method (1.5-log removal at 664 mg·min/L) was lower than the inactivation rate measured by the culture-based methods (4-log removal at 66 mg·min/L). Moreover, drastic changes in the live bacterial community structure were detected during monochloramine disinfection using PMA-pyrosequencing, while the community structure appeared to remain stable when pyrosequencing was performed on samples that were not subject to PMA treatment. Genera that increased in relative abundance during monochloramine treatment include Legionella, Escherichia, and Geobacter in the lab-scale system and Mycobacterium, Sphingomonas, and Coxiella in the full-scale system. These results demonstrate that bacterial populations in drinking water exhibit differential resistance to monochloramine, and that the disinfection process selects for resistant bacterial populations.

Comprehensive Analysis of Bacterial Flora in Postoperative Maxillary Cyst Fluid by 16S rRNA Gene and Culture Methods

PubMed Central

Sano, Naoto; Yamashita, Yoshio; Fukuda, Kazumasa; Taniguchi, Hatsumi; Goto, Masaaki; Miyamoto, Hiroshi

2012-01-01

Intracystic fluid was aseptically collected from 11 patients with postoperative maxillary cyst (POMC), and DNA was extracted from the POMC fluid. Bacterial species were identified by sequencing after cloning of approximately 580 bp of the 16S rRNA gene. Identification of pathogenic bacteria was also performed by culture methods. The phylogenetic identity was determined by sequencing 517–596 bp in each of the 1139 16S rRNA gene clones. A total of 1114 clones were classified while the remaining 25 clones were unclassified. A total of 103 bacterial species belonging to 42 genera were identified in POMC fluid samples by 16S rRNA gene analysis. Species of Prevotella (91%), Neisseria (73%), Fusobacterium (73%), Porphyromonas (73%), and Propionibacterium (73%) were found to be highly prevalent in all patients. Streptococcus mitis (64%), Fusobacterium nucleatum (55%), Propionibacterium acnes (55%), Staphylococcus capitis (55%), and Streptococcus salivarius (55%) were detected in more than 6 of the 11 patients. The results obtained by the culture method were different from those obtained by 16S rRNA gene analysis, but both approaches may be necessary for the identification of pathogens, especially of bacteria that are difficult to detect by culture methods, and the development of rational treatments for patients with POMC. PMID:22685668

Low Maternal Microbiota Sharing across Gut, Breast Milk and Vagina, as Revealed by 16S rRNA Gene and Reduced Metagenomic Sequencing.

PubMed

Avershina, Ekaterina; Angell, Inga Leena; Simpson, Melanie; Storrø, Ola; Øien, Torbjørn; Johnsen, Roar; Rudi, Knut

2018-05-01

The maternal microbiota plays an important role in infant gut colonization. In this work we have investigated which bacterial species are shared across the breast milk, vaginal and stool microbiotas of 109 women shortly before and after giving birth using 16S rRNA gene sequencing and a novel reduced metagenomic sequencing (RMS) approach in a subgroup of 16 women. All the species predicted by the 16S rRNA gene sequencing were also detected by RMS analysis and there was good correspondence between their relative abundances estimated by both approaches. Both approaches also demonstrate a low level of maternal microbiota sharing across the population and RMS analysis identified only two species common to most women and in all sample types ( Bifidobacterium longum and Enterococcus faecalis ). Breast milk was the only sample type that had significantly higher intra- than inter- individual similarity towards both vaginal and stool samples. We also searched our RMS dataset against an in silico generated reference database derived from bacterial isolates in the Human Microbiome Project. The use of this reference-based search enabled further separation of Bifidobacterium longum into Bifidobacterium longum ssp. longum and Bifidobacterium longum ssp. infantis . We also detected the Lactobacillus rhamnosus GG strain, which was used as a probiotic supplement by some women, demonstrating the potential of RMS approach for deeper taxonomic delineation and estimation.

Low Maternal Microbiota Sharing across Gut, Breast Milk and Vagina, as Revealed by 16S rRNA Gene and Reduced Metagenomic Sequencing

PubMed Central

Angell, Inga Leena; Storrø, Ola; Øien, Torbjørn; Johnsen, Roar; Rudi, Knut

2018-01-01

The maternal microbiota plays an important role in infant gut colonization. In this work we have investigated which bacterial species are shared across the breast milk, vaginal and stool microbiotas of 109 women shortly before and after giving birth using 16S rRNA gene sequencing and a novel reduced metagenomic sequencing (RMS) approach in a subgroup of 16 women. All the species predicted by the 16S rRNA gene sequencing were also detected by RMS analysis and there was good correspondence between their relative abundances estimated by both approaches. Both approaches also demonstrate a low level of maternal microbiota sharing across the population and RMS analysis identified only two species common to most women and in all sample types (Bifidobacterium longum and Enterococcus faecalis). Breast milk was the only sample type that had significantly higher intra- than inter- individual similarity towards both vaginal and stool samples. We also searched our RMS dataset against an in silico generated reference database derived from bacterial isolates in the Human Microbiome Project. The use of this reference-based search enabled further separation of Bifidobacterium longum into Bifidobacterium longum ssp. longum and Bifidobacterium longum ssp. infantis. We also detected the Lactobacillus rhamnosus GG strain, which was used as a probiotic supplement by some women, demonstrating the potential of RMS approach for deeper taxonomic delineation and estimation. PMID:29724017

Pneumocystis jirovecii multilocus genotyping in pooled DNA samples: a new approach for clinical and epidemiological studies.

PubMed

Esteves, F; Gaspar, J; de Sousa, B; Antunes, F; Mansinho, K; Matos, O

2012-06-01

Specific single-nucleotide polymorphisms (SNPs) are recognized as important DNA sequence variations influencing the pathogenesis of Pneumocystis jirovecii and the clinical outcome of Pneumocystis pneumonia, which is a major worldwide cause of illness among immunocompromised patients. Genotyping platforms for pooled DNA samples are promising methodologies for genetic characterization of infectious organisms. We have developed a new typing strategy for P. jirovecii, which consisted of DNA pools prepared according to clinical data (HIV diagnosis, microscopic and molecular detection of P. jirovecii, parasite burden, clinical diagnosis and follow-up of infection) from individual samples using quantitative real-time PCR followed by multiplex-PCR/single base extension (MPCR/SBE). The frequencies of multiple P. jirovecii SNPs (DHFR312, mt85, SOD215 and SOD110) encoded at three distinct loci, the dihydrofolate reductase (DHFR), the mitochondrial large-subunit rRNA (mtLSU rRNA) and the superoxide dismutase (SOD) loci, were estimated in seven DNA pooled samples, representing a total of 100 individual samples. The studied SNPs were confirmed to be associated with distinct clinical parameters of infection such as parasite burden and follow-up. The MPCR/SBE-DNA pooling methodology, described in the present study, was demonstrated to be a useful high-throughput procedure for large-scale P. jirovecii SNPs screening and a powerful tool for evaluation of clinically relevant SNPs potentially related to parasite burden, clinical diagnosis and follow-up of P. jirovecii infection. In further studies, the candidate SNPs mt85, SOD215 and SOD110 may be used as molecular markers in association with MPCR/SBE-DNA pooling to generate useful information for understanding the patterns and causes of Pneumocystis pneumonia. © 2012 The Authors. Clinical Microbiology and Infection © 2012 European Society of Clinical Microbiology and Infectious Diseases.

New microbes as causative agents of Ibuprofen degradation capabilities in the hyporheic zone of a lowland stream

NASA Astrophysics Data System (ADS)

Njeru, Cyrus; Posselt, Malte; Horn, Marcus A.

2017-04-01

Ibuprofen is a non-steroidal anti-inflammatory pain reliever and among pharmaceutical residues detected in aquatic environments. Widespread use of the drug and incomplete removal during waste water treatment results in its persistence in effluents and receiving waters. Potential total removal by microbial activity in the hyporheic zone (HZ) of rivers downstream of wastewater treatment plant discharge sites has been hypothesized. Ibuprofen degradation associated microbial communities in are essentially unknown. To address this hypothesis, two sets of oxic HZ sediment microcosms spiked with ibuprofen only (5, 40, 200 and 400 µM), or ibuprofen and 1 mM acetate were set up under laboratory conditions. Ibuprofen degradation in non-sterile relative to autoclaved sediments indicated removal by microbial degradation. Ibuprofen was completely consumed in the absence and presence of supplemental acetate after approximately 11 and 16 days, respectively. Refeeding of ibuprofen and acetate after the first depletion resulted in complete degradation within 24 hours in all treatments. Metabolites of ibuprofen included 1-, 2-, 3-hydroxy- and carboxyibuprofen. Quantitative real-time PCR revealed no pronounced differences in copy numbers of 16S rRNA gene or transcripts between non-spiked controls and treatments. Time resolved triplicate amplicon Illumina MiSeq sequencing targeting the 16S rRNA genes and transcripts revealed increased relative abundances of Proteobacteria, Acidobacteria, Actinobacteria and Firmicutes in treatments with compared to those without ibuprofen. Alpha-, Beta- and Deltaproteobacteria were most active as indicated by RNA based analyses. Enrichment and isolation yielded new Alphaproteobacteria utilizing ibuprofen as sole carbon and energy source. The collective results indicated that (i) HZ sediments sustain efficient biotic (micro-)pollutant removal and (ii) are a reservoir of hitherto unknown microbial diversity associated with such ecosystem services, including the genera Fodinicola, Hyphobacterium, and subgroup 6 Acidobacteria.

Separating Putative Pathogens from Background Contamination with Principal Orthogonal Decomposition: Evidence for Leptospira in the Ugandan Neonatal Septisome

PubMed Central

Schiff, Steven J.; Kiwanuka, Julius; Riggio, Gina; Nguyen, Lan; Mu, Kevin; Sproul, Emily; Bazira, Joel; Mwanga-Amumpaire, Juliet; Tumusiime, Dickson; Nyesigire, Eunice; Lwanga, Nkangi; Bogale, Kaleb T.; Kapur, Vivek; Broach, James R.; Morton, Sarah U.; Warf, Benjamin C.; Poss, Mary

2016-01-01

Neonatal sepsis (NS) is responsible for over 1 million yearly deaths worldwide. In the developing world, NS is often treated without an identified microbial pathogen. Amplicon sequencing of the bacterial 16S rRNA gene can be used to identify organisms that are difficult to detect by routine microbiological methods. However, contaminating bacteria are ubiquitous in both hospital settings and research reagents and must be accounted for to make effective use of these data. In this study, we sequenced the bacterial 16S rRNA gene obtained from blood and cerebrospinal fluid (CSF) of 80 neonates presenting with NS to the Mbarara Regional Hospital in Uganda. Assuming that patterns of background contamination would be independent of pathogenic microorganism DNA, we applied a novel quantitative approach using principal orthogonal decomposition to separate background contamination from potential pathogens in sequencing data. We designed our quantitative approach contrasting blood, CSF, and control specimens and employed a variety of statistical random matrix bootstrap hypotheses to estimate statistical significance. These analyses demonstrate that Leptospira appears present in some infants presenting within 48 h of birth, indicative of infection in utero, and up to 28 days of age, suggesting environmental exposure. This organism cannot be cultured in routine bacteriological settings and is enzootic in the cattle that often live in close proximity to the rural peoples of western Uganda. Our findings demonstrate that statistical approaches to remove background organisms common in 16S sequence data can reveal putative pathogens in small volume biological samples from newborns. This computational analysis thus reveals an important medical finding that has the potential to alter therapy and prevention efforts in a critically ill population. PMID:27379237

Methanotrophic bacteria in warm geothermal spring sediments identified using stable-isotope probing.

PubMed

Sharp, Christine E; Martínez-Lorenzo, Azucena; Brady, Allyson L; Grasby, Stephen E; Dunfield, Peter F

2014-10-01

We investigated methanotrophic bacteria in sediments of several warm geothermal springs ranging in temperature from 22 to 45 °C. Methane oxidation was measured at potential rates up to 141 μmol CH4 d(-1) g(-1) sediment. Active methanotrophs were identified using (13) CH4 stable-isotope probing (SIP) incubations performed at close to in situ temperatures for each site. Quantitative (q) PCR of pmoA genes identified the position of the heavy ((13) C-labelled) DNA fractions in density gradients, and 16S rRNA gene pyrotag sequencing of the heavy fractions was performed to identify the active methanotrophs. Methanotroph communities identified in heavy fractions of all samples were predominated by species similar (≥ 95% 16S rRNA gene identities) to previously characterized Gammaproteobacteria and Alphaproteobacteria methanotrophs. Among the five hottest samples (45 °C), members of the Gammaproteobacteria genus Methylocaldum dominated in two cases, while three others were dominated by an OTU closely related (96.8% similarity) to the Alphaproteobacteria genus Methylocapsa. These results suggest that diverse methanotroph groups are adapted to warm environments, including the Methylocapsa-Methylocella-Methyloferula group, which has previously only been detected in cooler sites. © 2014 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved.

Development of a quantitative PCR for rapid and sensitive diagnosis of an intranuclear coccidian parasite in Testudines (TINC), and detection in the critically endangered Arakan forest turtle (Heosemys depressa).

PubMed

Alvarez, W Alexander; Gibbons, Paul M; Rivera, Sam; Archer, Linda L; Childress, April L; Wellehan, James F X

2013-03-31

The intranuclear coccidian parasite of Testudines (TINC) is responsible for significant disease in turtles and tortoises causing high mortality and affecting several threatened species. Diagnostic testing has been limited to relatively labor intensive and expensive pan-coccidial PCR and sequencing techniques. A qPCR assay targeting a specific and conserved region of TINC 18S rRNA was designed. The qPCR reaction was run on samples known to be TINC positive and the results were consistent and analytically specific. The assay was able to detect as little as 10 copies of target DNA in a sample. Testing of soil and invertebrates was negative and did not provide any further insights into life cycles. This assay was used to identify TINC in a novel host species, the critically endangered Arakan forest turtle (Heosemys depressa). Copyright © 2012 Elsevier B.V. All rights reserved.

[A novel TaqMan® MGB probe for specifically detecting Streptococcus mutans].

PubMed

Zheng, Hui; Lin, Jiu-Xiang; DU, Ning; Chen, Feng

2013-10-18

To design a new TaqMan® MGB probe for improving the specificity of Streptococcus mutans's detection. We extracted six DNA samples from different streptococcal strains for PCR reaction. Conventional nested PCR and TaqMan® MGB real-time PCR were applied independently. The first round of nested PCR was carried out with the bacterial universal primers, while a second PCR was conducted by using primers specific for the 16S rRNA gene of Streptococcus mutans. The TaqMan® MGB probe for Streptococcus mutans was designed from sequence analyses, and the primers were the same as nested PCR. Streptococcus mutans DNA with 2.5 mg/L was sequentially diluted at 5-fold intervals to 0.16 μg/L. Standard DNA samples were used to generate standard curves by TaqMan® MGB real-time PCR. In the nested PCR, the primers specific for Streptococcus mutans also detected Streptococcus gordonii with visible band of 282 bp, giving false-positive results. In the TaqMan® MGB real-time PCR reaction, only Streptococcus mutans was detected. The detection limitation of TaqMan® MGB real-time PCR for Streptococcus mutans 16S rRNA gene was 20 μg/L. We designed a new TaqMan® MGB probe, and successfully set up a PCR based method for detecting oral Streptococcus mutans. TaqMan® MGB real-time PCR is a both specific and sensitive bacterial detection method.

Evaluation of PCR Approaches for Detection of Bartonella bacilliformis in Blood Samples.

PubMed

Gomes, Cláudia; Martinez-Puchol, Sandra; Pons, Maria J; Bazán, Jorge; Tinco, Carmen; del Valle, Juana; Ruiz, Joaquim

2016-03-01

The lack of an effective diagnostic tool for Carrion's disease leads to misdiagnosis, wrong treatments and perpetuation of asymptomatic carriers living in endemic areas. Conventional PCR approaches have been reported as a diagnostic technique. However, the detection limit of these techniques is not clear as well as if its usefulness in low bacteriemia cases. The aim of this study was to evaluate the detection limit of 3 PCR approaches. We determined the detection limit of 3 different PCR approaches: Bartonella-specific 16S rRNA, fla and its genes. We also evaluated the viability of dry blood spots to be used as a sample transport system. Our results show that 16S rRNA PCR is the approach with a lowest detection limit, 5 CFU/μL, and thus, the best diagnostic PCR tool studied. Dry blood spots diminish the sensitivity of the assay. From the tested PCRs, the 16S rRNA PCR-approach is the best to be used in the direct blood detection of acute cases of Carrion's disease. However its use in samples from dry blood spots results in easier management of transport samples in rural areas, a slight decrease in the sensitivity was observed. The usefulness to detect by PCR the presence of low-bacteriemic or asymptomatic carriers is doubtful, showing the need to search for new more sensible techniques.

The Resilience of Microbial Community under Drying and Rewetting Cycles of Three Forest Soils.

PubMed

Zhou, Xue; Fornara, Dario; Ikenaga, Makoto; Akagi, Isao; Zhang, Ruifu; Jia, Zhongjun

2016-01-01

Forest soil ecosystems are associated with large pools and fluxes of carbon (C) and nitrogen (N), which could be strongly affected by variation in rainfall events under current climate change. Understanding how dry and wet cycle events might influence the metabolic state of indigenous soil microbes is crucial for predicting forest soil responses to environmental change. We used 454 pyrosequencing and quantitative PCR to address how present (DNA-based) and potentially active (RNA-based) soil bacterial communities might response to the changes in water availability across three different forest types located in two continents (Africa and Asia) under controlled drying and rewetting cycles. Sequencing of rRNA gene and transcript indicated that Proteobacteria, Actinobacteria, and Acidobacteria were the most responsive phyla to changes in water availability. We defined the ratio of rRNA transcript to rRNA gene abundance as a key indicator of potential microbial activity and we found that this ratio was increased following soil dry-down process whereas it decreased after soil rewetting. Following rewetting Crenarchaeota-like 16S rRNA gene transcript increased in some forest soils and this was linked to increases in soil nitrate levels suggesting greater nitrification rates under higher soil water availability. Changes in the relative abundance of (1) different microbial phyla and classes, and (2) 16S and amoA genes were found to be site- and taxa-specific and might have been driven by different life-strategies. Overall, we found that, after rewetting, the structure of the present and potentially active bacterial community structure as well as the abundance of bacterial (16S), archaeal (16S) and ammonia oxidizers (amoA), all returned to pre-dry-down levels. This suggests that microbial taxa have the ability to recover from desiccation, a critical response, which will contribute to maintaining microbial biodiversity in harsh ecosystems under environmental perturbations, such as significant changes in water availability.

The Resilience of Microbial Community under Drying and Rewetting Cycles of Three Forest Soils

PubMed Central

Zhou, Xue; Fornara, Dario; Ikenaga, Makoto; Akagi, Isao; Zhang, Ruifu; Jia, Zhongjun

2016-01-01

Forest soil ecosystems are associated with large pools and fluxes of carbon (C) and nitrogen (N), which could be strongly affected by variation in rainfall events under current climate change. Understanding how dry and wet cycle events might influence the metabolic state of indigenous soil microbes is crucial for predicting forest soil responses to environmental change. We used 454 pyrosequencing and quantitative PCR to address how present (DNA-based) and potentially active (RNA-based) soil bacterial communities might response to the changes in water availability across three different forest types located in two continents (Africa and Asia) under controlled drying and rewetting cycles. Sequencing of rRNA gene and transcript indicated that Proteobacteria, Actinobacteria, and Acidobacteria were the most responsive phyla to changes in water availability. We defined the ratio of rRNA transcript to rRNA gene abundance as a key indicator of potential microbial activity and we found that this ratio was increased following soil dry-down process whereas it decreased after soil rewetting. Following rewetting Crenarchaeota-like 16S rRNA gene transcript increased in some forest soils and this was linked to increases in soil nitrate levels suggesting greater nitrification rates under higher soil water availability. Changes in the relative abundance of (1) different microbial phyla and classes, and (2) 16S and amoA genes were found to be site- and taxa-specific and might have been driven by different life-strategies. Overall, we found that, after rewetting, the structure of the present and potentially active bacterial community structure as well as the abundance of bacterial (16S), archaeal (16S) and ammonia oxidizers (amoA), all returned to pre-dry-down levels. This suggests that microbial taxa have the ability to recover from desiccation, a critical response, which will contribute to maintaining microbial biodiversity in harsh ecosystems under environmental perturbations, such as significant changes in water availability. PMID:27486444

Colorectal Cancer and the Human Gut Microbiome: Reproducibility with Whole-Genome Shotgun Sequencing

PubMed Central

Hua, Xing; Zeller, Georg; Sunagawa, Shinichi; Voigt, Anita Y.; Hercog, Rajna; Goedert, James J.; Shi, Jianxin; Bork, Peer; Sinha, Rashmi

2016-01-01

Accumulating evidence indicates that the gut microbiota affects colorectal cancer development, but previous studies have varied in population, technical methods, and associations with cancer. Understanding these variations is needed for comparisons and for potential pooling across studies. Therefore, we performed whole-genome shotgun sequencing on fecal samples from 52 pre-treatment colorectal cancer cases and 52 matched controls from Washington, DC. We compared findings from a previously published 16S rRNA study to the metagenomics-derived taxonomy within the same population. In addition, metagenome-predicted genes, modules, and pathways in the Washington, DC cases and controls were compared to cases and controls recruited in France whose specimens were processed using the same platform. Associations between the presence of fecal Fusobacteria, Fusobacterium, and Porphyromonas with colorectal cancer detected by 16S rRNA were reproduced by metagenomics, whereas higher relative abundance of Clostridia in cancer cases based on 16S rRNA was merely borderline based on metagenomics. This demonstrated that within the same sample set, most, but not all taxonomic associations were seen with both methods. Considering significant cancer associations with the relative abundance of genes, modules, and pathways in a recently published French metagenomics dataset, statistically significant associations in the Washington, DC population were detected for four out of 10 genes, three out of nine modules, and seven out of 17 pathways. In total, colorectal cancer status in the Washington, DC study was associated with 39% of the metagenome-predicted genes, modules, and pathways identified in the French study. More within and between population comparisons are needed to identify sources of variation and disease associations that can be reproduced despite these variations. Future studies should have larger sample sizes or pool data across studies to have sufficient power to detect associations that are reproducible and significant after correction for multiple testing. PMID:27171425

Detection of pork adulteration in processed meat by species-specific PCR-QIAxcel procedure based on D-loop and cytb genes.

PubMed

Barakat, Hassan; El-Garhy, Hoda A S; Moustafa, Mahmoud M A

2014-12-01

Detection of pork meat adulteration in "halal" meat products is a crucial issue in the fields of modern food inspection according to implementation of very strict procedures for halal food labelling. Present study aims at detecting and quantifying pork adulteration in both raw and cooked manufactured sausages. This is by applying an optimized species-specific PCR procedure followed by QIAxcel capillary electrophoresis system. Manufacturing experiment was designed by incorporating pork with beef meat at 0.01 to 10 % substitution levels beside beef and pork sausages as negative and positive controls, respectively. Subsequently, sausages were divided into raw and cooked sausages then subjected to DNA extraction. Results indicated that PCR amplifications of mitochondrial D-loop and cytochrome b (cytb) genes by porcine-specific primers produced 185 and 117 bp pork-specific DNA fragments in sausages, respectively. No DNA fragments were detected when PCR was applied on beef sausage DNA confirming primers specificity. For internal control, a 141-bp DNA fragment of eukaryotic 18S ribosomal RNA (rRNA) gene was amplified from pork and beef DNA templates. Although PCR followed by either QIAxcel or agarose techniques were efficient for targeted DNA fragments differentiation even as low as 0.01 % (pork/meat: w/w). For proficiency, adequacy, and performance, PCR-QIA procedure is highly sensitive, a time-saver, electronically documented, mutagenic-reagent free, of little manual errors, accurate in measuring PCR fragments length, and quantitative data supplier. In conclusion, it can be suggested that optimized PCR-QAI is considered as a rapid and sensitive method for routine pork detection and quantification in raw or processed meat.

Folate deficiency facilitates recruitment of upstream binding factor to hot spots of DNA double-strand breaks of rRNA genes and promotes its transcription.

PubMed

Xie, Qiu; Li, Caihua; Song, Xiaozhen; Wu, Lihua; Jiang, Qian; Qiu, Zhiyong; Cao, Haiyan; Yu, Kaihui; Wan, Chunlei; Li, Jianting; Yang, Feng; Huang, Zebing; Niu, Bo; Jiang, Zhengwen; Zhang, Ting

2017-03-17

The biogenesis of ribosomes in vivo is an essential process for cellular functions. Transcription of ribosomal RNA (rRNA) genes is the rate-limiting step in ribosome biogenesis controlled by environmental conditions. Here, we investigated the role of folate antagonist on changes of DNA double-strand breaks (DSBs) landscape in mouse embryonic stem cells. A significant DSB enhancement was detected in the genome of these cells and a large majority of these DSBs were found in rRNA genes. Furthermore, spontaneous DSBs in cells under folate deficiency conditions were located exclusively within the rRNA gene units, representing a H3K4me1 hallmark. Enrichment H3K4me1 at the hot spots of DSB regions enhanced the recruitment of upstream binding factor (UBF) to rRNA genes, resulting in the increment of rRNA genes transcription. Supplement of folate resulted in a restored UBF binding across DNA breakage sites of rRNA genes, and normal rRNA gene transcription. In samples from neural tube defects (NTDs) with low folate level, up-regulation of rRNA gene transcription was observed, along with aberrant UBF level. Our results present a new view by which alterations in folate levels affects DNA breakage through epigenetic control leading to the regulation of rRNA gene transcription during the early stage of development. © The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.

Analysis of Actinobacteria from mould-colonized water damaged building material.

PubMed

Schäfer, Jenny; Jäckel, Udo; Kämpfer, Peter

2010-08-01

Mould-colonized water damaged building materials are frequently co-colonized by actinomycetes. Here, we report the results of the analyses of Actinobacteria on different wall materials from water damaged buildings obtained by both cultivation-dependent and cultivation-independent methods. Actinobacteria were detected in all but one of the investigated materials by both methods. The detected concentrations of Actinobacteria ranged between 1.8 x 10(4) and 7.6 x 10(7) CFUg(-1) of investigated material. A total of 265 isolates from 17 materials could be assigned to 31 different genera of the class Actinobacteria on the basis of 16S rRNA gene sequence analyses. On the basis of the cultivation-independent approach, 16S rRNA gene inserts of 800 clones (50%) were assigned to 47 different genera. Representatives of the genera Streptomyces, Amycolatopsis, Nocardiopsis, Saccharopolyspora, Promicromonospora, and Pseudonocardia were found most frequently. The results derived from both methods indicated a high abundance and variety of Actinobacteria in water damaged buildings. Four bioaerosol samples were investigated by the cultivation-based approach in order to compare the communities of Actinobacteria in building material and associated air samples. A comparison of the detected genera of bioaerosol samples with those directly obtained from material samples resulted in a congruent finding of 9 of the overall 35 detected genera (25%), whereas four genera were only detected in bioaerosol samples. Copyright 2010 Elsevier GmbH. All rights reserved.

A low molecular weight artificial RNA of unique size with multiple probe target regions

NASA Technical Reports Server (NTRS)

Pitulle, C.; Dsouza, L.; Fox, G. E.

1997-01-01

Artificial RNAs (aRNAs) containing novel sequence segments embedded in a deletion mutant of Vibrio proteolyticus 5S rRNA have previously been shown to be expressed from a plasmid borne growth rate regulated promoter in E. coli. These aRNAs accumulate to high levels and their detection is a promising tool for studies in molecular microbial ecology and in environmental monitoring. Herein a new construct is described which illustrates the versatility of detection that is possible with aRNAs. This 3xPen aRNA construct carries a 72 nucleotide insert with three copies of a unique 17 base probe target sequence. This aRNA is 160 nucleotides in length and again accumulates to high levels in the E. coli cytoplasm without incorporating into ribosomes. The 3xPen aRNA illustrates two improvements in detection. First, by appropriate selection of insert size, we obtained an aRNA which provides a unique and hence, easily quantifiable peak, on a high resolution gel profile of low molecular weight RNAs. Second, the existence of multiple probe targets results in a nearly commensurate increase in signal when detection is by hybridization. These aRNAs are naturally amplified and carry sequence segments that are not found in known rRNA sequences. It thus may be possible to detect them directly. An experimental step involving RT-PCR or PCR amplification of the gene could therefore be avoided.

Performance and Specificity of the Covalently Linked Immunomagnetic Separation-ATP Method for Rapid Detection and Enumeration of Enterococci in Coastal Environments

PubMed Central

Zimmer-Faust, Amity G.; Thulsiraj, Vanessa; Ferguson, Donna

2014-01-01

The performance and specificity of the covalently linked immunomagnetic separation-ATP (Cov-IMS/ATP) method for the detection and enumeration of enterococci was evaluated in recreational waters. Cov-IMS/ATP performance was compared with standard methods: defined substrate technology (Enterolert; IDEXX Laboratories), membrane filtration (EPA Method 1600), and an Enterococcus-specific quantitative PCR (qPCR) assay (EPA Method A). We extend previous studies by (i) analyzing the stability of the relationship between the Cov-IMS/ATP method and culture-based methods at different field sites, (ii) evaluating specificity of the assay for seven ATCC Enterococcus species, (iii) identifying cross-reacting organisms binding the antibody-bead complexes with 16S rRNA gene sequencing and evaluating specificity of the assay to five nonenterococcus species, and (iv) conducting preliminary tests of preabsorption as a means of improving the assay. Cov-IMS/ATP was found to perform consistently and with strong agreement rates (based on exceedance/compliance with regulatory limits) of between 83% and 100% compared to the culture-based Enterolert method at a variety of sites with complex inputs. The Cov-IMS/ATP method is specific to five of seven different Enterococcus spp. tested. However, there is potential for nontarget bacteria to bind the antibody, which may be reduced by purification of the IgG serum with preabsorption at problematic sites. The findings of this study help to validate the Cov-IMS/ATP method, suggesting a predictable relationship between the Cov-IMS/ATP method and traditional culture-based methods, which will allow for more widespread application of this rapid and field-portable method for coastal water quality assessment. PMID:24561583

Molecular detection of feline hemoplasmas in feral cats in Korea.

PubMed

Yu, Do-Hyeon; Kim, Hyun-Wook; Desai, Atul R; Han, In-Ae; Li, Ying-Hua; Lee, Mi-Jin; Kim, In-Shik; Chae, Joon-Seok; Park, Jinho

2007-12-01

The purpose of this study was to determine if Mycoplasma haemofelis, 'Candidatus Mycoplasma haemominutum' exist in Korea. Three hundreds and thirty one feral cats were evaluated by using PCR assay targeting 16S rRNA gene sequence. Fourteen cats (4.2%) were positive for M. haemofelis, 34 cats (10.3%) were positive for 'Candidatus M. haemominutum' and 18 cats (5.4%) were positive for both species. Partial 16S rRNA gene sequences were closely (>98%) related to those from other countries. This is the first molecular detection of feline hemoplasmas in Korea.

Phytate degrading activities of lactic acid bacteria isolated from traditional fermented food

NASA Astrophysics Data System (ADS)

Damayanti, Ema; Ratisiwi, Febiyani Ndaru; Istiqomah, Lusty; Sembiring, Langkah; Febrisiantosa, Andi

2017-03-01

The objective of this study was to determine the potential of LAB with phytate degrading activity from fermented traditional food grain-based and legume-based. Lactic acid bacteria were isolated from different sources of traditional fermented food from Gunungkidul Yogyakarta Indonesia such as gembus tempeh (tofu waste), soybean tempeh, lamtoro tempeh (Leucaena bean) and kara tempeh. Isolation of LAB was performed using Total Plate Count (TPC) on de Man Rogosa Sharpe Agar (MRSA) medium supplemented with CaCO3. They were screened for their ability to degrade myo-inositol hexaphosphate or IP6 by using qualitative streak platemethod with modified de Man Rogosa-MorpholinoPropanesulfonic Acid Sharpe (MRS-MOPS) medium contained sodium salt of phytic acid as substrate and cobalt chloride staining (plate assay) method. The selected isolates were further assayed for phytase activities using quantitative method with spectrophotometer and the two selected isolates growth were optimized. Furthermore, thhe isolates that shown the highest phytase activity was characterized and identified using API 50 CH kitand 16S rRNA gene sequencing. The results showed that there were 18 LAB isolates obtained from samplesand 13 isolates were able to degrade sodium phytate based on qualitative screening. According to quantitative assay, the highest phytate degrading activities were found in TG-2(23.562 U/mL) and TG-1 (19.641 U/mL) isolated from gembus tempeh. The phytate activity of TG-2 was optimum at 37 °C with agitation, while the phytate activity of TG-1 was optimum at 45 °C without agitation. Characterization and identification of TG-2 isolate with the highest phytate degrading activity using API 50 CH and 16S rRNA showed that TG-2had homology with Lactobacillus fermentum. It could be concluded that LAB from from fermented traditional food grain-based and legume-based produced the extracellular phytase. Keywords: lactic acid bacteria, tempeh, phytatedegrading activity

Real-time label-free quantitative fluorescence microscopy-based detection of ATP using a tunable fluorescent nano-aptasensor platform.

PubMed

Shrivastava, Sajal; Sohn, Il-Yung; Son, Young-Min; Lee, Won-Il; Lee, Nae-Eung

2015-12-14

Although real-time label-free fluorescent aptasensors based on nanomaterials are increasingly recognized as a useful strategy for the detection of target biomolecules with high fidelity, the lack of an imaging-based quantitative measurement platform limits their implementation with biological samples. Here we introduce an ensemble strategy for a real-time label-free fluorescent graphene (Gr) aptasensor platform. This platform employs aptamer length-dependent tunability, thus enabling the reagentless quantitative detection of biomolecules through computational processing coupled with real-time fluorescence imaging data. We demonstrate that this strategy effectively delivers dose-dependent quantitative readouts of adenosine triphosphate (ATP) concentration on chemical vapor deposited (CVD) Gr and reduced graphene oxide (rGO) surfaces, thereby providing cytotoxicity assessment. Compared with conventional fluorescence spectrometry methods, our highly efficient, universally applicable, and rational approach will facilitate broader implementation of imaging-based biosensing platforms for the quantitative evaluation of a range of target molecules.

Diversity analysis of lactic acid bacteria in takju, Korean rice wine.

PubMed

Jin, Jianbo; Kim, So-Young; Jin, Qing; Eom, Hyun-Ju; Han, Nam Soo

2008-10-01

To investigate the lactic acid bacterial population in Korean traditional rice wines, biotyping was performed using cell morphology and whole-cell protein pattern analysis by SDSPAGE, and then the isolates were identified by 16S rRNA sequencing analysis. Based on the morphological characteristics, 103 LAB isolates were detected in wine samples, characterized by whole-cell protein pattern analysis, and they were then divided into 18 patterns. By gene sequencing of 16S rRNA, the isolates were identified as Lactobacillus paracasei, Lb. arizonensis, Lb. plantarum, Lb. harbinensis, Lb. parabuchneri, Lb. brevis, and Lb. hilgardii when listed by their frequency of occurrence. It was found that the difference in bacterial diversity between rice and grape wines depends on the raw materials, especially the composition of starch and glucose.

Real-time label-free quantitative fluorescence microscopy-based detection of ATP using a tunable fluorescent nano-aptasensor platform

NASA Astrophysics Data System (ADS)

Shrivastava, Sajal; Sohn, Il-Yung; Son, Young-Min; Lee, Won-Il; Lee, Nae-Eung

2015-11-01

Although real-time label-free fluorescent aptasensors based on nanomaterials are increasingly recognized as a useful strategy for the detection of target biomolecules with high fidelity, the lack of an imaging-based quantitative measurement platform limits their implementation with biological samples. Here we introduce an ensemble strategy for a real-time label-free fluorescent graphene (Gr) aptasensor platform. This platform employs aptamer length-dependent tunability, thus enabling the reagentless quantitative detection of biomolecules through computational processing coupled with real-time fluorescence imaging data. We demonstrate that this strategy effectively delivers dose-dependent quantitative readouts of adenosine triphosphate (ATP) concentration on chemical vapor deposited (CVD) Gr and reduced graphene oxide (rGO) surfaces, thereby providing cytotoxicity assessment. Compared with conventional fluorescence spectrometry methods, our highly efficient, universally applicable, and rational approach will facilitate broader implementation of imaging-based biosensing platforms for the quantitative evaluation of a range of target molecules.Although real-time label-free fluorescent aptasensors based on nanomaterials are increasingly recognized as a useful strategy for the detection of target biomolecules with high fidelity, the lack of an imaging-based quantitative measurement platform limits their implementation with biological samples. Here we introduce an ensemble strategy for a real-time label-free fluorescent graphene (Gr) aptasensor platform. This platform employs aptamer length-dependent tunability, thus enabling the reagentless quantitative detection of biomolecules through computational processing coupled with real-time fluorescence imaging data. We demonstrate that this strategy effectively delivers dose-dependent quantitative readouts of adenosine triphosphate (ATP) concentration on chemical vapor deposited (CVD) Gr and reduced graphene oxide (rGO) surfaces, thereby providing cytotoxicity assessment. Compared with conventional fluorescence spectrometry methods, our highly efficient, universally applicable, and rational approach will facilitate broader implementation of imaging-based biosensing platforms for the quantitative evaluation of a range of target molecules. Electronic supplementary information (ESI) available. See DOI: 10.1039/c5nr05839b

High-sensitivity stable-isotope probing by a quantitative terminal restriction fragment length polymorphism protocol.

PubMed

Andeer, Peter; Strand, Stuart E; Stahl, David A

2012-01-01

Stable-isotope probing (SIP) has proved a valuable cultivation-independent tool for linking specific microbial populations to selected functions in various natural and engineered systems. However, application of SIP to microbial populations with relatively minor buoyant density increases, such as populations that utilize compounds as a nitrogen source, results in reduced resolution of labeled populations. We therefore developed a tandem quantitative PCR (qPCR)-TRFLP (terminal restriction fragment length polymorphism) protocol that improves resolution of detection by quantifying specific taxonomic groups in gradient fractions. This method combines well-controlled amplification with TRFLP analysis to quantify relative taxon abundance in amplicon pools of FAM-labeled PCR products, using the intercalating dye EvaGreen to monitor amplification. Method accuracy was evaluated using mixtures of cloned 16S rRNA genes, DNA extracted from low- and high-G+C bacterial isolates (Escherichia coli, Rhodococcus, Variovorax, and Microbacterium), and DNA from soil microcosms amended with known amounts of genomic DNA from bacterial isolates. Improved resolution of minor shifts in buoyant density relative to TRFLP analysis alone was confirmed using well-controlled SIP analyses.

Isolation of novel bacteria within the Chloroflexi capable of reductive dechlorination of 1,2,3-trichloropropane.

PubMed

Yan, J; Rash, B A; Rainey, F A; Moe, W M

2009-04-01

Two strictly anaerobic bacterial strains were isolated from contaminated groundwater at a Superfund site located near Baton Rouge, LA, USA. These strains represent the first isolates reported to reductively dehalogenate 1,2,3-trichloropropane. Allyl chloride (3-chloro-1-propene), which is chemically unstable, was produced from 1,2,3-trichloropropane, and it was hydrolysed abiotically to allyl alcohol and also reacted with the sulfide- and cysteine-reducing agents in the medium to form various allyl sulfides. Both isolates also dehalogenated a variety of other vicinally chlorinated alkanes (1,2-dichloropropane, 1,2-dichloroethane, 1,1,2-trichloroethane, 1,1,2,2- tetrachloroethane) via dichloroelimination reactions. A quantitative real-time PCR (qPCR) approach targeting 16S rRNA genes indicated that both strains couple reductive dechlorination to cell growth. Growth was not observed in the absence of hydrogen (H2) as an electron donor and a polychlorinated alkane as an electron acceptor. Alkanes containing only a single chlorine substituent (1-chloropropane, 2-chloropropane), chlorinated alkenes (tetrachlorothene, trichlorothene, cisdichloroethene, trans-dichloroethene, vinyl chloride) and chlorinated benzenes (1-chlorobenzene and 1,2- dichlorobenzene) were not dechlorinated. Phylogenetic analysis based on 16S rRNA gene sequence data showed these isolates to represent a new lineage within the Chloroflexi. Their closest previously cultured relatives are 'Dehalococcoides' strains, with 16S rRNA gene sequence similarities of only 90%.

Microbial rRNA: rDNA gene ratios may be unexpectedly low due to extracellular DNA preservation in soils

USDA-ARS?s Scientific Manuscript database

We tested a method of estimating the activity of detectable individual bacterial and archaeal OTUs within a community by calculating ratios of absolute 16S rRNA to rDNA copy numbers. We investigated phylogenetically coherent patterns of activity among soil prokaryotes in non-growing soil communitie...

Phylogenetic Analysis of Bacteroidales 16S rRNA Genes Unveils Sequences Specific to Diverse Swine Fecal Sources

EPA Science Inventory

Two of the currently available methods to assess swine fecal pollution (Bac1 and PF163) target Bacteroidales 16S rRNA genes. However, these assays have been shown to exhibit poor host-specificity and low detection limits in environmental waters, in part due to the limited number...

Evaluation of bacterial pathogen diversity, abundance and health risks in urban recreational water by amplicon next-generation sequencing and quantitative PCR.

PubMed

Cui, Qijia; Fang, Tingting; Huang, Yong; Dong, Peiyan; Wang, Hui

2017-07-01

The microbial quality of urban recreational water is of great concern to public health. The monitoring of indicator organisms and several pathogens alone is not sufficient to accurately and comprehensively identify microbial risks. To assess the levels of bacterial pathogens and health risks in urban recreational water, we analyzed pathogen diversity and quantified four pathogens in 46 water samples collected from waterbodies in Beijing Olympic Forest Park in one year. The pathogen diversity revealed by 16S rRNA gene targeted next-generation sequencing (NGS) showed that 16 of 40 genera and 13 of 76 reference species were present. The most abundant species were Acinetobacter johnsonii, Mycobacterium avium and Aeromonas spp. Quantitative polymerase chain reaction (qPCR) of Escherichia coli (uidA), Aeromonas (aerA), M. avium (16S rRNA), Pseudomonas aeruginosa (oaa) and Salmonella (invA) showed that the aerA genes were the most abundant, occurring in all samples with concentrations of 10 4-6 genome copies/100mL, followed by oaa, invA and M. avium. In total, 34.8% of the samples harbored all genes, indicating the prevalence of these pathogens in this recreational waterbody. Based on the qPCR results, a quantitative microbial risk assessment (QMRA) showed that the annual infection risks of Salmonella, M. avium and P. aeruginosa in five activities were mostly greater than the U.S. EPA risk limit for recreational contacts, and children playing with water may be exposed to the greatest infection risk. Our findings provide a comprehensive understanding of bacterial pathogen diversity and pathogen abundance in urban recreational water by applying both NGS and qPCR. Copyright © 2016. Published by Elsevier B.V.

Molecular detection and phylogenetic analysis of Hepatozoon spp. in questing Ixodes ricinus ticks and rodents from Slovakia and Czech Republic.

PubMed

Hamšíková, Zuzana; Silaghi, Cornelia; Rudolf, Ivo; Venclíková, Kristýna; Mahríková, Lenka; Slovák, Mirko; Mendel, Jan; Blažejová, Hana; Berthová, Lenka; Kocianová, Elena; Hubálek, Zdeněk; Schnittger, Leonhard; Kazimírová, Mária

2016-10-01

By amplification and sequencing of 18S rRNA gene fragments, Hepatozoon spp. DNA was detected in 0.08 % (4/5057) and 0.04 % (1/2473) of questing Ixodes ricinus ticks from Slovakia and Czech Republic, respectively. Hepatozoon spp. DNA was also detected in spleen and/or lungs of 4.45 % (27/606) of rodents from Slovakia. Prevalence of infection was significantly higher in Myodes glareolus (11.45 %) than in Apodemus spp. (0.28 %) (P < 0.001). Sequencing of 18S rRNA Hepatozoon spp. gene amplicons from I. ricinus showed 100 % identity with Hepatozoon canis isolates from red foxes or dogs in Europe. Phylogenetic analysis showed that at least two H. canis 18S rRNA genotypes exist in Slovakia of which one was identified also in the Czech Republic. The finding of H. canis in questing I. ricinus suggests the geographical spread of the parasite and a potential role of other ticks as its vectors in areas where Rhipicephalus sanguineus is not endemic. Sequencing of 18S rRNA gene amplicons from M. glareolus revealed the presence of two closely related genetic variants, Hepatozoon sp. SK1 and Hepatozoon sp. SK2, showing 99-100 % identity with isolates from M. glareolus from other European countries. Phylogenetic analysis demonstrates that 18S rRNA variants SK1 and SK2 correspond to previously described genotypes UR1 and UR2 of H. erhardovae, respectively. The isolate from Apodemus flavicollis (Hepatozoon sp. SK3b) was 99 % identical with isolates from reptiles in Africa and Asia. Further studies are necessary to identify the taxonomic status of Hepatozoon spp. parasitizing rodents in Europe and the host-parasite interactions in natural foci.

Archaeal and bacterial communities respond differently to environmental gradients in anoxic sediments of a California hypersaline lake, the Salton Sea.

PubMed

Swan, Brandon K; Ehrhardt, Christopher J; Reifel, Kristen M; Moreno, Lilliana I; Valentine, David L

2010-02-01

Sulfidic, anoxic sediments of the moderately hypersaline Salton Sea contain gradients in salinity and carbon that potentially structure the sedimentary microbial community. We investigated the abundance, community structure, and diversity of Bacteria and Archaea along these gradients to further distinguish the ecologies of these domains outside their established physiological range. Quantitative PCR was used to enumerate 16S rRNA gene abundances of Bacteria, Archaea, and Crenarchaeota. Community structure and diversity were evaluated by terminal restriction fragment length polymorphism (T-RFLP), quantitative analysis of gene (16S rRNA) frequencies of dominant microorganisms, and cloning and sequencing of 16S rRNA. Archaea were numerically dominant at all depths and exhibited a lesser response to environmental gradients than that of Bacteria. The relative abundance of Crenarchaeota was low (0.4 to 22%) at all depths but increased with decreased carbon content and increased salinity. Salinity structured the bacterial community but exerted no significant control on archaeal community structure, which was weakly correlated with total carbon. Partial sequencing of archaeal 16S rRNA genes retrieved from three sediment depths revealed diverse communities of Euryarchaeota and Crenarchaeota, many of which were affiliated with groups previously described from marine sediments. The abundance of these groups across all depths suggests that many putative marine archaeal groups can tolerate elevated salinity (5.0 to 11.8% [wt/vol]) and persist under the anaerobic conditions present in Salton Sea sediments. The differential response of archaeal and bacterial communities to salinity and carbon patterns is consistent with the hypothesis that adaptations to energy stress and availability distinguish the ecologies of these domains.

Time-course monitoring of urban bioaerosol bacterial communities and its use in microbial hazard identification during Asian Dust events in Seoul, Korea

NASA Astrophysics Data System (ADS)

Park, J.

2015-12-01

The microbial communities transported by Asian dust events have attracted much attention as bioaerosols because the transported airborne microbes may strongly influence the downwind ecosystems and potentially human health in East Asia. Bioaerosol study has received relatively little attention and their characterization and risk assessments remain poorly developed. We used high throughput 16S rRNA gene targeted pyrosequencing and real-time quantitative PCR (qPCR) to monitor airborne bacterial communities and assess their potential risk. We monitored microbial communities in bioaerosol in Seoul between 2011 and 2013 using high volume air samplers. Six samples were collected during Asian dust (AD) events and the other 34 samples were urban air collected during non-Asian dust (non-AD) events. According to the qPCR result, the gene copy numbers of 16S rRNA genes were significantly higher during the AD events (P < 0.05) and their abundances were positively correlated with PM10 concentrations and bacterial diversities. The most abundant bacterial members (genus level) in the AD samples were Bacillus, Neisseria and E.coli/Shigella. To identify pathogenic populations, multilocus sequence typing (MLST) and virulence tests were applied using culture methods. 16S rRNA gene sequences of several pathogens were detected and their relative abundances appeared to have increased with increased concentrations of PM10. About 1% of Bacillus isolates were identified as known pathogenic B. cereus, confirming their presence in Asian dust samples. The qPCR detection of bceT gene, which codes for an enterotoxin in B. cereus group, was significantly increased in the AD dust samples over the non-AD samples. The following MLST assessment and virulence test of cultivated Bacillus isolates showed that B. cereus, B. licheniformis and B. mycoides were identified as pathogenic bacteria, and these pathogenic bacteria were usually more abundant during AD events. To assess the possible associations of identified pathogens on the hospital stroke admissions of residents in Seoul, we identified sixteen bioaerosol episodes using Poisson regression and calculated relative risk. The findings are useful in building a database for bacterial pathogens in AD events.

[Screening and identification of an endophytic bacterium with 1-aminocyclopropane-1-carboxylate deaminase activity from Panax ginseng and its effect on host growth].

PubMed

Tian, Lei; Jiang, Yun; Chen, Changqing; Zhang, Guanjun; Li, Tong; Tong, Bin; Xu, Peng

2014-07-04

This study aimed to screen endophytic bacteria with 1-aminocyclopropane-1-carboxylate deaminase activity from Panax ginseng and test the capability of growth promotion to its host. In total 120 endophytic bacterial strains isolated from Panax ginseng were screened for 1-aminocyclopropane-1-carboxylate deaminase activity using the qualitative and quantitative methods. The obtained strain was also tested for its ability of nitrogen fixation using the Ashby agar plates and the gene of nifH, for its ability of phosphate solubilization using the Pikovaskaia's plates and quantitative analysis of Mo-Sb-Ascrobiology acid colorimetry, for its ability of producing siderophores using the method of Chrome azurol S detecting, and its effect on promoting growth of Panax ginseng by laboratory and field experiments. The bacterial strain with ACC deaminase was identified based on morphology, physiological and biochemical traits, and 16S rRNA sequence analysis. The bacterial stain JJ8-3 with the ability of producing ACC deaminase activity was obtained through screening, which its ACC deaminase activity was alpha-ketobutyric acid 6.7 micromol/(mg x h). Strain JJ8-3 had other traits of phosphate solubilizing, nitrogen fixation, producing siderophores, and the ability of promoting growth of Panax ginseng. Strain JJ8-3 was identified as Pseudomonas fluorescens. Strain JJ8-3 of endophytic bacterium with ACC deaminase activity from Panax ginseng was obtained and would lay the foundation for its further study and application on plant growth promotion.

Comparison of Sanger and next generation sequencing performance for genotyping Cryptosporidium isolates at the 18S rRNA and actin loci.

PubMed

Paparini, Andrea; Gofton, Alexander; Yang, Rongchang; White, Nicole; Bunce, Michael; Ryan, Una M

2015-01-01

Cryptosporidium is an important enteric pathogen that infects a wide range of humans and animals. Rapid and reliable detection and characterisation methods are essential for understanding the transmission dynamics of the parasite. Sanger sequencing, and high-throughput sequencing (HTS) on an Ion Torrent platform, were compared with each other for their sensitivity and accuracy in detecting and characterising 25 Cryptosporidium-positive human and animal faecal samples. Ion Torrent reads (n = 123,857) were obtained at both 18S rRNA and actin loci for 21 of the 25 samples. Of these, one isolate at the actin locus (Cattle 05) and three at the 18S rRNA locus (HTS 10, HTS 11 and HTS 12), suffered PCR drop-out (i.e. PCR failures) when using fusion-tagged PCR. Sanger sequences were obtained for both loci for 23 of the 25 samples and showed good agreement with Ion Torrent-based genotyping. Two samples both from pythons (SK 02 and SK 05) produced mixed 18S and actin chromatograms by Sanger sequencing but were clearly identified by Ion Torrent sequencing as C. muris. One isolate (SK 03) was typed as C. muris by Sanger sequencing but was identified as a mixed C. muris and C. tyzzeri infection by HTS. 18S rRNA Type B sequences were identified in 4/6 C. parvum isolates when deep sequenced but were undetected in Sanger sequencing. Sanger was cheaper than Ion Torrent when sequencing a small numbers of samples, but when larger numbers of samples are considered (n = 60), the costs were comparative. Fusion-tagged amplicon based approaches are a powerful way of approaching mixtures, the only draw-back being the loss of PCR efficiency on low-template samples when using primers coupled to MID tags and adaptors. Taken together these data show that HTS has excellent potential for revealing the "true" composition of species/types in a Cryptosporidium infection, but that HTS workflows need to be carefully developed to ensure sensitivity, accuracy and contamination are controlled. Copyright © 2015 Elsevier Inc. All rights reserved.

Rapid detection of Mannheimia haemolytica in lung tissues of sheep and from bacterial culture.

PubMed

Kumar, Jyoti; Dixit, Shivendra Kumar; Kumar, Rajiv

2015-09-01

This study was aimed to detect Mannheimia haemolytica in lung tissues of sheep and from a bacterial culture. M. haemolytica is one of the most important and well-established etiological agents of pneumonia in sheep and other ruminants throughout the world. Accurate diagnosis of M. haemolytica primarily relies on bacteriological examination, biochemical characteristics and, biotyping and serotyping of the isolates. In an effort to facilitate rapid M. haemolytica detection, polymerase chain reaction assay targeting Pasteurella haemolytica serotype-1 specific antigens (PHSSA), Rpt2 and 12S ribosomal RNA (rRNA) genes were used to detect M. haemolytica directly from lung tissues and from bacterial culture. A total of 12 archived lung tissues from sheep that died of pneumonia on an organized farm were used. A multiplex polymerase chain reaction (mPCR) based on two-amplicons targeted PHSSA and Rpt2 genes of M. haemolytica were used for identification of M. haemolytica isolates in culture from the lung samples. All the 12 lung tissue samples were tested for the presence M. haemolytica by PHSSA and Rpt2 genes based PCR and its confirmation by sequencing of the amplicons. All the 12 lung tissue samples tested for the presence of PHSSA and Rpt2 genes of M. haemolytica by mPCR were found to be positive. Amplification of 12S rRNA gene fragment as internal amplification control was obtained with each mPCR reaction performed from DNA extracted directly from lung tissue samples. All the M. haemolytica were also positive for mPCR. No amplified DNA bands were observed for negative control reactions. All the three nucleotide sequences were deposited in NCBI GenBank (Accession No. KJ534629, KJ534630 and KJ534631). Sequencing of the amplified products revealed the identity of 99-100%, with published sequence of PHSSA and Rpt2 genes of M. haemolytica available in the NCBI database. Sheep specific mitochondrial 12S rRNA gene sequence also revealed the identity of 98% with published sequences in the NCBI database. The present study emphasized the PCR as a valuable tool for rapid detection of M. haemolytica in clinical samples from animals. In addition, it offers the opportunity to perform large-scale epidemiological studies regarding the role of M. haemolytica in clinical cases of pneumonia and other disease manifestations in sheep and other ruminants, thereby providing the basis for effective preventive strategies.

Comparing culture and molecular methods for the identification of microorganisms involved in necrotizing soft tissue infections.

PubMed

Rudkjøbing, Vibeke Børsholt; Thomsen, Trine Rolighed; Xu, Yijuan; Melton-Kreft, Rachael; Ahmed, Azad; Eickhardt, Steffen; Bjarnsholt, Thomas; Poulsen, Steen Seier; Nielsen, Per Halkjær; Earl, Joshua P; Ehrlich, Garth D; Moser, Claus

2016-11-08

Necrotizing soft tissue infections (NSTIs) are a group of infections affecting all soft tissues. NSTI involves necrosis of the afflicted tissue and is potentially life threatening due to major and rapid destruction of tissue, which often leads to septic shock and organ failure. The gold standard for identification of pathogens is culture; however molecular methods for identification of microorganisms may provide a more rapid result and may be able to identify additional microorganisms that are not detected by culture. In this study, tissue samples (n = 20) obtained after debridement of 10 patients with NSTI were analyzed by standard culture, fluorescence in situ hybridization (FISH) and multiple molecular methods. The molecular methods included analysis of microbial diversity by 1) direct 16S and D2LSU rRNA gene Microseq 2) construction of near full-length 16S rRNA gene clone libraries with subsequent Sanger sequencing for most samples, 3) the Ibis T5000 biosensor and 4) 454-based pyrosequencing. Furthermore, quantitative PCR (qPCR) was used to verify and determine the relative abundance of Streptococcus pyogenes in samples. For 70 % of the surgical samples it was possible to identify microorganisms by culture. Some samples did not result in growth (presumably due to administration of antimicrobial therapy prior to sampling). The molecular methods identified microorganisms in 90 % of the samples, and frequently detected additional microorganisms when compared to culture. Although the molecular methods generally gave concordant results, our results indicate that Microseq may misidentify or overlook microorganisms that can be detected by other molecular methods. Half of the patients were found to be infected with S. pyogenes, but several atypical findings were also made including infection by a) Acinetobacter baumannii, b) Streptococcus pneumoniae, and c) fungi, mycoplasma and Fusobacterium necrophorum. The study emphasizes that many pathogens can be involved in NSTIs, and that no specific "NSTI causing" combination of species exists. This means that clinicians should be prepared to diagnose and treat any combination of microbial pathogens. Some of the tested molecular methods offer a faster turnaround time combined with a high specificity, which makes supplemental use of such methods attractive for identification of microorganisms, especially for fulminant life-threatening infections such as NSTI.

[Automated RNA amplification for the rapid identification of Mycobacterium tuberculosis complex in respiratory specimens].

PubMed

Drouillon, V; Houriez, F; Buze, M; Lagrange, P; Herrmann, J-L

2006-01-01

Rapid and sensitive detection of Mycobacterium tuberculosis complex (MTB) directly on clinical respiratory specimens is essential for a correct management of patients suspected of tuberculosis. For this purpose PCR-based kits are available to detect MTB in respiratory specimen but most of them need at least 4 hours to be completed. New methods, based on TRC method (TRC: Transcription Reverse transcription Concerted--TRCRapid M. Tuberculosis--Tosoh Bioscience, Tokyo, Japon) and dedicated monitor have been developed. A new kit (TRC Rapid M. tuberculosis and Real-time monitor TRCRapid-160, Tosoh Corporation, Japan) enabling one step amplification and real-time detection of MTB 16S rRNA by a combination of intercalative dye oxazole yellow-linked DNA probe and isothermal RNA amplification directly on respiratory specimens has been tested in our laboratory. 319 respiratory specimens were tested in this preliminary study and results were compared to smear and culture. Fourteen had a positive culture for MTB. Among theses samples, smear was positive in 11 cases (78.6%) and TRC process was positive in 8 cases (57.1%). Overall sensitivity of TRC compared to smear positive samples is 73%. Theses first results demonstrated that a rapid identification of MTB was possible (less than 2 processing hours for 14 specimens and about 1 hour for 1 specimen) in most cases of smear positive samples using ready to use reagents for real time detection of MTB rRNA in clinical samples. New pretreatment and extraction reagents kits to increase the stability of the sputum RNA and the extraction efficiency are now tested in our laboratory.

Electrophoretically deposited multiwalled carbon nanotube based amperometric genosensor for E.coli detection

NASA Astrophysics Data System (ADS)

Bhardwaj, Hema; Solanki, Shipra; Sumana, Gajjala

2016-04-01

This work reports on a sensitive and selective genosensor fabrication method for Escherichia coli (E.coli) detection. The functionalized multiwalled carbon nanotubes (MWCNT) synthesized via chemical vapour deposition have been deposited electrophoretically onto indium tin oxide coated glass surface and have been utilized as matrices for the covalent immobilization of E.coli specific probe oligonucleotide that was identified from the 16s rRNA coding region of the E.coli genome. This fabricated functionalized MWCNT based platform sought to provide improved fundamental characteristics to electrode interface in terms of electro-active surface area and diffusion coefficient. Electrochemical cyclic voltammetry revealed that this genosensor exhibits a linear response to complementary DNA in the concentration range of 10-7 to 10-12 M with a detection limit of 1×10-12 M.

Estimation of total bacteria by real-time PCR in patients with periodontal disease.

PubMed

Brajović, Gavrilo; Popović, Branka; Puletić, Miljan; Kostić, Marija; Milasin, Jelena

2016-01-01

Periodontal diseases are associated with the presence of elevated levels of bacteria within the gingival crevice. The aim of this study was to evaluate a total amount of bacteria in subgingival plaque samples in patients with a periodontal disease. A quantitative evaluation of total bacteria amount using quantitative real-time polymerase chain reaction (qRT-PCR) was performed on 20 samples of patients with ulceronecrotic periodontitis and on 10 samples of healthy subjects. The estimation of total bacterial amount was based on gene copy number for 16S rRNA that was determined by comparing to Ct values/gene copy number of the standard curve. A statistically significant difference between average gene copy number of total bacteria in periodontal patients (2.55 x 10⁷) and healthy control (2.37 x 10⁶) was found (p = 0.01). Also, a trend of higher numbers of the gene copy in deeper periodontal lesions (> 7 mm) was confirmed by a positive value of coefficient of correlation (r = 0.073). The quantitative estimation of total bacteria based on gene copy number could be an important additional tool in diagnosing periodontitis.

Molecular prevalence and characterization of Hepatozoon ursi infection in Indian sloth bears (Melursus ursinus).

PubMed

Pawar, Rahul Mohanchandra; Poornachandar, Anantula; Arun, Attur Shanmugam; Manikandan, Santhanam; Shivaji, Sisinthy

2011-12-15

Hepatozoon species are parasites that infect a wide variety of domestic and wild animals. The objective of the study was to detect the occurrence of Hepatozoon ursi in Indian sloth bears and to characterize the parasite based on phylogenetic analysis of the partial 18S rRNA gene sequence. Hepatozoon infection could be detected in 38 (70%) out of fifty-four blood samples of Indian sloth bears (captive and wild), suggestive of high prevalence of Hepatozoon infection in Indian sloth bears. Sequencing of partial 18S rRNA gene of the positive samples and BLAST analysis indicated that the nearest phylogenetic neighbour was H. ursi with which they exhibited 99-100% similarity. Additionally, Hepatozoon sp. isolated from wild sloth bears of India were identical to those in captive sloth bears and phylogenetically related to H. ursi reported from Japanese black bears from Japan. To our knowledge, this is the first report on the molecular characterization of H. ursi infection in Indian sloth bears. Copyright © 2011 Elsevier B.V. All rights reserved.

Exploiting 16S rRNA gene for the detection and quantification of fish as a potential allergenic food: A comparison of two real-time PCR approaches.

PubMed

Fernandes, Telmo J R; Costa, Joana; Oliveira, M Beatriz P P; Mafra, Isabel

2018-04-15

Fish is one of the most common allergenic foods that should be accurately labelled to protect the health of allergic consumers. In this work, two real-time PCR systems based on the EvaGreen dye and a TaqMan probe are proposed and compared. New primers were designed to target the 16S rRNA gene, as a universal maker for fish detection, with fully demonstrated specificity for a wide range of fish species. Both systems showed similar absolute sensitivities, down to 0.01 pg of fish DNA, and adequate real-time PCR performance parameters. The probe system showed higher relative sensitivity and dynamic range (0.0001-50%) than the EvaGreen (0.05-50%). They were both precise, but trueness was compromised at the highest tested level with the EvaGreen assay. Therefore, both systems were successful, although the probe one exhibited the best performance. Its application to verify labelling compliance of foodstuffs suggested a high level of mislabelling and/or fraudulent practices. Copyright © 2017 Elsevier Ltd. All rights reserved.

A Label-Free, Quantitative Fecal Hemoglobin Detection Platform for Colorectal Cancer Screening

PubMed Central

Soraya, Gita V.; Nguyen, Thanh C.; Abeyrathne, Chathurika D.; Huynh, Duc H.; Chan, Jianxiong; Nguyen, Phuong D.; Nasr, Babak; Chana, Gursharan; Kwan, Patrick; Skafidas, Efstratios

2017-01-01

The early detection of colorectal cancer is vital for disease management and patient survival. Fecal hemoglobin detection is a widely-adopted method for screening and early diagnosis. Fecal Immunochemical Test (FIT) is favored over the older generation chemical based Fecal Occult Blood Test (FOBT) as it does not require dietary or drug restrictions, and is specific to human blood from the lower digestive tract. To date, no quantitative FIT platforms are available for use in the point-of-care setting. Here, we report proof of principle data of a novel low cost quantitative fecal immunochemical-based biosensor platform that may be further developed into a point-of-care test in low-resource settings. The label-free prototype has a lower limit of detection (LOD) of 10 µg hemoglobin per gram (Hb/g) of feces, comparable to that of conventional laboratory based quantitative FIT diagnostic systems. PMID:28475117

Evaluation of two main RNA-seq approaches for gene quantification in clinical RNA sequencing: polyA+ selection versus rRNA depletion.

PubMed

Zhao, Shanrong; Zhang, Ying; Gamini, Ramya; Zhang, Baohong; von Schack, David

2018-03-19

To allow efficient transcript/gene detection, highly abundant ribosomal RNAs (rRNA) are generally removed from total RNA either by positive polyA+ selection or by rRNA depletion (negative selection) before sequencing. Comparisons between the two methods have been carried out by various groups, but the assessments have relied largely on non-clinical samples. In this study, we evaluated these two RNA sequencing approaches using human blood and colon tissue samples. Our analyses showed that rRNA depletion captured more unique transcriptome features, whereas polyA+ selection outperformed rRNA depletion with higher exonic coverage and better accuracy of gene quantification. For blood- and colon-derived RNAs, we found that 220% and 50% more reads, respectively, would have to be sequenced to achieve the same level of exonic coverage in the rRNA depletion method compared with the polyA+ selection method. Therefore, in most cases we strongly recommend polyA+ selection over rRNA depletion for gene quantification in clinical RNA sequencing. Our evaluation revealed that a small number of lncRNAs and small RNAs made up a large fraction of the reads in the rRNA depletion RNA sequencing data. Thus, we recommend that these RNAs are specifically depleted to improve the sequencing depth of the remaining RNAs.

Transcriptional analysis of nucleolar dominance in polyploid plants: Biased expression/silencing of progenitor rRNA genes is developmentally regulated in Brassica

PubMed Central

Chen, Z. Jeffrey; Pikaard, Craig S.

1997-01-01

Nucleolar dominance is an epigenetic phenomenon that describes the formation of nucleoli around rRNA genes inherited from only one parent in the progeny of an interspecific hybrid. Despite numerous cytogenetic studies, little is known about nucleolar dominance at the level of rRNA gene expression in plants. We used S1 nuclease protection and primer extension assays to define nucleolar dominance at a molecular level in the plant genus Brassica. rRNA transcription start sites were mapped in three diploids and in three allotetraploids (amphidiploids) and one allohexaploid species derived from these diploid progenitors. rRNA transcripts of only one progenitor were detected in vegetative tissues of each polyploid. Dominance was independent of maternal effect, ploidy, or rRNA gene dosage. Natural and newly synthesized amphidiploids yielded the same results, arguing against substantial evolutionary effects. The hypothesis that nucleolar dominance in plants is correlated with physical characteristics of rRNA gene intergenic spacers is not supported in Brassica. Furthermore, in Brassica napus, rRNA genes silenced in vegetative tissues were found to be expressed in all floral organs, including sepals and petals, arguing against the hypothesis that passage through meiosis is needed to reactivate suppressed genes. Instead, the transition of inflorescence to floral meristem appears to be a developmental stage when silenced genes can be derepressed. PMID:9096413

Toxic Cyanobacterial Bloom Triggers in Missisquoi Bay, Lake Champlain, as Determined by Next-Generation Sequencing and Quantitative PCR

PubMed Central

Fortin, Nathalie; Munoz-Ramos, Valentina; Bird, David; Lévesque, Benoît; Whyte, Lyle G.; Greer, Charles W.

2015-01-01

Missisquoi Bay (MB) is a temperate eutrophic freshwater lake that frequently experiences toxic Microcystis-dominated cyanobacterial blooms. Non-point sources are responsible for the high concentrations of phosphorus and nitrogen in the bay. This study combined data from environmental parameters, E. coli counts, high-throughput sequencing of 16S rRNA gene amplicons, quantitative PCR (16S rRNA and mcyD genes) and toxin analyses to identify the main bloom-promoting factors. In 2009, nutrient concentrations correlated with E. coli counts, abundance of total cyanobacterial cells, Microcystis 16S rRNA and mcyD genes and intracellular microcystin. Total and dissolved phosphorus also correlated significantly with rainfall. The major cyanobacterial taxa were members of the orders Chroococcales and Nostocales. The genus Microcystis was the main mcyD-carrier and main microcystin producer. Our results suggested that increasing nutrient concentrations and total nitrogen:total phosphorus (TN:TP) ratios approaching 11:1, coupled with an increase in temperature, promoted Microcystis-dominated toxic blooms. Although the importance of nutrient ratios and absolute concentrations on cyanobacterial and Microcystis dynamics have been documented in other laboratories, an optimum TN:TP ratio for Microcystis dominance has not been previously observed in situ. This observation provides further support that nutrient ratios are an important determinant of species composition in natural phytoplankton assemblages. PMID:25984732

Leuconostoc pseudomesenteroides WCFur3 partial 16S rRNA gene

USDA-ARS?s Scientific Manuscript database

This study used a partial 535 base pair 16S rRNA gene sequence to identify a bacterial isolate. Fatty acid profiles are consistent with the 16S rRNA gene sequence identification of this bacterium. The isolate was obtained from a compost bin in Fort Collins, Colorado, USA. The 16S rRNA gene sequen...

Development of duplex SYBR Green I-based real-time quantitative reverse-transcription PCR for detection and discrimination of grapevine viruses

USDA-ARS?s Scientific Manuscript database

A SYBR® Green-based real-time quantitative reverse transcription PCR (qRT-PCR) assay in combination with melt curve analysis (MCA) was developed for the detection of nine grapevine viruses. The detection limits for singleplex qRT-PCR for all nine grapevine viruses were determined to be in the range ...

Multiplex PCR for the Detection of Lactobacillus pontis and Two Related Species in a Sourdough Fermentation

PubMed Central

Müller, Martin R. A.; Ehrmann, Matthias A.; Vogel, Rudi F.

2000-01-01

A specific multiplex PCR assay based on the amplification of parts of the 16S rRNA molecule was designed. Primers derived from variable regions of the 16S rRNA provided a means of easily differentiating the species Lactobacillus pontis and Lactobacillus panis. They could be clearly discriminated from the phylogenetically related species Lactobacillus vaginalis, Lactobacillus oris, and Lactobacillus reuteri and from other lactobacilli commonly known to be present in sourdough. Other strains isolated together with L. pontis from an industrial sourdough fermentation could be clearly separated from these species by comparative sequence analysis and construction of a specific PCR primer. For a fast identification a DNA isolation protocol based on the ultrasonic lysis of cells from single colonies was developed. To demonstrate the potential of such techniques for tracking these organisms in a laboratory-scale fermentation, we combined the specific PCR assay with direct DNA extraction from the organisms in the sourdough without previous cultivation. PMID:10788389

Molecular Quantification of Zooplankton Gut Content: The Case For qPCR

NASA Astrophysics Data System (ADS)

Frischer, M. E.; Walters, T. L.; Gibson, D. M.; Nejstgaard, J. C.; Troedsson, C.

2016-02-01

The ability to obtain information about feeding selectivity and rates in situ for zooplankton is vital for understanding the mechanisms structuring marine ecosystems. However, directly estimating feeding selection and rates of zooplankton, without bias, associated with culturing conditions has been notoriously difficult. A potential approach for addressing this problem is to target prey-specific DNA as a marker for prey ingestion and selection. In this study we report the development of a differential length amplification quantitative PCR (dla-qPCR) assay targeting the 18S rRNA gene to validate the use of a DNA-based approach to quantify consumption of specific plankton prey by the pelagic tunicate (doliolid) Dolioletta gegenbauri. Compared to copepods and other marine animals, the digestion of prey genomic DNA inside the gut of doliolids is low. This method minimizes potential underestimations, and therefore allows prey DNA to be used as an effective indicator of prey consumption. We also present an initial application of a qPCR-assay to estimate consumption of specific prey species on the southeastern continental shelf of the U.S., where doliolids stochastically bloom in response to upwelling events. Estimated feeding rates, based on qPCR, were in the same range as those estimated from clearance rates in laboratory feeding studies. In the field, consumption of specific prey, including the centric diatom Thalassiosira spp. was detected in the gut of wild caught D. gegenbauri at the levels consistent with their abundance in the water column at the time of collection. Thus, both experimental and field investigations support the hypothesis that a qPCR approach will be useful for the quantitative investigation of the in situ diet of D. gegenbauri without introduced bias' associated with cultivation.

Cross-reactions in specific Brachyspira spp. PCR assays caused by "Brachyspira hampsonii" isolates: implications for detection.

PubMed

Aller-Morán, Luis M; Martínez-Lobo, F Javier; Rubio, Pedro; Carvajal, Ana

2016-11-01

An emerging novel spirochete in swine, provisionally designated "Brachyspira hampsonii," has been detected worldwide. It has been associated with swine dysentery and cannot be differentiated from B. hyodysenteriae, the classical etiologic agent of this disease, using standard phenotypic methods. We evaluated cross-reactions of "B. hampsonii" isolates recovered from avian species in some of the currently available species-specific polymerase chain reaction (PCR) assays for the identification of swine Brachyspira species. Ten avian "B. hampsonii" isolates recovered from wild waterfowl were used. No false-positive results were recorded with a B. pilosicoli-specific PCR based on the amplification of a fragment of the 16S rRNA gene. However, the percentage of false-positive results varied, with a range of 10-80%, in the evaluated B. hyodysenteriae-specific assays based on the amplification of the 23S rRNA, nox, and tlyA genes. Similarly, results of the B. intermedia-specific PCR assays yielded poor specificity, with up to 80% of the "B. hampsonii" isolates tested giving false-positive results. Finally, 2 "B. hampsonii" avian isolates yielded a positive result in a B. innocens- and B. murdochii-specific PCR. This result should be interpreted very cautiously as these 2 isolates could represent a recombinant genotype. © 2016 The Author(s).

Sequence heterogeneities of genes encoding 16S rRNAs in Paenibacillus polymyxa detected by temperature gradient gel electrophoresis.

PubMed Central

Nübel, U; Engelen, B; Felske, A; Snaidr, J; Wieshuber, A; Amann, R I; Ludwig, W; Backhaus, H

1996-01-01

Sequence heterogeneities in 16S rRNA genes from individual strains of Paenibacillus polymyxa were detected by sequence-dependent separation of PCR products by temperature gradient gel electrophoresis (TGGE). A fragment of the 16S rRNA genes, comprising variable regions V6 to V8, was used as a target sequence for amplifications. PCR products from P. polymyxa (type strain) emerged as a well-defined pattern of bands in the gradient gel. Six plasmids with different inserts, individually demonstrating the migration characteristics of single bands of the pattern, were obtained by cloning the PCR products. Their sequences were analyzed as a representative sample of the total heterogeneity. An amount of 10 variant nucleotide positions in the fragment of 347 bp was observed, with all substitutions conserving the relevant secondary structures of the V6 and V8 regions in the RNA molecules. Hybridizations with specifically designed probes demonstrated different chromosomal locations of the respective rRNA genes. Amplifications of reverse-transcribed rRNA from ribosome preparations, as well as whole-cell hybridizations, revealed a predominant representation of particular sequences in ribosomes of exponentially growing laboratory cultures. Different strains of P. polymyxa showed not only remarkably differing patterns of PCR products in TGGE analysis but also discriminative whole-cell labeling with the designed oligonucleotide probes, indicating the different representation of individual sequences in active ribosomes. Our results demonstrate the usefulness of TGGE for the structural analysis of heterogeneous rRNA genes together with their expression, stress problems of the generation of meaningful data for 16S rRNA sequences and probe designs, and might have consequences for evolutionary concepts. PMID:8824607

Comparison of culture-based, vital stain and PMA-qPCR methods for the quantitative detection of viable hookworm ova.

PubMed

Gyawali, P; Sidhu, J P S; Ahmed, W; Jagals, P; Toze, S

2017-06-01

Accurate quantitative measurement of viable hookworm ova from environmental samples is the key to controlling hookworm re-infections in the endemic regions. In this study, the accuracy of three quantitative detection methods [culture-based, vital stain and propidium monoazide-quantitative polymerase chain reaction (PMA-qPCR)] was evaluated by enumerating 1,000 ± 50 Ancylostoma caninum ova in the laboratory. The culture-based method was able to quantify an average of 397 ± 59 viable hookworm ova. Similarly, vital stain and PMA-qPCR methods quantified 644 ± 87 and 587 ± 91 viable ova, respectively. The numbers of viable ova estimated by the culture-based method were significantly (P < 0.05) lower than vital stain and PMA-qPCR methods. Therefore, both PMA-qPCR and vital stain methods appear to be suitable for the quantitative detection of viable hookworm ova. However, PMA-qPCR would be preferable over the vital stain method in scenarios where ova speciation is needed.

Novel TaqMan real-time polymerase chain reaction assay for verifying the authenticity of meat and commercial meat products from game birds.

PubMed

Rojas, María; González, Isabel; Pavón, Miguel Angel; Pegels, Nicolette; Lago, Adriana; Hernández, Pablo E; García, Teresa; Martín, Rosario

2010-06-01

Species-specific real-time polymerase chain reaction (PCR) assays using TaqMan probes have been developed for verifying the labeling of meat and commercial meat products from game birds, including quail, pheasant, partridge, guinea fowl, pigeon, Eurasian woodcock and song thrush. The method combines the use of species-specific primers and TaqMan probes that amplify small fragments (amplicons <150 base pairs) of the mitochondrial 12S rRNA gene, and an endogenous control primer pair that amplifies a 141-bp fragment of the nuclear 18S rRNA gene from eukaryotic DNA. Analysis of experimental raw and heat-treated binary mixtures as well as of commercial meat products from the target species demonstrated the suitability of the assay for the detection of the target DNAs.

Evidence for a conserved microbiota across the different developmental stages of Plodia interpunctella.

PubMed

Mereghetti, Valeria; Chouaia, Bessem; Limonta, Lidia; Locatelli, Daria Patrizia; Montagna, Matteo

2017-11-01

Diversity and composition of lepidopteran microbiotas are poorly investigated, especially across the different developmental stages. To improve this knowledge, we characterize the microbiota among different developmental stages of the Indian meal moth, Plodia interpunctella, which is considered one of the major pest of commodities worldwide. Using culture-independent approach based on Illumina 16S rRNA gene sequencing we characterized the microbiota of four developmental stages: eggs, first-, and last-instar larvae, and adult. A total of 1022 bacterial OTUs were obtained, showing a quite diversified microbiota associated to all the analyzed stages. The microbiotas associated with P. interpunctella resulted almost constant throughout the developmental stages, with approximately 77% of bacterial OTUs belonging to the phylum of Proteobacteria. The dominant bacterial genus is represented by Burkholderia (∼64%), followed by Propionibacterium, Delftia, Pseudomonas, and Stenotrophomonas. A core bacterial community, composed of 139 OTUs, was detected in all the developmental stages, among which 112 OTUs were assigned to the genus Burkholderia. A phylogenetic reconstruction, based on the 16S rRNA, revealed that our Burkholderia OTUs clustered with Burkholderia cepacia complex, in the same group of those isolated from the hemipterans Gossyparia spuria and Acanthococcus aceris. The functional profiling, predicted on the base of the bacterial 16S rRNA, indicates differences in the metabolic pathways related to metabolism of amino acids between preimaginal and adult stages. We can hypothesize that bacteria may support the insect host during preimaginal stages. © 2017 Institute of Zoology, Chinese Academy of Sciences.

Quantitative Detection of Cracks in Steel Using Eddy Current Pulsed Thermography.

PubMed

Shi, Zhanqun; Xu, Xiaoyu; Ma, Jiaojiao; Zhen, Dong; Zhang, Hao

2018-04-02

Small cracks are common defects in steel and often lead to catastrophic accidents in industrial applications. Various nondestructive testing methods have been investigated for crack detection; however, most current methods focus on qualitative crack identification and image processing. In this study, eddy current pulsed thermography (ECPT) was applied for quantitative crack detection based on derivative analysis of temperature variation. The effects of the incentive parameters on the temperature variation were analyzed in the simulation study. The crack profile and position are identified in the thermal image based on the Canny edge detection algorithm. Then, one or more trajectories are determined through the crack profile in order to determine the crack boundary through its temperature distribution. The slope curve along the trajectory is obtained. Finally, quantitative analysis of the crack sizes was performed by analyzing the features of the slope curves. The experimental verification showed that the crack sizes could be quantitatively detected with errors of less than 1%. Therefore, the proposed ECPT method was demonstrated to be a feasible and effective nondestructive approach for quantitative crack detection.

Gold Nanoparticle Labeling Based ICP-MS Detection/Measurement of Bacteria, and Their Quantitative Photothermal Destruction

PubMed Central

Lin, Yunfeng

2015-01-01

Bacteria such as Salmonella and E. coli present a great challenge in public health care in today’s society. Protection of public safety against bacterial contamination and rapid diagnosis of infection require simple and fast assays for the detection and elimination of bacterial pathogens. After utilizing Salmonella DT104 as an example bacterial strain for our investigation, we report a rapid and sensitive assay for the qualitative and quantitative detection of bacteria by using antibody affinity binding, popcorn shaped gold nanoparticle (GNPOPs) labeling, surfance enchanced Raman spectroscopy (SERS), and inductively coupled plasma mass spectrometry (ICP-MS) detection. For qualitative analysis, our assay can detect Salmonella within 10 min by Raman spectroscopy; for quantitative analysis, our assay has the ability to measure as few as 100 Salmonella DT104 in a 1 mL sample (100 CFU/mL) within 40 min. Based on the quantitative detection, we investigated the quantitative destruction of Salmonella DT104, and the assay’s photothermal efficiency in order to reduce the amount of GNPOPs in the assay to ultimately to eliminate any potential side effects/toxicity to the surrounding cells in vivo. Results suggest that our assay may serve as a promising candidate for qualitative and quantitative detection and elimination of a variety of bacterial pathogens. PMID:26417447

A novel RT-PCR for the detection of Helicobacter pylori and identification of clarithromycin resistance mediated by mutations in the 23S rRNA gene.

PubMed

Redondo, Javier Jareño; Keller, Peter M; Zbinden, Reinhard; Wagner, Karoline

2018-01-01

In this study we evaluated the commercially available LightMix® RT-PCR assay for Helicobacter pylori detection and identification of clarithromycin (CLR) resistance in culture and clinical specimens (gastric biopsies and stool). The H. pylori LightMix® RT-PCR detects a 97bp long fragment of the 23S rRNA gene and allows the identification of 3 distinct point mutations conferring CLR resistance via melting curve analysis. The performance of the H. pylori LightMix® RT-PCR was evaluated using a set of 60 H. pylori strains showing phenotypical CLR susceptibility or CLR resistance (Minimum inhibitory concentrations from 0.016 to 256mg/L). We found high concordance (95%) between phenotypical CLR resistance screening by E-Test® and the Lightmix® RT-PCR. Discrepant results were verified by sequencing of the 23S rRNA gene that always confirmed the results obtained by Lightmix® RT-PCR. Furthermore, H. pylori was detected in clinical biopsy and stool specimens by Lightmix® RT-PCR that identified the correct H. pylori genotype. The LightMix® RT-PCR is an accurate, sensitive and easy to use test for H. pylori and CLR resistance detection and can therefore be readily implemented in any diagnostic laboratory. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

Archaeal community diversity and abundance changes along a natural salinity gradient in estuarine sediments

PubMed Central

Webster, Gordon; O'Sullivan, Louise A.; Meng, Yiyu; Williams, Angharad S.; Sass, Andrea M.; Watkins, Andrew J.; Parkes, R. John; Weightman, Andrew J.

2014-01-01

Archaea are widespread in marine sediments, but their occurrence and relationship with natural salinity gradients in estuarine sediments is not well understood. This study investigated the abundance and diversity of Archaea in sediments at three sites [Brightlingsea (BR), Alresford (AR) and Hythe (HY)] along the Colne Estuary, using quantitative real-time PCR (qPCR) of 16S rRNA genes, DNA hybridization, Archaea 16S rRNA and mcrA gene phylogenetic analyses. Total archaeal 16S rRNA abundance in sediments were higher in the low-salinity brackish sediments from HY (2–8 × 107 16S rRNA gene copies cm−3) than the high-salinity marine sites from BR and AR (2 × 104–2 × 107 and 4 × 106–2 × 107 16S rRNA gene copies cm−3, respectively), although as a proportion of the total prokaryotes Archaea were higher at BR than at AR or HY. Phylogenetic analysis showed that members of the ‘Bathyarchaeota’ (MCG), Thaumarchaeota and methanogenic Euryarchaeota were the dominant groups of Archaea. The composition of Thaumarchaeota varied with salinity, as only ‘marine’ group I.1a was present in marine sediments (BR). Methanogen 16S rRNA genes from low-salinity sediments at HY were dominated by acetotrophic Methanosaeta and putatively hydrogentrophic Methanomicrobiales, whereas the marine site (BR) was dominated by mcrA genes belonging to methylotrophic Methanococcoides, versatile Methanosarcina and methanotrophic ANME-2a. Overall, the results indicate that salinity and associated factors play a role in controlling diversity and distribution of Archaea in estuarine sediments. PMID:25764553

Community succession of bacteria and eukaryotes in dune ecosystems of Gurbantünggüt Desert, Northwest China.

PubMed

Li, Ke; Bai, Zhihui; Zhang, Hongxun

2015-01-01

Pyrosequencing and quantitative polymerase chain reaction of small subunit rRNA genes were used to provide a comprehensive examination of bacterial, cyanobacterial, and eukaryotic communities in the biological soil crusts (BSCs) of Gurbantünggüt Desert sand dunes (China). Three succession stages were recognized based on the analyses of eukaryotic communities: a late succession stage of BSCs in a swale with eukaryotes mainly related to the Bryophyta clade, an initial succession stage in a slope with barely any eukaryotic phototrophic microorganisms detected, and an intermediate succession type detected from both the swale and slope BSCs dominated by the phylum Chlorophyta. Moreover, the cyanobacterial community dominated all of the BSCs (48.2-69.5% of the total bacteria) and differed among the three succession stages: sequences related to Microcoleus steenstrupii and the genus Scytonema were abundant in the later succession stage, whereas both the initial and intermediate stages were dominated by Microcoleus vaginatus. Compared with swales, BSCs from slopes are exposed to a harsher environment, e.g., higher irradiance and lower water availability, and thus may be restricted from developing to a higher succession stage. Other disturbances such as wind and grazing may explain the different succession stages observed in swales or slopes. However, no clear differences were detected from non-phototrophic bacterial communities of the three succession stages, and sequences related to Alphaproteobacteria and Actinobacteria were most abundant in all the BSCs. The closest matches for the most frequent non-phototrophic bacterial genera were mainly derived from harsh environments, indicating the robustness of these genera.

Molecular detection and genetic diversity of Babesia gibsoni in dogs in Bangladesh.

PubMed

Terao, Masashi; Akter, Shirin; Yasin, Md Golam; Nakao, Ryo; Kato, Hirotomo; Alam, Mohammad Zahangir; Katakura, Ken

2015-04-01

Babesia gibsoni is a tick-borne hemoprotozoan parasite of dogs that often causes fever and hemolytic illness. Detection of B. gibsoni has been predominantly reported in Asian countries, including Japan, Korea, Taiwan, Malaysia, Bangladesh and India. The present study shows the first molecular characterization of B. gibsoni detected from dogs in Bangladesh. Blood samples were collected on FTA® Elute cards from 50 stray dogs in Mymensingh District in Bangladesh. DNA eluted from the cards was subjected to nested PCR for the 18S rRNA gene of Babesia species. Approximately 800bp PCR products were detected in 15 of 50 dogs (30%). Based on restriction fragment length polymorphism (RFLP) and direct sequencing of the PCR products, all parasite isolates were identified as B. gibsoni. Furthermore, the BgTRAP (B. gibsoni thrombospondin-related adhesive protein) gene fragments were detected in 13 of 15 18S rRNA gene PCR positive blood samples. Phylogenetic analysis of the BgTRAP gene revealed that B. gibsoni parasites in Bangladesh formed a cluster, which was genetically different from other Asian B. gibsoni isolates. In addition, tandem repeat analysis of the BgTRAP gene clearly showed considerable genetic variation among Bangladeshi isolates. These results suggested that B. gibsoni parasites in a different genetic clade are endemic in dogs in Bangladesh. Further studies are required to elucidate the origin, distribution, vector and pathogenesis of B. gibsoni parasites circulating in dogs in Bangladesh. Copyright © 2015 Elsevier B.V. All rights reserved.

Diversity of thermophiles in a Malaysian hot spring determined using 16S rRNA and shotgun metagenome sequencing.

PubMed

Chan, Chia Sing; Chan, Kok-Gan; Tay, Yea-Ling; Chua, Yi-Heng; Goh, Kian Mau

2015-01-01

The Sungai Klah (SK) hot spring is the second hottest geothermal spring in Malaysia. This hot spring is a shallow, 150-m-long, fast-flowing stream, with temperatures varying from 50 to 110°C and a pH range of 7.0-9.0. Hidden within a wooded area, the SK hot spring is continually fed by plant litter, resulting in a relatively high degree of total organic content (TOC). In this study, a sample taken from the middle of the stream was analyzed at the 16S rRNA V3-V4 region by amplicon metagenome sequencing. Over 35 phyla were detected by analyzing the 16S rRNA data. Firmicutes and Proteobacteria represented approximately 57% of the microbiome. Approximately 70% of the detected thermophiles were strict anaerobes; however, Hydrogenobacter spp., obligate chemolithotrophic thermophiles, represented one of the major taxa. Several thermophilic photosynthetic microorganisms and acidothermophiles were also detected. Most of the phyla identified by 16S rRNA were also found using the shotgun metagenome approaches. The carbon, sulfur, and nitrogen metabolism within the SK hot spring community were evaluated by shotgun metagenome sequencing, and the data revealed diversity in terms of metabolic activity and dynamics. This hot spring has a rich diversified phylogenetic community partly due to its natural environment (plant litter, high TOC, and a shallow stream) and geochemical parameters (broad temperature and pH range). It is speculated that symbiotic relationships occur between the members of the community.

NAIMA: target amplification strategy allowing quantitative on-chip detection of GMOs.

PubMed

Morisset, Dany; Dobnik, David; Hamels, Sandrine; Zel, Jana; Gruden, Kristina

2008-10-01

We have developed a novel multiplex quantitative DNA-based target amplification method suitable for sensitive, specific and quantitative detection on microarray. This new method named NASBA Implemented Microarray Analysis (NAIMA) was applied to GMO detection in food and feed, but its application can be extended to all fields of biology requiring simultaneous detection of low copy number DNA targets. In a first step, the use of tailed primers allows the multiplex synthesis of template DNAs in a primer extension reaction. A second step of the procedure consists of transcription-based amplification using universal primers. The cRNA product is further on directly ligated to fluorescent dyes labelled 3DNA dendrimers allowing signal amplification and hybridized without further purification on an oligonucleotide probe-based microarray for multiplex detection. Two triplex systems have been applied to test maize samples containing several transgenic lines, and NAIMA has shown to be sensitive down to two target copies and to provide quantitative data on the transgenic contents in a range of 0.1-25%. Performances of NAIMA are comparable to singleplex quantitative real-time PCR. In addition, NAIMA amplification is faster since 20 min are sufficient to achieve full amplification.

NAIMA: target amplification strategy allowing quantitative on-chip detection of GMOs

PubMed Central

Morisset, Dany; Dobnik, David; Hamels, Sandrine; Žel, Jana; Gruden, Kristina

2008-01-01

We have developed a novel multiplex quantitative DNA-based target amplification method suitable for sensitive, specific and quantitative detection on microarray. This new method named NASBA Implemented Microarray Analysis (NAIMA) was applied to GMO detection in food and feed, but its application can be extended to all fields of biology requiring simultaneous detection of low copy number DNA targets. In a first step, the use of tailed primers allows the multiplex synthesis of template DNAs in a primer extension reaction. A second step of the procedure consists of transcription-based amplification using universal primers. The cRNA product is further on directly ligated to fluorescent dyes labelled 3DNA dendrimers allowing signal amplification and hybridized without further purification on an oligonucleotide probe-based microarray for multiplex detection. Two triplex systems have been applied to test maize samples containing several transgenic lines, and NAIMA has shown to be sensitive down to two target copies and to provide quantitative data on the transgenic contents in a range of 0.1–25%. Performances of NAIMA are comparable to singleplex quantitative real-time PCR. In addition, NAIMA amplification is faster since 20 min are sufficient to achieve full amplification. PMID:18710880

Development of a Flow Cytometry-Based Method for Rapid Detection of Escherichia coli and Shigella Spp. Using an Oligonucleotide Probe

PubMed Central

Xue, Yong; Wilkes, Jon G.; Moskal, Ted J.; Williams, Anna J.; Cooper, Willie M.; Nayak, Rajesh; Rafii, Fatemeh; Buzatu, Dan A.

2016-01-01

Standard methods to detect Escherichia coli contamination in food use the polymerase chain reaction (PCR) and agar culture plates. These methods require multiple incubation steps and take a long time to results. An improved rapid flow-cytometry based detection method was developed, using a fluorescence-labeled oligonucleotide probe specifically binding a16S rRNA sequence. The method positively detected 51 E. coli isolates as well as 4 Shigella species. All 27 non-E. coli strains tested gave negative results. Comparison of the new genetic assay with a total plate count (TPC) assay and agar plate counting indicated similar sensitivity, agreement between cytometry cell and colony counts. This method can detect a small number of E.coli cells in the presence of large numbers of other bacteria. This method can be used for rapid, economical, and stable detection of E. coli and Shigella contamination in the food industry and other contexts. PMID:26913737

Development of a Flow Cytometry-Based Method for Rapid Detection of Escherichia coli and Shigella Spp. Using an Oligonucleotide Probe.

PubMed

Xue, Yong; Wilkes, Jon G; Moskal, Ted J; Williams, Anna J; Cooper, Willie M; Nayak, Rajesh; Rafii, Fatemeh; Buzatu, Dan A

2016-01-01

Standard methods to detect Escherichia coli contamination in food use the polymerase chain reaction (PCR) and agar culture plates. These methods require multiple incubation steps and take a long time to results. An improved rapid flow-cytometry based detection method was developed, using a fluorescence-labeled oligonucleotide probe specifically binding a16S rRNA sequence. The method positively detected 51 E. coli isolates as well as 4 Shigella species. All 27 non-E. coli strains tested gave negative results. Comparison of the new genetic assay with a total plate count (TPC) assay and agar plate counting indicated similar sensitivity, agreement between cytometry cell and colony counts. This method can detect a small number of E.coli cells in the presence of large numbers of other bacteria. This method can be used for rapid, economical, and stable detection of E. coli and Shigella contamination in the food industry and other contexts.

Determining Fungi rRNA Copy Number by PCR

EPA Science Inventory

The goal of this project is to improve the quantification of indoor fungal pollutants via the specific application of quantitative PCR (qPCR). Improvement will be made in the controls used in current qPCR applications. This work focuses on the use of two separate controls within ...

The microbial ecology of anaerobic cellulose degradation in municipal waste landfill sites: evidence of a role for fibrobacters.

PubMed

McDonald, James E; Houghton, James N I; Rooks, David J; Allison, Heather E; McCarthy, Alan J

2012-04-01

Cellulose is reputedly the most abundant organic polymer in the biosphere, yet despite the fundamental role of cellulolytic microorganisms in global carbon cycling and as potential sources of novel enzymes for biotechnology, their identity and ecology is not well established. Cellulose is a major component of landfill waste and its degradation is therefore a key feature of the anaerobic microbial decomposition process. Here, we targeted a number of taxa containing known cellulolytic anaerobes (members of the bacterial genus Fibrobacter, lineages of Clostridium clusters I, III, IV and XIV, and anaerobic fungi of the Neocallimastigales) in landfill leachate and colonized cellulose 'baits' via PCR and quantitative PCR (qPCR). Fibrobacter spp. and Clostridium clusters III, IV and XIV were detected in almost all leachate samples and cluster III and XIV clostridia were the most abundant (1-6% and 1-17% of total bacterial 16S rRNA gene copies respectively). Two landfill leachate microcosms were constructed to specifically assess those microbial communities that colonize and degrade cellulose substrates in situ. Scanning electron microscopy (SEM) of colonized cotton revealed extensive cellulose degradation in one microcosm, and Fibrobacter spp. and Clostridium cluster III represented 29% and 17%, respectively, of total bacterial 16S rRNA gene copies in the biofilm. Visible cellulose degradation was not observed in the second microcosm, and this correlated with negligible relative abundances of Clostridium cluster III and Fibrobacter spp. (≤ 0.1%), providing the first evidence that the novel fibrobacters recently detected in landfill sites and other non-gut environments colonize and degrade cellulose substrates in situ. © 2012 Society for Applied Microbiology and Blackwell Publishing Ltd.

Gene copy number variation and its significance in cyanobacterial phylogeny

PubMed Central

2012-01-01

Background In eukaryotes, variation in gene copy numbers is often associated with deleterious effects, but may also have positive effects. For prokaryotes, studies on gene copy number variation are rare. Previous studies have suggested that high numbers of rRNA gene copies can be advantageous in environments with changing resource availability, but further association of gene copies and phenotypic traits are not documented. We used one of the morphologically most diverse prokaryotic phyla to test whether numbers of gene copies are associated with levels of cell differentiation. Results We implemented a search algorithm that identified 44 genes with highly conserved copies across 22 fully sequenced cyanobacterial taxa. For two very basal cyanobacterial species, Gloeobacter violaceus and a thermophilic Synechococcus species, distinct phylogenetic positions previously found were supported by identical protein coding gene copy numbers. Furthermore, we found that increased ribosomal gene copy numbers showed a strong correlation to cyanobacteria capable of terminal cell differentiation. Additionally, we detected extremely low variation of 16S rRNA sequence copies within the cyanobacteria. We compared our results for 16S rRNA to three other eubacterial phyla (Chroroflexi, Spirochaetes and Bacteroidetes). Based on Bayesian phylogenetic inference and the comparisons of genetic distances, we could confirm that cyanobacterial 16S rRNA paralogs and orthologs show significantly stronger conservation than found in other eubacterial phyla. Conclusions A higher number of ribosomal operons could potentially provide an advantage to terminally differentiated cyanobacteria. Furthermore, we suggest that 16S rRNA gene copies in cyanobacteria are homogenized by both concerted evolution and purifying selection. In addition, the small ribosomal subunit in cyanobacteria appears to evolve at extraordinary slow evolutionary rates, an observation that has been made previously for morphological characteristics of cyanobacteria. PMID:22894826

23S rRNA gene-based enterococci community signatures in Lake Pontchartrain, Louisiana, USA, following urban runoff inputs after Hurricane Katrina.

PubMed

Bae, Hee-Sung; Hou, Aixin

2013-02-01

Little is known about the impacts of fecal polluted urban runoff inputs on the structure of enterococci communities in estuarine waters. This study employed a 23S rRNA gene-based polymerase chain reaction (PCR) assay with newly designed genus-specific primers, Ent127F-Ent907R, to determine the possible impacts of Hurricane Katrina floodwaters via the 17th Street Canal discharge on the community structure of enterococci in Lake Pontchartrain. A total of 94 phylotypes were identified through the restriction fragment length polymorphism (RFLP) screening of 494 clones while only 8 phylotypes occurred among 88 cultivated isolates. Sequence analyses of representative phylotypes and their temporal and spatial distribution in the lake and the canal indicated the Katrina floodwater input introduced a large portion of Enterococcus flavescens, Enterococcus casseliflavus, and Enterococcus dispar into the lake; typical fecal groups Enterococcus faecium, Enterococcus durans, Enterococcus hirae, and Enterococcus mundtii were detected primarily in the floodwater-impacted waters. This study provides a global picture of enterococci in estuarine waters impacted by Hurricane Katrina-derived urban runoff. It also demonstrates the culture-independent PCR approach using 23S rRNA gene as a molecular marker could be a good alternative in ecological studies of enterococci in natural environments to overcome the limitation of conventional cultivation methods.

Analysis of Chromobacterium sp. natural isolates from different Brazilian ecosystems

PubMed Central

Lima-Bittencourt, Cláudia I; Astolfi-Filho, Spartaco; Chartone-Souza, Edmar; Santos, Fabrício R; Nascimento, Andréa MA

2007-01-01

Background Chromobacterium violaceum is a free-living bacterium able to survive under diverse environmental conditions. In this study we evaluate the genetic and physiological diversity of Chromobacterium sp. isolates from three Brazilian ecosystems: Brazilian Savannah (Cerrado), Atlantic Rain Forest and Amazon Rain Forest. We have analyzed the diversity with molecular approaches (16S rRNA gene sequences and amplified ribosomal DNA restriction analysis) and phenotypic surveys of antibiotic resistance and biochemistry profiles. Results In general, the clusters based on physiological profiles included isolates from two or more geographical locations indicating that they are not restricted to a single ecosystem. The isolates from Brazilian Savannah presented greater physiologic diversity and their biochemical profile was the most variable of all groupings. The isolates recovered from Amazon and Atlantic Rain Forests presented the most similar biochemical characteristics to the Chromobacterium violaceum ATCC 12472 strain. Clusters based on biochemical profiles were congruent with clusters obtained by the 16S rRNA gene tree. According to the phylogenetic analyses, isolates from the Amazon Rain Forest and Savannah displayed a closer relationship to the Chromobacterium violaceum ATCC 12472. Furthermore, 16S rRNA gene tree revealed a good correlation between phylogenetic clustering and geographic origin. Conclusion The physiological analyses clearly demonstrate the high biochemical versatility found in the C. violaceum genome and molecular methods allowed to detect the intra and inter-population diversity of isolates from three Brazilian ecosystems. PMID:17584942

The Bacterial Communities of Full-Scale Biologically Active, Granular Activated Carbon Filters Are Stable and Diverse and Potentially Contain Novel Ammonia-Oxidizing Microorganisms

PubMed Central

Hope Wilkinson, Katheryn; Strait, Jacqueline M.; Hozalski, Raymond M.; Sadowksy, Michael J.; Hamilton, Matthew J.

2015-01-01

The bacterial community composition of the full-scale biologically active, granular activated carbon (BAC) filters operated at the St. Paul Regional Water Services (SPRWS) was investigated using Illumina MiSeq analysis of PCR-amplified 16S rRNA gene fragments. These bacterial communities were consistently diverse (Shannon index, >4.4; richness estimates, >1,500 unique operational taxonomic units [OTUs]) throughout the duration of the 12-month study period. In addition, only modest shifts in the quantities of individual bacterial populations were observed; of the 15 most prominent OTUs, the most highly variable population (a Variovorax sp.) modulated less than 13-fold over time and less than 8-fold from filter to filter. The most prominent population in the profiles was a Nitrospira sp., representing 13 to 21% of the community. Interestingly, very few of the known ammonia-oxidizing bacteria (AOB; <0.07%) and no ammonia-oxidizing Archaea were detected in the profiles. Quantitative PCR of amoA genes, however, suggested that AOB were prominent in the bacterial communities (amoA/16S rRNA gene ratio, 1 to 10%). We conclude, therefore, that the BAC filters at the SPRWS potentially contained significant numbers of unidentified and novel ammonia-oxidizing microorganisms that possess amoA genes similar to those of previously described AOB. PMID:26209671

Growth of ammonia-oxidizing archaea in soil microcosms is inhibited by acetylene.

PubMed

Offre, Pierre; Prosser, James I; Nicol, Graeme W

2009-10-01

Autotrophic ammonia-oxidizing bacteria were considered to be responsible for the majority of ammonia oxidation in soil until the recent discovery of the autotrophic ammonia-oxidizing archaea. To assess the relative contributions of bacterial and archaeal ammonia oxidizers to soil ammonia oxidation, their growth was analysed during active nitrification in soil microcosms incubated for 30 days at 30 degrees C, and the effect of an inhibitor of ammonia oxidation (acetylene) on their growth and soil nitrification kinetics was determined. Denaturing gradient gel electrophoresis (DGGE) analysis of bacterial ammonia oxidizer 16S rRNA genes did not detect any change in their community composition during incubation, and quantitative PCR (qPCR) analysis of bacterial amoA genes indicated a small decrease in abundance in control and acetylene-containing microcosms. DGGE fingerprints of archaeal amoA and 16S rRNA genes demonstrated changes in the relative abundance of specific crenarchaeal phylotypes during active nitrification. Growth was also indicated by increases in crenarchaeal amoA gene copy number, determined by qPCR. In microcosms containing acetylene, nitrification and growth of the crenarchaeal phylotypes were suppressed, suggesting that these crenarchaea are ammonia oxidizers. Growth of only archaeal but not bacterial ammonia oxidizers occurred in microcosms with active nitrification, indicating that ammonia oxidation was mostly due to archaea in the conditions of the present study.

3M™ Molecular detection system versus MALDI-TOF mass spectrometry and molecular techniques for the identification of Escherichia coli 0157:H7, Salmonella spp. &Listeria spp.

PubMed

Loff, Marché; Mare, Louise; de Kwaadsteniet, Michele; Khan, Wesaal

2014-06-01

The aim of this study was to compare standard selective plating, conventional PCR (16S rRNA and species specific primers), MALDI-TOF MS and the 3M™ Molecular Detection System for the routine detection of the pathogens Listeria, Salmonella and Escherichia coli 0157:H7 in wastewater and river water samples. MALDI-TOF MS was able to positively identify 20/21 (95%) of the E. coli isolates obtained at genus and species level, while 16S rRNA sequencing only correctly identified 6/21 (28%) as E. coli strains. None of the presumptive positive Listeria spp. and Salmonella spp. isolates obtained by culturing on selective media were positively identified by MALDI-TOF and 16S rRNA analysis. The species-specific E. coli 0157:H7 PCR described in this present study, was not able to detect any E. coli 0157:H7 strains in the wastewater and river water samples analysed. However, E. coli strains, Listeria spp., L. monocytogenes and Salmonella spp. were detected using species specific PCR. Escherichia coli 0157:H7, Listeria spp. and Salmonella spp. were also sporadically detected throughout the sampling period in the wastewater and river water samples analysed by the 3M™ Molecular Detection System. MALDI-TOF MS, which is a simple, accurate and cost-effective detection method, efficiently identified the culturable organisms, while in the current study both species specific PCR (Listeria spp. and Salmonella spp.) and 3M™ Molecular Detection System could be utilised for the direct routine analysis of pathogens in water sources. Copyright © 2014 Elsevier B.V. All rights reserved.

Seasonal distribution of potentially pathogenic Acanthamoeba species from drinking water reservoirs in Taiwan.

PubMed

Kao, Po-Min; Hsu, Bing-Mu; Hsu, Tsui-Kang; Liu, Jorn-Hon; Chang, Hsiang-Yu; Ji, Wen-Tsai; Tzeng, Kai-Jiun; Huang, Shih-Wei; Huang, Yu-Li

2015-03-01

In order to detect the presence/absence of Acanthamoeba along with geographical variations, water quality variations and seasonal change of Acanthamoeba in Taiwan was investigated by 18S ribosomal RNA (rRNA) gene TaqMan quantitative real-time PCR. Samples were collected quarterly at 19 drinking water reservoir sites from November 2012 to August 2013. Acanthamoeba was detected in 39.5 % (30/76) of the water sample, and the detection rate was 63.2 % (12/19) from samples collected in autumn. The average concentration of Acanthamoeba was 3.59 × 10(4) copies/L. For geographic distribution, the detection rate for Acanthamoeba at the northern region was higher than the central and southern regions in all seasons. Results of Spearman rank test revealed that heterotrophic plate count (HPC) had a negative correlation (R = -0.502), while dissolved oxygen (DO) had a positive correlation (R = 0.463) in summer. Significant differences were found only between the presence/absence of Acanthamoeba and HPC in summer (Mann-Whitney U test, P < 0.05). T2 and T4 genotypes of Acanthamoeba were identified, and T4 was the most commonly identified Acanthamoeba genotypes. The presence of Acanthamoeba in reservoirs presented a potential public health threat and should be further examined.

The applicability of TaqMan-based quantitative real-time PCR assays for detecting and enumeratIng Cryptosporidium spp. oocysts in the environment

EPA Science Inventory

Molecular detection methods such as PCR have been extensively used to type Cryptosporidium oocysts detected in the environment. More recently, studies have developed quantitative real-time PCR assays for detection and quantification of microbial contaminants in water as well as ...

Development and Evaluation of a Quantitative PCR Assay Targeting Sandhill Crane (Grus canadensis) Fecal Pollution

EPA Science Inventory

While the microbial water quality in the Platte River is seasonally impacted by excreta from migrating cranes, there are no methods available to study crane fecal contamination. Here we characterized microbial populations in crane feces using phylogenetic analysis of 16S rRNA gen...

Performance evaluation of canine-associated Bacteroidales assays in a multi-laboratory comparison study

EPA Science Inventory

The contribution of fecal pollution from dogs in urbanized areas can be significant and is an often underestimated problem. Microbial source tracking methods (MST) utilizing quantitative PCR of dog-associated gene sequences encoding 16S rRNA of Bacteroidales are a useful tool to ...

Rapid Detection & Identification of Bacillus Species using MALDI-TOF/TOF and Biomarker Database

DTIC Science & Technology

2006-06-01

rRNA sequence analysis. Multilocus enzyme electrophoresis ( MEE ) and comparative DNA sequence analysis suggest that they may represent a single species...adaptation of the MEE method [63] but with greater discrimination [64]. All of these new PCR-based subtyping methods are certainly superior and more...Demirev, P.A., Lin, J.S., Pineda , F.J., and Fenselau, C. (2001). Bioinformatics and mass spectrometry for microorganism identification: proteome-wide

N6-Methylation Assessment in Escherichia coli 23S rRNA Utilizing a Bulge Loop in an RNA-DNA Hybrid.

PubMed

Yoshioka, Kyoko; Kurita, Ryoji

2018-06-07

We propose a sequence-selective assay of N6-methyl-adenosine (m6A) in RNA without PCR or reverse transcription, by employing a hybridization assay with a DNA probe designed to form a bulge loop at the position of a target modified nucleotide. The m6A in the bulge in the RNA-DNA hybrid was assumed to be sufficiently mobile to be selectively recognized by an anti-m6A antibody with a high affinity. By employing a surface-plasmon-resonance measurement or using a microtiter-plate immunoassay method, a specific m6A in the Escherichia coli 23S rRNA sequence could be detected at the nanomolar level when synthesized and purified oligo-RNA fragments were used for measurement. We have successfully achieved the first selective detection of m6A 2030 specifically in 23S rRNA from real samples of E. coli total RNA by using our immunochemical approach.

Hepatozoon canis and Leishmania spp. coinfection in dogs diagnosed with visceral leishmaniasis.

PubMed

Morgado, Fernanda Nazaré; Cavalcanti, Amanda Dos Santos; Miranda, Luisa Helena de; O'Dwyer, Lúcia Helena; Silva, Maria Regina Lucas da; Menezes, Rodrigo Caldas; Andrade da Silva, Aurea Virgínia; Boité, Mariana Côrtes; Cupolillo, Elisa; Porrozzi, Renato

2016-01-01

This study describes the occurrence of dogs naturally co-infected with Hepatozoon canis and two Leishmania species: L. infantum or L. braziliensis. Four dogs serologically diagnosed with Visceral Leishmaniasis were euthanized. Liver and spleen samples were collected for histopathological analysis and DNA isolation. H. canis meronts were observed in tissues from all four dogs. H. canis infection was confirmed by PCR followed by sequencing of a fragment of 18S rRNA gene. Leishmania detection and typing was confirmed by ITS1' PCR-RFLP and parasite burden was calculated using ssrRNA quantitative qPCR. A DPP - Dual Path platform test was performed. One out (Dog #2) of four animals was asymptomatic. Dogs #1 and #4 were infected by L. infantum and were DPP test positive. Dogs #2 and #3 were infected by L. braziliensis and were DPP test negative. Furthermore, visceral dissemination was observed in Dogs #2 and #3, since L. braziliensis was detected in liver and spleen samples. The visceral dissemination of L. braziliensis associated with systemic signs suggested that this co-infection could influence the parasite burden and disease progression.

Genetic divergence and phylogenetic relationships in grey mullets (Teleostei: Mugilidae) based on PCR-RFLP analysis of mtDNA segments.

PubMed

Papasotiropoulos, V; Klossa-Kilia, E; Kilias, G; Alahiotis, S

2002-04-01

The genetic differentiation and phylogenetic relationships among five species of the Mugilidae family (Mugil cephalus, Chelon labrosus, Liza aurata, Liza ramada, and Liza saliens) were investigated at the mtDNA level, on samples taken from Messolongi lagoon-Greece. RFLP analysis of three PCR-amplified mtDNA gene segments (12s rRNA, 16s rRNA, and CO I) was used. Ten, eight, and nine restriction enzymes were found to have at least one recognition site at 12s rRNA, 16s rRNA, and CO I genes, respectively. Several fragment patterns were revealed to be species-specific, and thus they could be useful in species taxonomy as diagnostic markers, as well as for further evolutionary studies. Seven different haplotypes were detected. The greatest amount of genetic differentiation was observed at the interspecific level, while little variation was revealed at the intraspecific level. The highest values of nucleotide sequence divergence were observed between M. cephalus and all the other species, while the lowest was found between C. labrosus and L. saliens. Dendrograms obtained by the three different methods (UPGMA, Neighbor-Joining, and Dollo parsimony), were found to exhibit in all cases the same topology. According to this, the most distinct species is M. cephalus, while the other species are clustered in two separate groups, thefirst one containing L. aurata and L. ramada, the other L. saliens and C. labrosus. This last clustering makes the monophyletic origin of the genus Liza questionable.

Bacteria evade immune recognition via TLR13 and binding of their 23S rRNA by MLS antibiotics by the same mechanisms

PubMed Central

Hochrein, Hubertus; Kirschning, Carsten J.

2013-01-01

The immune system recognizes pathogens and other danger by means of pattern recognition receptors. Recently, we have demonstrated that the orphan Toll-like receptor 13 (TLR13) senses a defined sequence of the bacterial rRNA and that bacteria use specific mechanisms to evade macrolide lincosamide streptogramin (MLS) antibiotics detection via TLR13. PMID:23802068

Quantitative Real-Time Legionella PCR for Environmental Water Samples: Data Interpretation

PubMed Central

Joly, Philippe; Falconnet, Pierre-Alain; André, Janine; Weill, Nicole; Reyrolle, Monique; Vandenesch, François; Maurin, Max; Etienne, Jerome; Jarraud, Sophie

2006-01-01

Quantitative Legionella PCRs targeting the 16S rRNA gene (specific for the genus Legionella) and the mip gene (specific for the species Legionella pneumophila) were applied to a total of 223 hot water system samples (131 in one laboratory and 92 in another laboratory) and 37 cooling tower samples (all in the same laboratory). The PCR results were compared with those of conventional culture. 16S rRNA gene PCR results were nonquantifiable for 2.8% of cooling tower samples and up to 39.1% of hot water system samples, and this was highly predictive of Legionella CFU counts below 250/liter. PCR cutoff values for identifying hot water system samples containing >103 CFU/liter legionellae were determined separately in each laboratory. The cutoffs differed widely between the laboratories and had sensitivities from 87.7 to 92.9% and specificities from 77.3 to 96.5%. The best specificity was obtained with mip PCR. PCR cutoffs could not be determined for cooling tower samples, as the results were highly variable and often high for culture-negative samples. Thus, quantitative Legionella PCR appears to be applicable to samples from hot water systems, but the positivity cutoff has to be determined in each laboratory. PMID:16597985

Successional changes in the chicken cecal microbiome during 42 days of growth are independent of organic acid feed additives.

PubMed

Oakley, Brian B; Buhr, R Jeff; Ritz, Casey W; Kiepper, Brian H; Berrang, Mark E; Seal, Bruce S; Cox, Nelson A

2014-11-27

Poultry remains a major source of foodborne bacterial infections. A variety of additives with presumed anti-microbial and/or growth-promoting effects are commonly added to poultry feed during commercial grow-out, yet the effects of these additives on the gastrointestinal microbial community (the GI microbiome) as the bird matures remain largely unknown. Here we compared temporal changes in the cecal microbiome to the effects of formic acid, propionic acid, and medium-chain fatty acids (MCFA) added to feed and/or drinking water. Cecal bacterial communities at day of hatch (n = 5 birds), 7d (n = 32), 21d (n = 27), and 42d (n = 36) post-hatch were surveyed using direct 454 sequencing of 16S rRNA gene amplicons from each bird in combination with cultivation-based recovery of a Salmonella Typhimurium marker strain and quantitative-PCR targeting Clostridium perfringens. Treatment effects on specific pathogens were generally non-significant. S. Typhimurium introduced by oral gavage at day of hatch was recovered by cultivation from nearly all birds sampled across treatments at 7d and 21d, but by 42d, S. Typhimurium was only recovered from ca. 25% of birds, regardless of treatment. Sequencing data also revealed non-significant treatment effects on genera containing known pathogens and on the cecal microbiome as a whole. In contrast, temporal changes in the cecal microbiome were dramatic, highly significant, and consistent across treatments. At 7d, the cecal community was dominated by three genera (Flavonifractor, Pseudoflavonifractor, and a Lachnospiracea sequence type) that accounted for more than half of sequences. By 21d post-hatch, a single genus (Faecalibacterium) accounted for 23-55% of sequences, and the number of Clostridium 16S rRNA gene copies detected by quantitative-PCR reached a maximum. Over the 42 d experiment, the cecal bacterial community changed significantly as measured by a variety of ecological metrics and increases in the complexity of co-occurrence networks. Management of poultry to improve animal health, nutrition, or food safety may need to consider the interactive effects of any treatments with the dramatic temporal shifts in the taxonomic composition of the cecal microbiome as described here.

The rRNA evolution and procaryotic phylogeny

NASA Technical Reports Server (NTRS)

Fox, G. E.

1986-01-01

Studies of ribosomal RNA primary structure allow reconstruction of phylogenetic trees for prokaryotic organisms. Such studies reveal major dichotomy among the bacteria that separates them into eubacteria and archaebacteria. Both groupings are further segmented into several major divisions. The results obtained from 5S rRNA sequences are essentially the same as those obtained with the 16S rRNA data. In the case of Gram negative bacteria the ribosomal RNA sequencing results can also be directly compared with hybridization studies and cytochrome c sequencing studies. There is again excellent agreement among the several methods. It seems likely then that the overall picture of microbial phylogeny that is emerging from the RNA sequence studies is a good approximation of the true history of these organisms. The RNA data allow examination of the evolutionary process in a semi-quantitative way. The secondary structures of these RNAs are largely established. As a result it is possible to recognize examples of local structural evolution. Evolutionary pathways accounting for these events can be proposed and their probability can be assessed.

A versatile quantitation platform based on platinum nanoparticles incorporated volumetric bar-chart chip for highly sensitive assays.

PubMed

Wang, Yuzhen; Zhu, Guixian; Qi, Wenjin; Li, Ying; Song, Yujun

2016-11-15

Platinum nanoparticles incorporated volumetric bar-chart chip (PtNPs-V-Chip) is able to be used for point-of-care tests by providing quantitative and visualized readout without any assistance from instruments, data processing, or graphic plotting. To improve the sensitivity of PtNPs-V-Chip, hybridization chain reaction was employed in this quantitation platform for highly sensitive assays that can detect as low as 16 pM Ebola Virus DNA, 0.01ng/mL carcinoembryonic antigen (CEA), and the 10 HER2-expressing cancer cells. Based on this amplified strategy, a 100-fold decrease of detection limit was achieved for DNA by improving the number of platinum nanoparticle catalyst for the captured analyte. This quantitation platform can also distinguish single base mismatch of DNA hybridization and observe the concentration threshold of CEA. The new strategy lays the foundation for this quantitation platform to be applied in forensic analysis, biothreat detection, clinical diagnostics and drug screening. Copyright © 2016 Elsevier B.V. All rights reserved.

Grape pomace compost harbors organohalide-respiring Dehalogenimonas species with novel reductive dehalogenase genes

DOE PAGES

Yang, Yi; Higgins, Steven A.; Yan, Jun; ...

2017-08-15

Here, organohalide-respiring bacteria play key roles in the natural chlorine cycle; however, most of the current knowledge is based on cultures from contaminated environments. We demonstrate that grape pomace compost without prior exposure to chlorinated solvents harbors a Dehalogenimonas ( Dhgm) species capable of using chlorinated ethenes, including the human carcinogen and common groundwater pollutant vinyl chloride (VC) as electron acceptors. Grape pomace microcosms and derived solid-free enrichment cultures were able to dechlorinate trichloroethene (TCE) to less chlorinated daughter products including ethene. 16S rRNA gene amplicon and qPCR analyses revealed the predominance of Dhgm sequences, but no Dehalococcoides mccartyi (more » Dhc) biomarker genes were detected. The enumeration of Dhgm 16S rRNA genes demonstrated VC-dependent growth, and 6.55 ± 0.64 x 10 8 cells were produced per µmole of chloride released. Metagenome sequencing enabled the assembly of a Dhgm draft genome, and 52 putative reductive dehalogenase (RDase) genes were identified. Proteomic workflows identified a putative VC RDase with 49% and 56.1% amino acid similarity to the known VC RDases VcrA and BvcA, respectively. A survey of 1,173 groundwater samples collected from 111 chlorinated solvent-contaminated sites revealed that Dhgm 16S rRNA genes were frequently detected and outnumbered Dhc in 65% of the samples. Dhgm may be more relevant contributors to chlorinated solvent reductive dechlorination in contaminated aquifers than is currently recognized, and non-polluted environments are a source of strictly organohalide-respiring bacteria with novel RDase genes.« less

Grape pomace compost harbors organohalide-respiring Dehalogenimonas species with novel reductive dehalogenase genes

DOE Office of Scientific and Technical Information (OSTI.GOV)

Yang, Yi; Higgins, Steven A.; Yan, Jun

Here, organohalide-respiring bacteria play key roles in the natural chlorine cycle; however, most of the current knowledge is based on cultures from contaminated environments. We demonstrate that grape pomace compost without prior exposure to chlorinated solvents harbors a Dehalogenimonas ( Dhgm) species capable of using chlorinated ethenes, including the human carcinogen and common groundwater pollutant vinyl chloride (VC) as electron acceptors. Grape pomace microcosms and derived solid-free enrichment cultures were able to dechlorinate trichloroethene (TCE) to less chlorinated daughter products including ethene. 16S rRNA gene amplicon and qPCR analyses revealed the predominance of Dhgm sequences, but no Dehalococcoides mccartyi (more » Dhc) biomarker genes were detected. The enumeration of Dhgm 16S rRNA genes demonstrated VC-dependent growth, and 6.55 ± 0.64 x 10 8 cells were produced per µmole of chloride released. Metagenome sequencing enabled the assembly of a Dhgm draft genome, and 52 putative reductive dehalogenase (RDase) genes were identified. Proteomic workflows identified a putative VC RDase with 49% and 56.1% amino acid similarity to the known VC RDases VcrA and BvcA, respectively. A survey of 1,173 groundwater samples collected from 111 chlorinated solvent-contaminated sites revealed that Dhgm 16S rRNA genes were frequently detected and outnumbered Dhc in 65% of the samples. Dhgm may be more relevant contributors to chlorinated solvent reductive dechlorination in contaminated aquifers than is currently recognized, and non-polluted environments are a source of strictly organohalide-respiring bacteria with novel RDase genes.« less

First molecular characterization of canine hepatozoonosis in Argentina: evaluation of asymptomatic Hepatozoon canis infection in dogs from Buenos Aires.

PubMed

Eiras, Diego Fernando; Basabe, Julia; Scodellaro, Carla F; Banach, Diana B; Matos, María L; Krimer, Alejandro; Baneth, Gad

2007-11-10

Canine hepatozoonosis is an expanding tick-borne disease in Argentina. Hepatozoonosis was studied during 1 year in six dogs from the same household in Buenos Aires. Blood parasitemia with Hepatozoon gamonts was found in five dogs and all six were positive by PCR for Hepatozoon sp. Although the levels of parasitemia fluctuated during the year, no clinical signs of disease were detected during the follow up period. Amplification and sequencing of a 650 bases fragment of the 18S rRNA gene from all six dogs yielded fragments that were 99% identical to H. canis. The results of the partial 18S rRNA genotyping with the sub-clinical course of infection and lack of severe hematological abnormalities are compatible with clinical and molecular descriptions of Hepatozoon canis infection from other areas of the world. This is the first molecular characterization of Hepatozoon from Argentina.

Archaeal community diversity and abundance changes along a natural salinity gradient in estuarine sediments.

PubMed

Webster, Gordon; O'Sullivan, Louise A; Meng, Yiyu; Williams, Angharad S; Sass, Andrea M; Watkins, Andrew J; Parkes, R John; Weightman, Andrew J

2015-02-01

Archaea are widespread in marine sediments, but their occurrence and relationship with natural salinity gradients in estuarine sediments is not well understood. This study investigated the abundance and diversity of Archaea in sediments at three sites [Brightlingsea (BR), Alresford (AR) and Hythe (HY)] along the Colne Estuary, using quantitative real-time PCR (qPCR) of 16S rRNA genes, DNA hybridization, Archaea 16S rRNA and mcrA gene phylogenetic analyses. Total archaeal 16S rRNA abundance in sediments were higher in the low-salinity brackish sediments from HY (2-8 × 10(7) 16S rRNA gene copies cm(-3)) than the high-salinity marine sites from BR and AR (2 × 10(4)-2 × 10(7) and 4 × 10(6)-2 × 10(7) 16S rRNA gene copies cm(-3), respectively), although as a proportion of the total prokaryotes Archaea were higher at BR than at AR or HY. Phylogenetic analysis showed that members of the 'Bathyarchaeota' (MCG), Thaumarchaeota and methanogenic Euryarchaeota were the dominant groups of Archaea. The composition of Thaumarchaeota varied with salinity, as only 'marine' group I.1a was present in marine sediments (BR). Methanogen 16S rRNA genes from low-salinity sediments at HY were dominated by acetotrophic Methanosaeta and putatively hydrogentrophic Methanomicrobiales, whereas the marine site (BR) was dominated by mcrA genes belonging to methylotrophic Methanococcoides, versatile Methanosarcina and methanotrophic ANME-2a. Overall, the results indicate that salinity and associated factors play a role in controlling diversity and distribution of Archaea in estuarine sediments. © The Author 2014. Published by Oxford University Press on behalf of Federation of European Microbiological Society.

Limitations of commonly used internal controls for real-time RT-PCR analysis of renal epithelial-mesenchymal cell transition.

PubMed

Elberg, Gerard; Elberg, Dorit; Logan, Charlotte J; Chen, Lijuan; Turman, Martin A

2006-01-01

Progressive renal fibrotic disease is accompanied by the massive accumulation of myofibroblasts as defined by alpha smooth muscle actin (alphaSMA) expression. We quantitated gene expression using real-time RT-PCR analysis during conversion of primary cultured human renal tubular cells (RTC) to myofibroblasts after treatment with transforming growth factor-beta1 (TGF-beta1). We report herein the limitations of commonly used reference genes for mRNA quantitation. We determined the expression of alphaSMA and megakaryoblastic leukemia-1 (MKL1), a transcriptional regulator of alphaSMA, by quantitative real-time PCR using three common internal controls, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), cyclophilin A and 18S rRNA. Expression of GAPDH mRNA and cyclophilin A mRNA, and to a lesser extent, 18S rRNA levels varied over time in culture and with exposure to TGF-beta1. Thus, depending on which reference gene was used, TGF-beta1 appeared to have different effects on expression of MKL1 and alphaSMA. RTC converting to myofibroblasts in primary culture is a valuable system to study renal fibrosis in humans. However, variability in expression of reference genes with TGF-beta1 treatment illustrates the need to validate mRNA quantitation with multiple reference genes to provide accurate interpretation of fibrosis studies in the absence of a universal internal standard for mRNA expression. 2006 S. Karger AG, Basel.

Diversity of thermophiles in a Malaysian hot spring determined using 16S rRNA and shotgun metagenome sequencing

PubMed Central

Chan, Chia Sing; Chan, Kok-Gan; Tay, Yea-Ling; Chua, Yi-Heng; Goh, Kian Mau

2015-01-01

The Sungai Klah (SK) hot spring is the second hottest geothermal spring in Malaysia. This hot spring is a shallow, 150-m-long, fast-flowing stream, with temperatures varying from 50 to 110°C and a pH range of 7.0–9.0. Hidden within a wooded area, the SK hot spring is continually fed by plant litter, resulting in a relatively high degree of total organic content (TOC). In this study, a sample taken from the middle of the stream was analyzed at the 16S rRNA V3-V4 region by amplicon metagenome sequencing. Over 35 phyla were detected by analyzing the 16S rRNA data. Firmicutes and Proteobacteria represented approximately 57% of the microbiome. Approximately 70% of the detected thermophiles were strict anaerobes; however, Hydrogenobacter spp., obligate chemolithotrophic thermophiles, represented one of the major taxa. Several thermophilic photosynthetic microorganisms and acidothermophiles were also detected. Most of the phyla identified by 16S rRNA were also found using the shotgun metagenome approaches. The carbon, sulfur, and nitrogen metabolism within the SK hot spring community were evaluated by shotgun metagenome sequencing, and the data revealed diversity in terms of metabolic activity and dynamics. This hot spring has a rich diversified phylogenetic community partly due to its natural environment (plant litter, high TOC, and a shallow stream) and geochemical parameters (broad temperature and pH range). It is speculated that symbiotic relationships occur between the members of the community. PMID:25798135

An Insight Into the Microbiome of the Amblyomma maculatum (Acari: Ixodidae)

PubMed Central

BUDACHETRI, KHEMRAJ; BROWNING, REBECCA E.; ADAMSON, STEVEN W.; DOWD, SCOT E.; CHAO, CHIEN-CHUNG; CHING, WEI-MEI; KARIM, SHAHID

2014-01-01

The aim of this study was to survey the bacterial diversity of Amblyomma maculatum Koch, 1844, and characterize its infection with Rickettsia parkeri. Pyrosequencing of the bacterial 16S rRNA was used to determine the total bacterial population in A. maculatum. Pyrosequencing analysis identified Rickettsia in A. maculatum midguts, salivary glands, and saliva, which indicates successful trafficking in the arthropod vector. The identity of Rickettsia spp. was determined based on sequencing the rickettsial outer membrane protein A (rompA) gene. The sequence homology search revealed the presence of R. parkeri, Rickettsia amblyommii, and Rickettsia endosymbiont of A. maculatum in midgut tissues, whereas the only rickettsia detected in salivary glands was R. parkeri, suggesting it is unique in its ability to migrate from midgut to salivary glands, and colonize this tissue before dissemination to the host. Owing to its importance as an emerging infectious disease, the R. parkeri pathogen burden was quantified by a rompB-based quantitative polymerase chain reaction (qPCR) assay and the diagnostic effectiveness of using R. parkeri polyclonal antibodies in tick tissues was tested. Together, these data indicate that field-collected A. maculatum had a R. parkeri infection rate of 12–32%. This study provides an insight into the A. maculatum microbiome and confirms the presence of R. parkeri, which will serve as the basis for future tick and microbiome interaction studies. PMID:24605461

A gene-targeted approach to investigate the intestinal butyrate-producing bacterial community

PubMed Central

2013-01-01

Background Butyrate, which is produced by the human microbiome, is essential for a well-functioning colon. Bacteria that produce butyrate are phylogenetically diverse, which hinders their accurate detection based on conventional phylogenetic markers. As a result, reliable information on this important bacterial group is often lacking in microbiome research. Results In this study we describe a gene-targeted approach for 454 pyrotag sequencing and quantitative polymerase chain reaction for the final genes in the two primary bacterial butyrate synthesis pathways, butyryl-CoA:acetate CoA-transferase (but) and butyrate kinase (buk). We monitored the establishment and early succession of butyrate-producing communities in four patients with ulcerative colitis who underwent a colectomy with ileal pouch anal anastomosis and compared it with three control samples from healthy colons. All patients established an abundant butyrate-producing community (approximately 5% to 26% of the total community) in the pouch within the 2-month study, but patterns were distinctive among individuals. Only one patient harbored a community profile similar to the healthy controls, in which there was a predominance of but genes that are similar to reference genes from Acidaminococcus sp., Eubacterium sp., Faecalibacterium prausnitzii and Roseburia sp., and an almost complete absence of buk genes. Two patients were greatly enriched in buk genes similar to those of Clostridium butyricum and C. perfringens, whereas a fourth patient displayed abundant communities containing both genes. Most butyrate producers identified in previous studies were detected and the general patterns of taxa found were supported by 16S rRNA gene pyrotag analysis, but the gene-targeted approach provided more detail about the potential butyrate-producing members of the community. Conclusions The presented approach provides quantitative and genotypic insights into butyrate-producing communities and facilitates a more specific functional characterization of the intestinal microbiome. Furthermore, our analysis refines but and buk reference annotations found in central databases. PMID:24451334

Fetal Tissues Tested for Microbial Sterility by Culture- and PCR-Based Methods Can be Safely Used in Clinics.

PubMed

Vitrenko, Yakov; Kostenko, Iryna; Kulebyakina, Kateryna; Duda, Alla; Klunnyk, Mariya; Sorochynska, Khrystyna

2017-02-16

Cell preparations to be used in clinical practice must be free of infectious agents. Safety concerns are especially elevated upon the use of human fetal tissues, which are otherwise highly advantageous in cell therapy. We demonstrate that treating fetal samples with antibiotic, extensive washing, and homogenization prior to cryoconservation efficiently removes microbes in general. Screening a large collection by an automatic culture system showed that 89.2% fetal tissue samples were sterile, while contamination was detected in 10.8% samples. Liver and chorion were contaminated more than the brain, kidney, lung, and soft tissues. Broad-range PCR from the bacterial 16s rRNA gene was adopted as a confirmatory assay; however, the concordance between the culture-based and PCR assays was weak. Taxonomic identification was done for contaminated samples by bacteriological methods and sequencing 16s rRNA PCR products. The two approaches revealed different spectra of taxonomic groups sharing only Lactobacillus, the most frequently found genus. In addition, other representatives of vaginal microbiota were detected by culture-based identification, while PCR product sequencing has also revealed a subset of nosocomial microorganisms. Importantly, species known to cause sepsis were identified by both techniques, arguing for their indispensability and mutual complementarity. We suggest that most contaminations are taken up during collection of fetal material rather than originating from an in utero infection. In conclusion, a rigorous microbiological control by culture and PCR is a prerequisite for safe clinical use of fetal tissue suspensions.

Antimicrobial Use and Resistance in Swine Waste Treatment Systems▿

PubMed Central

Jindal, Archana; Kocherginskaya, Svetlana; Mehboob, Asma; Robert, Matthew; Mackie, Roderick I.; Raskin, Lutgarde; Zilles, Julie L.

2006-01-01

Chlortetracycline and the macrolide tylosin were identified as commonly used antimicrobials for growth promotion and prophylaxis in swine production. Resistance to these antimicrobials was measured throughout the waste treatment processes at five swine farms by culture-based and molecular methods. Conventional farm samples had the highest levels of resistance with both culture-based and molecular methods and had similar levels of resistance despite differences in antimicrobial usage. The levels of resistance in organic farm samples, where no antimicrobials were used, were very low by a culture-based method targeting fecal streptococci. However, when the same samples were analyzed with a molecular method detecting methylation of a specific nucleotide in the 23S rRNA that results in resistance to macrolides, lincosamides, and streptogramin B (MLSB), an unexpectedly high level of resistant rRNA (approximately 50%) was observed, suggesting that the fecal streptococci were not an appropriate target group to evaluate resistance in the overall microbial community and that background levels of MLSB resistance may be substantial. All of the feed samples tested, including those from the organic farm, contained tetracycline resistance genes. Generally, the same tetracycline resistance genes and frequency of detection were found in the manure and lagoon samples for each commercial farm. The levels of tetracycline and MLSB resistance remained high throughout the waste treatment systems, suggesting that the potential impact of land application of treated wastes and waste treatment by-products on environmental levels of resistance should be investigated further. PMID:17041160

Bellerophon: A program to detect chimeric sequences in multiple sequence alignments

DOE Office of Scientific and Technical Information (OSTI.GOV)

Huber, Thomas; Faulkner, Geoffrey; Hugenholtz, Philip

2003-12-23

Bellerophon is a program for detecting chimeric sequences in multiple sequence datasets by an adaption of partial treeing analysis. Bellerophon was specifically developed to detect 16S rRNA gene chimeras in PCR-clone libraries of environmental samples but can be applied to other nucleotide sequence alignments.

Phylogenetic analysis of a biofilm bacterial population in a water pipeline in the Gulf of Mexico.

PubMed

López, Miguel A; Zavala-Díaz de la Serna, F Javier; Jan-Roblero, Janet; Romero, Juan M; Hernández-Rodríguez, César

2006-10-01

The aim of this study was to assess the bacterial diversity associated with a corrosive biofilm in a steel pipeline from the Gulf of Mexico used to inject marine water into the oil reservoir. Several aerobic and heterotrophic bacteria were isolated and identified by 16S rRNA gene sequence analysis. Metagenomic DNA was also extracted to perform a denaturing gradient gel electrophoresis analysis of ribosomal genes and to construct a 16S rRNA gene metagenomic library. Denaturing gradient gel electrophoresis profiles and ribosomal libraries exhibited a limited bacterial diversity. Most of the species detected in the ribosomal library or isolated from the pipeline were assigned to Proteobacteria (Halomonas spp., Idiomarina spp., Marinobacter aquaeolei, Thalassospira sp., Silicibacter sp. and Chromohalobacter sp.) and Bacilli (Bacillus spp. and Exiguobacterium spp.). This is the first report that associates some of these bacteria with a corrosive biofilm. It is relevant that no sulfate-reducing bacteria were isolated or detected by a PCR-based method. The diversity and relative abundance of bacteria from water pipeline biofilms may contribute to an understanding of the complexity and mechanisms of metal corrosion during marine water injection in oil secondary recovery.

Novel Detection of Coxiella spp., Theileria luwenshuni, and T. ovis Endosymbionts in Deer Keds (Lipoptena fortisetosa).

PubMed

Lee, Seung-Hun; Kim, Kyoo-Tae; Kwon, Oh-Deog; Ock, Younsung; Kim, Taeil; Choi, Donghag; Kwak, Dongmi

2016-01-01

We describe for the first time the detection of Coxiella-like bacteria (CLB), Theileria luwenshuni, and T. ovis endosymbionts in blood-sucking deer keds. Eight deer keds attached to a Korean water deer were identified as Lipoptena fortisetosa (Diptera: Hippoboscidae) by morphological and genetic analyses. Among the endosymbionts assessed, CLB, Theileria luwenshuni, and T. ovis were identified in L. fortisetosa by PCR and nucleotide sequencing. Based on phylogeny, CLB 16S rRNA sequences were classified into clade B, sharing 99.4% identity with CLB from Haemaphysalis longicornis in South Korea. Although the virulence of CLB to vertebrates is still controversial, several studies have reported clinical symptoms in birds due to CLB infections. The 18S rRNA sequences of T. luwenshuni and T. ovis in this study were 98.8-100% identical to those in GenBank, and all of the obtained sequences of T. ovis and T. luwenshuni in this study were 100% identical to each other, respectively. Although further studies are required to positively confirm L. fortisetosa as a biological vector of these pathogens, strong genetic relationships among sequences from this and previous studies suggest potential transmission among mammalian hosts by ticks and keds.

Effects of Histone Deacetylase Inhibitor (HDACi); Trichostatin-A (TSA) on the expression of housekeeping genes.

PubMed

Mogal, Ashish; Abdulkadir, Sarki A

2006-04-01

In quantitative RT-PCR (qRT-PCR), analysis of gene expression is dependent on normalization using housekeeping genes such as 18S rRNA, GAPDH and beta actin. However, variability in their expression has been reported to be caused by factors like drug treatment, pathological states and cell-cycle phase. An emerging area of cancer research focuses on identifying the role of epigenetic alterations such as histone modifications and DNA methylation in the initiation and progression of cancer. Histone acetylation is the best studied modification so far and has been probed through the use of histone deacetylase inhibitors (HDACi). Further, modulation of histone acetylation is currently being explored as a therapeutic strategy in the treatment of cancer and HDACis have shown promise in inhibiting tumorigenesis and metastasis. Trichostatin-A (TSA) is the most widely used HDACi. Therefore, we were driven to identify a suitable internal control for RT-PCR following TSA treatment. We performed quantitative RT-PCR analysis using mouse prostate tissue explants, human prostate cancer (LNCaP) cells and human breast cancer (T-47D and ZR-75-1) cells following TSA treatment. Expression of housekeeping genes including 18S rRNA, beta actin, GAPDH and ribosomal highly-basic 23-kDa protein (rb 23-kDa, RPL13A) were compared in vehicle versus TSA treated samples. Our results showed marked variations in 18S rRNA, beta actin mRNA and GAPDH mRNA levels in mouse prostate explants and a human prostate cancer (LNCaP) cell line following TSA treatment. Furthermore, in two human breast cancer cell lines (T-47D and ZR-75-1) 18S rRNA, beta actin mRNA and GAPDH mRNA levels varied significantly. However, RPL13A mRNA levels remained constant in all the conditions tested. Therefore, we recommend use of RPL13A as a standard for normalization during TSA treatment.

Comparison of PCR and quantitative real-time PCR methods for the characterization of ruminant and cattle fecal pollution sources.

PubMed

Raith, Meredith R; Kelty, Catherine A; Griffith, John F; Schriewer, Alexander; Wuertz, Stefan; Mieszkin, Sophie; Gourmelon, Michele; Reischer, Georg H; Farnleitner, Andreas H; Ervin, Jared S; Holden, Patricia A; Ebentier, Darcy L; Jay, Jennifer A; Wang, Dan; Boehm, Alexandria B; Aw, Tiong Gim; Rose, Joan B; Balleste, E; Meijer, W G; Sivaganesan, Mano; Shanks, Orin C

2013-11-15

The State of California has mandated the preparation of a guidance document on the application of fecal source identification methods for recreational water quality management. California contains the fifth highest population of cattle in the United States, making the inclusion of cow-associated methods a logical choice. Because the performance of these methods has been shown to change based on geography and/or local animal feeding practices, laboratory comparisons are needed to determine which assays are best suited for implementation. We describe the performance characterization of two end-point PCR assays (CF128 and CF193) and five real-time quantitative PCR (qPCR) assays (Rum2Bac, BacR, BacCow, CowM2, and CowM3) reported to be associated with either ruminant or cattle feces. Each assay was tested against a blinded set of 38 reference challenge filters (19 duplicate samples) containing fecal pollution from 12 different sources suspected to impact water quality. The abundance of each host-associated genetic marker was measured for qPCR-based assays in both target and non-target animals and compared to quantities of total DNA mass, wet mass of fecal material, as well as Bacteroidales, and enterococci determined by 16S rRNA qPCR and culture-based approaches (enterococci only). Ruminant- and cow-associated genetic markers were detected in all filters containing a cattle fecal source. However, some assays cross-reacted with non-target pollution sources. A large amount of variability was evident across laboratories when protocols were not fixed suggesting that protocol standardization will be necessary for widespread implementation. Finally, performance metrics indicate that the cattle-associated CowM2 qPCR method combined with either the BacR or Rum2Bac ruminant-associated methods are most suitable for implementation. Published by Elsevier Ltd.

Molecular and phenotypic analyses reveal the non-identity of the Phaeobacter gallaeciensis type strain deposits CIP 105210T and DSM 17395.

PubMed

Buddruhs, Nora; Pradella, Silke; Göker, Markus; Päuker, Orsola; Pukall, Rüdiger; Spröer, Cathrin; Schumann, Peter; Petersen, Jörn; Brinkhoff, Thorsten

2013-11-01

The marine genus Phaeobacter currently comprises six species, some of which were intensively studied mainly due to their ability to produce secondary metabolites. The type strain of the type species, Phaeobacter gallaeciensis BS107(T), has been deposited at several public culture collections worldwide. Based on differences in plasmid profiles, we detected that the alleged P. gallaeciensis type strains deposited at the Collection Institute Pasteur (CIP; Paris, France) as CIP 105210 and at the German Collection of Microorganisms and Cell Cultures (DSMZ; Braunschweig, Germany) as DSM 17395 are not identical. To determine the identity of these strains, we conducted DNA-DNA hybridization, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF), 16S rRNA gene and internal transcribed spacer (ITS) sequence analyses, as well as physiological experiments. Based on the detailed 16S rRNA gene reanalysis we showed that strain CIP 105210 most likely corresponds to the original P. gallaeciensis type strain BS107(T). In contrast, the Phaeobacter strain DSM 17395 exhibits a much closer affiliation to Phaeobacter inhibens DSM 16374(T) ( = T5(T)) and should thus be allocated to this species. The detection of the dissimilarity of strains CIP 105210(T) and DSM 17395 will influence future comparative studies within the genus Phaeobacter.

Visual loop-mediated isothermal amplification (LAMP) for the rapid diagnosis of Enterocytozoon hepatopenaei (EHP) infection.

PubMed

T, Sathish Kumar; A, Navaneeth Krishnan; J, Joseph Sahaya Rajan; M, Makesh; K P, Jithendran; S V, Alavandi; K K, Vijayan

2018-05-01

The emerging microsporidian parasite Enterocytozoon hepatopenaei (EHP), the causative agent of hepatopancreatic microsporidiosis, has been widely reported in shrimp-farming countries. EHP infection can be detected by light microscopy observation of spores (1.7 × 1 μm) in stained hepatopancreas (HP) tissue smears, HP tissue sections, and fecal samples. EHP can also be detected by polymerase chain reaction (PCR) targeting the small subunit (SSU) ribosomal RNA (rRNA) gene or the spore wall protein gene (SWP). In this study, a rapid, sensitive, specific, and closed tube visual loop-mediated isothermal amplification (LAMP) protocol combined with FTA cards was developed for the diagnosis of EHP. LAMP primers were designed based on the SSU rRNA gene of EHP. The target sequence of EHP was amplified at constant temperature of 65 °C for 45 min and amplified LAMP products were visually detected in a closed tube system by using SYBR™ green I dye. Detection limit of this LAMP protocol was ten copies. Field and clinical applicability of this assay was evaluated using 162 field samples including 106 HP tissue samples and 56 fecal samples collected from shrimp farms. Out of 162 samples, EHP could be detected in 62 samples (47 HP samples and 15 fecal samples). When compared with SWP-PCR as the gold standard, this EHP LAMP assay had 95.31% sensitivity, 98.98% specificity, and a kappa value of 0.948. This simple, closed tube, clinically evaluated visual LAMP assay has great potential for diagnosing EHP at the farm level, particularly under low-resource circumstances.

Nucleation by rRNA Dictates the Precision of Nucleolus Assembly.

PubMed

Falahati, Hanieh; Pelham-Webb, Bobbie; Blythe, Shelby; Wieschaus, Eric

2016-02-08

Membrane-less organelles are intracellular compartments specialized to carry out specific cellular functions. There is growing evidence supporting the possibility that such organelles form as a new phase, separating from cytoplasm or nucleoplasm. However, a main challenge to such phase separation models is that the initial assembly, or nucleation, of the new phase is typically a highly stochastic process and does not allow for the spatiotemporal precision observed in biological systems. Here, we investigate the initial assembly of the nucleolus, a membrane-less organelle involved in different cellular functions including ribosomal biogenesis. We demonstrate that the nucleolus formation is precisely timed in D. melanogaster embryos and follows the transcription of rRNA. We provide evidence that transcription of rRNA is necessary for overcoming the highly stochastic nucleation step in the formation of the nucleolus, through a seeding mechanism. In the absence of rDNA, the nucleolar proteins studied are able to form high-concentration assemblies. However, unlike the nucleolus, these assemblies are highly variable in number, location, and time at which they form. In addition, quantitative study of the changes in the nucleoplasmic concentration and distribution of these nucleolar proteins in the wild-type embryos is consistent with the role of rRNA in seeding the nucleolus formation. Copyright © 2016 Elsevier Ltd. All rights reserved.

Molecular detection of Theileria, Babesia, and Hepatozoon spp. in ixodid ticks from Palestine.

PubMed

Azmi, Kifaya; Ereqat, Suheir; Nasereddin, Abedelmajeed; Al-Jawabreh, Amer; Baneth, Gad; Abdeen, Ziad

2016-07-01

Ixodid ticks transmit various infectious agents that cause disease in humans and livestock worldwide. A cross-sectional survey on the presence of protozoan pathogens in ticks was carried out to assess the impact of tick-borne protozoa on domestic animals in Palestine. Ticks were collected from herds with sheep, goats and dogs in different geographic districts and their species were determined using morphological keys. The presence of piroplasms and Hepatozoon spp. was determined by PCR amplification of a 460-540bp fragment of the 18S rRNA gene followed by RFLP or DNA sequencing. A PCR-RFLP method based on the 18S rRNA was used in order to detect and to identify Hepatozoon, Babesia and Theileria spp. A total of 516 ticks were collected from animals in six Palestinian localities. Five tick species were found: Rhipicephalus sanguineus sensu lato, Rhipicephalus turanicus, Rhipicephalus bursa, Haemaphysalis parva and Haemaphysalis adleri. PCR-based analyses of the ticks revealed Theileria ovis (5.4%), Hepatozoon canis (4.3%), Babesia ovis (0.6%), and Babesia vogeli (0.4%). Theileria ovis was significantly associated with ticks from sheep and with R. turanicus ticks (p<0.01). H. canis was detected only in R. sanguineus s.l. and was significantly associated with ticks from dogs (p<0.01). To our knowledge, this is the first report describing the presence of these pathogens in ticks collected from Palestine. Communicating these findings with health and veterinary professionals will increase their awareness, and contribute to improved diagnosis and treatment of tick-borne diseases. Copyright © 2016. Published by Elsevier GmbH.

Yeast diversity during the fermentation of Andean chicha: A comparison of high-throughput sequencing and culture-dependent approaches.

PubMed

Mendoza, Lucía M; Neef, Alexander; Vignolo, Graciela; Belloch, Carmela

2017-10-01

Diversity and dynamics of yeasts associated with the fermentation of Argentinian maize-based beverage chicha was investigated. Samples taken at different stages from two chicha productions were analyzed by culture-dependent and culture-independent methods. Five hundred and ninety six yeasts were isolated by classical microbiological methods and 16 species identified by RFLPs and sequencing of D1/D2 26S rRNA gene. Genetic typing of isolates from the dominant species, Saccharomyces cerevisiae, by PCR of delta elements revealed up to 42 different patterns. High-throughput sequencing (HTS) of D1/D2 26S rRNA gene amplicons from chicha samples detected more than one hundred yeast species and almost fifty filamentous fungi taxa. Analysis of the data revealed that yeasts dominated the fermentation, although, a significant percentage of filamentous fungi appeared in the first step of the process. Statistical analysis of results showed that very few taxa were represented by more than 1% of the reads per sample at any step of the process. S. cerevisiae represented more than 90% of the reads in the fermentative samples. Other yeast species dominated the pre-fermentative steps and abounded in fermented samples when S. cerevisiae was in percentages below 90%. Most yeasts species detected by pyrosequencing were not recovered by cultivation. In contrast, the cultivation-based methodology detected very few yeast taxa, and most of them corresponded with very few reads in the pyrosequencing analysis. Copyright © 2017 Elsevier Ltd. All rights reserved.

Modified RNA-seq method for microbial community and diversity analysis using rRNA in different types of environmental samples

PubMed Central

Yan, Yong-Wei; Zou, Bin; Zhu, Ting; Hozzein, Wael N.

2017-01-01

RNA-seq-based SSU (small subunit) rRNA (ribosomal RNA) analysis has provided a better understanding of potentially active microbial community within environments. However, for RNA-seq library construction, high quantities of purified RNA are typically required. We propose a modified RNA-seq method for SSU rRNA-based microbial community analysis that depends on the direct ligation of a 5’ adaptor to RNA before reverse-transcription. The method requires only a low-input quantity of RNA (10–100 ng) and does not require a DNA removal step. The method was initially tested on three mock communities synthesized with enriched SSU rRNA of archaeal, bacterial and fungal isolates at different ratios, and was subsequently used for environmental samples of high or low biomass. For high-biomass salt-marsh sediments, enriched SSU rRNA and total nucleic acid-derived RNA-seq datasets revealed highly consistent community compositions for all of the SSU rRNA sequences, and as much as 46.4%-59.5% of 16S rRNA sequences were suitable for OTU (operational taxonomic unit)-based community and diversity analyses with complete coverage of V1-V2 regions. OTU-based community structures for the two datasets were also highly consistent with those determined by all of the 16S rRNA reads. For low-biomass samples, total nucleic acid-derived RNA-seq datasets were analyzed, and highly active bacterial taxa were also identified by the OTU-based method, notably including members of the previously underestimated genus Nitrospira and phylum Acidobacteria in tap water, members of the phylum Actinobacteria on a shower curtain, and members of the phylum Cyanobacteria on leaf surfaces. More than half of the bacterial 16S rRNA sequences covered the complete region of primer 8F, and non-coverage rates as high as 38.7% were obtained for phylum-unclassified sequences, providing many opportunities to identify novel bacterial taxa. This modified RNA-seq method will provide a better snapshot of diverse microbial communities, most notably by OTU-based analysis, even communities with low-biomass samples. PMID:29016661

Isolation of temperature-sensitive mutants of 16 S rRNA in Escherichia coli.

PubMed

Triman, K; Becker, E; Dammel, C; Katz, J; Mori, H; Douthwaite, S; Yapijakis, C; Yoast, S; Noller, H F

1989-10-20

Temperature-sensitive mutants have been isolated following hydroxylamine mutagenesis of a plasmid containing Escherichia coli rRNA genes carrying selectable markers for spectinomycin resistance (U1192 in 16 S rRNA) and erythromycin resistance (G2058 in 23 S rRNA). These antibiotic resistance alleles, originally identified by Morgan and co-workers, enable us to follow expression of cloned rRNA genes in vivo. Recessive mutations causing the loss of expression of the cloned 16 S rRNA gene were identified by the loss of the ability of cells to survive on media containing spectinomycin. The mutations were localized by in vitro restriction fragment replacement followed by in vivo marker rescue and were identified by DNA sequence analysis. We report here seven single-base alterations in 16 S rRNA (A146, U153, A350, A359, A538, A1292 and U1293), five of which produce temperature-sensitive spectinomycin resistance and two that produce unconditional loss of resistance. In each case, loss of ribosomal function can be accounted for by disruption of base-pairing in the secondary structure of 16 S rRNA. For the temperature-sensitive mutants, there is a lag period of about two generations between a shift to the restrictive temperature and cessation of growth, implying that the structural defects cause impairment of ribosome assembly.

ICP-MS as a novel detection system for quantitative element-tagged immunoassay of hidden peanut allergens in foods.

PubMed

Careri, Maria; Elviri, Lisa; Mangia, Alessandro; Mucchino, Claudio

2007-03-01

A novel ICP-MS-based ELISA immunoassay via element-tagged determination was devised for quantitative analysis of hidden allergens in food. The method was able to detect low amounts of peanuts (down to approximately 2 mg peanuts kg(-1) cereal-based matrix) by using a europium-tagged antibody. Selectivity was proved by the lack of detectable cross-reaction with a number of protein-rich raw materials.

Gold nanoparticle-based RT-PCR and real-time quantitative RT-PCR assays for detection of Japanese encephalitis virus

NASA Astrophysics Data System (ADS)

Huang, Su-Hua; Yang, Tsuey-Ching; Tsai, Ming-Hong; Tsai, I.-Shou; Lu, Huang-Chih; Chuang, Pei-Hsin; Wan, Lei; Lin, Ying-Ju; Lai, Chih-Ho; Lin, Cheng-Wen

2008-10-01

Virus isolation and antibody detection are routinely used for diagnosis of Japanese encephalitis virus (JEV) infection, but the low level of transient viremia in some JE patients makes JEV isolation from clinical and surveillance samples very difficult. We describe the use of gold nanoparticle-based RT-PCR and real-time quantitative RT-PCR assays for detection of JEV from its RNA genome. We tested the effect of gold nanoparticles on four different PCR systems, including conventional PCR, reverse-transcription PCR (RT-PCR), and SYBR green real-time PCR and RT-PCR assays for diagnosis in the acute phase of JEV infection. Gold nanoparticles increased the amplification yield of the PCR product and shortened the PCR time compared to the conventional reaction. In addition, nanogold-based real-time RT-PCR showed a linear relationship between Ct and template amount using ten-fold dilutions of JEV. The nanogold-based RT-PCR and real-time quantitative RT-PCR assays were able to detect low levels (1-10 000 copies) of the JEV RNA genomes extracted from culture medium or whole blood, providing early diagnostic tools for the detection of low-level viremia in the acute-phase infection. The assays described here were simple, sensitive, and rapid approaches for detection and quantitation of JEV in tissue cultured samples as well as clinical samples.

Aggregation-Induced Emission-Active Ruthenium(II) Complex of 4,7-Dichloro Phenanthroline for Selective Luminescent Detection and Ribosomal RNA Imaging.

PubMed

Sheet, Sanjoy Kumar; Sen, Bhaskar; Patra, Sumit Kumar; Rabha, Monosh; Aguan, Kripamoy; Khatua, Snehadrinarayan

2018-05-02

The development of red emissive aggregation-induced emission (AIE) active probes for organelle-specific imaging is of great importance. Construction of metal complex-based AIE-active materials with metal-to-ligand charge transfer (MLCT), ligand-to-metal charge transfer (LMCT) emission together with the ligand-centered and intraligand (LC/ILCT) emission is a challenging task. We developed a red emissive ruthenium(II) complex, 1[PF 6 ] 2 , and its perchlorate analogues of the 4,7-dichloro phenanthroline ligand. 1[PF 6 ] 2 has been characterized by spectroscopic and single-crystal X-ray diffraction. Complex 1 showed AIE enhancement in water, highly dense polyethylene glycol media, and also in the solid state. The possible reason behind the AIE property may be the weak supramolecular π···π, C-H···π, and C-Cl···H interactions between neighboring phen ligands as well as C-Cl···O halogen bonding (XB). The crystal structures of the two perchlorate analogues revealed C-Cl···O distances shorter than the sum of the van der Waals radii, which confirmed the XB interaction. The AIE property was supported by scanning electron microscopy, transmission electron microscopy, dynamic light scattering, and atomic force microscopy studies. Most importantly, the probe was found to be low cytotoxicity and to efficiently permeate the cell membrane. The cell-imaging experiments revealed rapid staining of the nucleolus in HeLa cells via the interaction with nucleolar ribosomal ribonucleic acid (rRNA). It is expected that the supramolecular interactions as well as C-Cl···O XB interaction with rRNA is the origin of aggregation and possible photoluminescence enhancement. To the best of our knowledge, this is the first report of red emissive ruthenium(II) complex-based probes with AIE characteristics for selective rRNA detection and nucleolar imaging.

Vertical stratification of bacteria and archaea in sediments of a boreal stratified humic lake

NASA Astrophysics Data System (ADS)

Rissanen, Antti J.; Mpamah, Promise; Peura, Sari; Taipale, Sami; Biasi, Christina; Nykänen, Hannu

2015-04-01

Boreal stratified humic lakes, with steep redox gradients in the water column and in the sediment, are important sources of methane (CH4) to the atmosphere. CH4 flux from these lakes is largely controlled by the balance between CH4-production (methanogenesis), which takes place in the organic rich sediment and in the deepest water layers, and CH4-consumption (methanotrophy), which takes place mainly in the water column. While there is already some published information on the activity, diversity and community structure of bacteria in the water columns of these lakes, such information on sediment microbial communities is very scarce. This study aims to characterize the vertical variation patterns in the diversity and the structure of microbial communities in sediment of a boreal stratified lake. Particular focus is on microbes with the potential to contribute to methanogenesis (fermentative bacteria and methanogenic archaea) and to methanotrophy (methanotrophic bacteria and archaea). Two sediment cores (26 cm deep), collected from the deepest point (~6 m) of a small boreal stratified lake during winter-stratification, were divided into depth sections of 1 to 2 cm for analyses. Communities were studied from DNA extracted from sediment samples by next-generation sequencing (Ion Torrent) of polymerase chain reaction (PCR) - amplified bacterial and archaeal 16S rRNA gene amplicons. The abundance of methanogenic archaea was also specifically studied by quantitative-PCR of methyl coenzyme-M reductase gene (mcrA) amplicons. Furthermore, the community structure and the abundance of bacteria were studied by phospholipid fatty acid (PLFA) analysis. Dominant potential fermentative bacteria belonged to families Syntrophaceae, Clostridiaceae and Peptostreptococcaceae. There were considerable differences in the vertical distribution among these groups. The relative abundance of Syntrophaceae started to increase from the sediment surface, peaked at depth layer from 5 to 10 cm (up to 21 % of bacterial 16S rRNA gene amplicons) and decreased gradually towards deeper layers while the relative abundances of Clostridiaceae and Peptostreptococcaceae started to increase at deeper depths, at 5 cm and 10 cm, respectively, both peaking at depth layer from 20 to 26 cm (Clostridiaceae up to 13 % and Peptostreptococcaceae up to 11 % of bacterial 16S rRNA amplicons). Methanogenic community was dominated by acetoclastic methanogens (genus Methanosaeta), which were most abundant at depth layer from sediment surface to 10 cm (up to 87 % of archaeal 16S rRNA gene amplicons) and decreased drastically until the depth of 18 cm having quite stable relative abundance from 18 to 26 cm (5 to 11 % of archaeal 16S rRNA gene amplicons). Hydrogenotrophic methanogens (Methanoregula, Methanolinea, Methanospirillum, Methanocella) (3 to 11 % of archaeal 16S rRNA gene amplicons) did not show any specific depth patterns. The proportion of methanotrophic microbes was very low and they consisted almost completely of type II methanotrophic bacteria (family Methylocystaceae), which had highest relative abundance at depth layer from 5 to 10 cm (up to 3 % of bacterial 16S rRNA gene amplicons) and were almost absent below 15 cm. Anaerobic methanotrophic archaea were not detected. These findings will be discussed with results from PLFA and q-PCR analyses.

Temporal changes in soil bacterial and archaeal communities with different fertilizers in tea orchards.

PubMed

Wang, Hua; Yang, Shao-hui; Yang, Jing-ping; Lv, Ya-min; Zhao, Xing; Pang, Ji-liang

2014-11-01

It is important to understand the effects of temporal changes in microbial communities in the acidic soils of tea orchards with different fertilizers. A field experiment involving organic fertilizer (OF), chemical fertilizer (CF), and unfertilized control (CK) treatments was arranged to analyze the temporal changes in the bacterial and archaeal communities at bimonthly intervals based on the 16S ribosomal RNA (rRNA) gene using terminal restriction fragment length polymorphism (T-RFLP) profiling. The abundances of total bacteria, total archaea, and selected functional genes (bacterial and archaeal amoA, bacterial narG, nirK, nirS, and nosZ) were determined by quantitative polymerase chain reaction (qPCR). The results indicate that the structures of bacterial and archaeal communities varied significantly with time and fertilization based on changes in the relative abundance of dominant T-RFs. The abundancy of the detected genes changed with time. The total bacteria, total archaea, and archaeal amoA were less abundant in July. The bacterial amoA and denitrifying genes were less abundant in September, except the nirK gene. The OF treatment increased the abundance of the observed genes, while the CF treatment had little influence on them. The soil temperature significantly affected the bacterial and archaeal community structures. The soil moisture was significantly correlated with the abundance of denitrifying genes. Of the soil chemical properties, soil organic carbon was the most important factor and was significantly correlated with the abundance of the detected genes, except the nirK gene. Overall, this study demonstrated the effects of both temporal alteration and organic fertilizer on the structures of microbial communities and the abundance of genes involved in the nitrogen cycle.

Temporal changes in soil bacterial and archaeal communities with different fertilizers in tea orchards* #

PubMed Central

Wang, Hua; Yang, Shao-hui; Yang, Jing-ping; Lv, Ya-min; Zhao, Xing; Pang, Ji-liang

2014-01-01

It is important to understand the effects of temporal changes in microbial communities in the acidic soils of tea orchards with different fertilizers. A field experiment involving organic fertilizer (OF), chemical fertilizer (CF), and unfertilized control (CK) treatments was arranged to analyze the temporal changes in the bacterial and archaeal communities at bimonthly intervals based on the 16S ribosomal RNA (rRNA) gene using terminal restriction fragment length polymorphism (T-RFLP) profiling. The abundances of total bacteria, total archaea, and selected functional genes (bacterial and archaeal amoA, bacterial narG, nirK, nirS, and nosZ) were determined by quantitative polymerase chain reaction (qPCR). The results indicate that the structures of bacterial and archaeal communities varied significantly with time and fertilization based on changes in the relative abundance of dominant T-RFs. The abundancy of the detected genes changed with time. The total bacteria, total archaea, and archaeal amoA were less abundant in July. The bacterial amoA and denitrifying genes were less abundant in September, except the nirK gene. The OF treatment increased the abundance of the observed genes, while the CF treatment had little influence on them. The soil temperature significantly affected the bacterial and archaeal community structures. The soil moisture was significantly correlated with the abundance of denitrifying genes. Of the soil chemical properties, soil organic carbon was the most important factor and was significantly correlated with the abundance of the detected genes, except the nirK gene. Overall, this study demonstrated the effects of both temporal alteration and organic fertilizer on the structures of microbial communities and the abundance of genes involved in the nitrogen cycle. PMID:25367788

U14 small nucleolar RNA makes multiple contacts with the pre-ribosomal RNA.

PubMed

Morrissey, J P; Tollervey, D

1997-06-01

The small nucleolar RNA (snoRNA) U14 has two regions of extended primary sequence complementarity to the 18S rRNA. The 3' region (domain B) shows the consensus structure for the methylation guide class of snoRNAs, whereas base-pairing between the 5' region (domain A) and the 18S rRNA sequence is required for the formation of functional ribosomes. Between domains A and B lies another essential region (domain Y). Here we report that yeast U14 can be cross-linked in vivo to the pre-rRNA; cross-linking is detected exclusively with the 35S primary transcript. Many nucleotides in U14 that lie outside of domains A and B are cross-linked to the pre-rRNA; in particular the essential domain Y region is cross-linked at several sites. U14 is, therefore, in far more extensive contact with the pre-rRNA than predicted from simple base-pairing models. Moreover, U14 can be cross-linked to other small RNA species. The functional interactions made by U14 during ribosome synthesis are likely to be very complex.

A PCR detection method for rapid identification of Melissococcus pluton in honeybee larvae.

PubMed

Govan, V A; Brözel, V; Allsopp, M H; Davison, S

1998-05-01

Melissococcus pluton is the causative agent of European foulbrood, a disease of honeybee larvae. This bacterium is particularly difficult to isolate because of its stringent growth requirements and competition from other bacteria. PCR was used selectively to amplify specific rRNA gene sequences of M. pluton from pure culture, from crude cell lysates, and directly from infected bee larvae. The PCR primers were designed from M. pluton 16S rRNA sequence data. The PCR products were visualized by agarose gel electrophoresis and confirmed as originating from M. pluton by sequencing in both directions. Detection was highly specific, and the probes did not hybridize with DNA from other bacterial species tested. This method enabled the rapid and specific detection and identification of M. pluton from pure cultures and infected bee larvae.

A PCR Detection Method for Rapid Identification of Melissococcus pluton in Honeybee Larvae

PubMed Central

Govan, V. A.; Brözel, V.; Allsopp, M. H.; Davison, S.

1998-01-01

Melissococcus pluton is the causative agent of European foulbrood, a disease of honeybee larvae. This bacterium is particularly difficult to isolate because of its stringent growth requirements and competition from other bacteria. PCR was used selectively to amplify specific rRNA gene sequences of M. pluton from pure culture, from crude cell lysates, and directly from infected bee larvae. The PCR primers were designed from M. pluton 16S rRNA sequence data. The PCR products were visualized by agarose gel electrophoresis and confirmed as originating from M. pluton by sequencing in both directions. Detection was highly specific, and the probes did not hybridize with DNA from other bacterial species tested. This method enabled the rapid and specific detection and identification of M. pluton from pure cultures and infected bee larvae. PMID:9572987

Phylogenetic diversity of Flavobacteria isolated from the North Sea on solid media.

PubMed

Hahnke, Richard L; Harder, Jens

2013-10-01

Flavobacteria are abundant in the North Sea, an epeiric sea on the continental shelf of Europe. However, this abundance has so far not been reflected by the number of strains in culture collections. In this study, Flavobacteria were isolated from pelagic and benthic samples, such as seawater, phytoplankton, sediment and its porewater, and from surfaces of animals and seaweeds on agar plates with a variety of carbon sources. Dilution cultivation with a new medium, incubation at low temperatures and with long incubation times, and colony screening by a Flavobacteria-Cytophagia-specific PCR detecting 16S rRNA gene sequences led to a collection of phylogenetically diverse strains. Two strains affiliated with Flammeovirgaceae and seven strains affiliated with Cyclobacteriaceae, whereas within the Flavobacteriaceae 20 isolated strains presumably represented seven novel candidate genera and 355 strains affiliated with 26 of 80 validly described marine Flavobacteriaceae genera, based on a genus boundary of 95.0% 16S rRNA gene sequence identity. The majority of strains (276) affiliated with 37 known species in 16 genera (based on a boundary of 98.7% 16S rRNA gene sequence identity), whereas 79 strains likely represented 42 novel species in 22 established Flavobacteriaceae genera. Pigmentation, iridescence, gliding motility, agar lysis, and flexirubin as a chemical marker supported the taxonomy at the species level. This study demonstrated the culturability on solid medium of phylogenetically diverse Flavobacteria originating from the North Sea. Copyright © 2013 Elsevier GmbH. All rights reserved.

[Abundances of ammonia-oxidizing archaeal accA and amoA genes in response to NO2 - and NO3 - of hot springs in Yunnan province].

PubMed

Song, Zhaoqi; Wang, Li; Zhou, Enmin; Wang, Fengping; Xiao, Xiang; Zhang, Chuanlun; Li, Wenjun

2014-12-04

Yunnan hot springs have highly diverseammonia-oxidizing archaea (AOA), which are autotrophic and can fix CO2 using the 3-hydroxypropionate/ 4-hydroxybutyrate (HP/HD) pathway. In this study, we investigated the abundances of prokaryotic 16S rRNA gene and archaeal accA and amoA genes in the sediments of hot springs of Yunnan Province, and analysed the correlations between the above gene abundances and environmental factors. We selected the sediments of twenty representative hot springs, and detected the gene abundances by quantitative polymerase chain reaction (qPCR). The principal component analysis (PCA) and the Mantel test in the R software package were performed for the correlations of gene abundance and environmental variables. The bacterial and archaeal 16S rRNA gene abundances were from 6.6 x 10(7) to 4.19 x 10(11) and from 1.27 x 10(6) to 1.51 x 10(11) copies/g sediment, respectively; Archaeal accA and amoA genes were from 8.89 x 10(3) to 6.49 x 10(5) and from 7.64 x 10(3) to 4.36 x 10(5) copies/g sediment, respectively. The results of mantel test showed that accA gene was significantly (R = 0.98, P < 0.001) correlated with amoA gene; Both of them also were correlated significantly with NO2- and NO3 -, but not with pH. The abundances of bacterial and archaeal 16S rRNA genes and the ratio between them varied significantly among Yunnan hot springs. The archaealaccA and amoA genes showed significant correlation with each other, validating our previous finding that AOA in terrestrial hot springs might acquire energy from ammonia oxidation coupled with CO2 fixation using the 3-hydroxypropionate/4-hydroxybutyrate pathway.

Development of duplex PCR for simultaneous detection of Theileria spp. and Anaplasma spp. in sheep and goats.

PubMed

Cui, Yanyan; Zhang, Yan; Jian, Fuchun; Zhang, Longxian; Wang, Rongjun; Cao, Shuxuan; Wang, Xiaoxing; Yan, Yaqun; Ning, Changshen

2017-05-01

Theileria spp. and Anaplasma spp., which are important tick-borne pathogens (TBPs), impact the health of humans and animals in tropical and subtropical areas. Theileria and Anaplasma co-infections are common in sheep and goats. Following alignment of the relevant DNA sequences, two primer sets were designed to specifically target the Theileria spp. 18S rRNA and Anaplasma spp. 16S rRNA gene sequences. Genomic DNA from the two genera was serially diluted tenfold for testing the sensitivities of detection of the primer sets. The specificities of the primer sets were confirmed when DNA from Anaplasma and Theileria (positive controls), other related hematoparasites (negative controls) and ddH 2 O were used as templates. Fifty field samples were also used to evaluate the utility of single PCR and duplex PCR assays, and the detection results were compared with those of the PCR methods previously published. An optimized duplex PCR assay was established from the two primer sets based on the relevant genes from the two TBPs, and this assay generated products of 298-bp (Theileria spp.) and 139-bp (Anaplasma spp.). The detection limit of the assay was 29.4 × 10 -3  ng per μl, and there was no cross-reaction with the DNA from other hematoparasites. The results showed that the newly developed duplex PCR assay had an efficiency of detection (P > 0.05) similar to other published PCR methods. In this study, a duplex PCR assay was developed that can simultaneously identify Theileria spp. and Anaplasma spp. in sheep and goats. This duplex PCR is a potentially valuable assay for epidemiological studies of TBPs in that it can detect cases of mixed infections of the pathogens. Copyright © 2017 Elsevier Inc. All rights reserved.

Occurrence and Phylogenetic Diversity of Sphingomonas Strains in Soils Contaminated with Polycyclic Aromatic Hydrocarbons

PubMed Central

Leys, Natalie M. E. J.; Ryngaert, Annemie; Bastiaens, Leen; Verstraete, Willy; Top, Eva M.; Springael, Dirk

2004-01-01

Bacterial strains of the genus Sphingomonas are often isolated from contaminated soils for their ability to use polycyclic aromatic hydrocarbons (PAH) as the sole source of carbon and energy. The direct detection of Sphingomonas strains in contaminated soils, either indigenous or inoculated, is, as such, of interest for bioremediation purposes. In this study, a culture-independent PCR-based detection method using specific primers targeting the Sphingomonas 16S rRNA gene combined with denaturing gradient gel electrophoresis (DGGE) was developed to assess Sphingomonas diversity in PAH-contaminated soils. PCR using the new primer pair on a set of template DNAs of different bacterial genera showed that the method was selective for bacteria belonging to the family Sphingomonadaceae. Single-band DGGE profiles were obtained for most Sphingomonas strains tested. Strains belonging to the same species had identical DGGE fingerprints, and in most cases, these fingerprints were typical for one species. Inoculated strains could be detected at a cell concentration of 104 CFU g of soil−1. The analysis of Sphingomonas population structures of several PAH-contaminated soils by the new PCR-DGGE method revealed that soils containing the highest phenanthrene concentrations showed the lowest Sphingomonas diversity. Sequence analysis of cloned PCR products amplified from soil DNA revealed new 16S rRNA gene Sphingomonas sequences significantly different from sequences from known cultivated isolates (i.e., sequences from environmental clones grouped phylogenetically with other environmental clone sequences available on the web and that possibly originated from several potential new species). In conclusion, the newly designed Sphingomonas-specific PCR-DGGE detection technique successfully analyzed the Sphingomonas communities from polluted soils at the species level and revealed different Sphingomonas members not previously detected by culture-dependent detection techniques. PMID:15066784

Toolbox Approaches Using Molecular Markers and 16S rRNA Gene Amplicon Data Sets for Identification of Fecal Pollution in Surface Water

PubMed Central

Staley, C.; Sadowsky, M. J.; Gyawali, P.; Sidhu, J. P. S.; Palmer, A.; Beale, D. J.; Toze, S.

2015-01-01

In this study, host-associated molecular markers and bacterial 16S rRNA gene community analysis using high-throughput sequencing were used to identify the sources of fecal pollution in environmental waters in Brisbane, Australia. A total of 92 fecal and composite wastewater samples were collected from different host groups (cat, cattle, dog, horse, human, and kangaroo), and 18 water samples were collected from six sites (BR1 to BR6) along the Brisbane River in Queensland, Australia. Bacterial communities in the fecal, wastewater, and river water samples were sequenced. Water samples were also tested for the presence of bird-associated (GFD), cattle-associated (CowM3), horse-associated, and human-associated (HF183) molecular markers, to provide multiple lines of evidence regarding the possible presence of fecal pollution associated with specific hosts. Among the 18 water samples tested, 83%, 33%, 17%, and 17% were real-time PCR positive for the GFD, HF183, CowM3, and horse markers, respectively. Among the potential sources of fecal pollution in water samples from the river, DNA sequencing tended to show relatively small contributions from wastewater treatment plants (up to 13% of sequence reads). Contributions from other animal sources were rarely detected and were very small (<3% of sequence reads). Source contributions determined via sequence analysis versus detection of molecular markers showed variable agreement. A lack of relationships among fecal indicator bacteria, host-associated molecular markers, and 16S rRNA gene community analysis data was also observed. Nonetheless, we show that bacterial community and host-associated molecular marker analyses can be combined to identify potential sources of fecal pollution in an urban river. This study is a proof of concept, and based on the results, we recommend using bacterial community analysis (where possible) along with PCR detection or quantification of host-associated molecular markers to provide information on the sources of fecal pollution in waterways. PMID:26231650

Simple Real-Time PCR and Amplicon Sequencing Method for Identification of Plasmodium Species in Human Whole Blood.

PubMed

Lefterova, Martina I; Budvytiene, Indre; Sandlund, Johanna; Färnert, Anna; Banaei, Niaz

2015-07-01

Malaria is the leading identifiable cause of fever in returning travelers. Accurate Plasmodium species identification has therapy implications for P. vivax and P. ovale, which have dormant liver stages requiring primaquine. Compared to microscopy, nucleic acid tests have improved specificity for species identification and higher sensitivity for mixed infections. Here, we describe a SYBR green-based real-time PCR assay for Plasmodium species identification from whole blood, which uses a panel of reactions to detect species-specific non-18S rRNA gene targets. A pan-Plasmodium 18S rRNA target is also amplified to allow species identification or confirmation by sequencing if necessary. An evaluation of assay accuracy, performed on 76 clinical samples (56 positives using thin smear microscopy as the reference method and 20 negatives), demonstrated clinical sensitivities of 95.2% for P. falciparum (20/21 positives detected) and 100% for the Plasmodium genus (52/52), P. vivax (20/20), P. ovale (9/9), and P. malariae (6/6). The sensitivity of the P. knowlesi-specific PCR was evaluated using spiked whole blood samples (100% [10/10 detected]). The specificities of the real-time PCR primers were 94.2% for P. vivax (49/52) and 100% for P. falciparum (51/51), P. ovale (62/62), P. malariae (69/69), and P. knowlesi (52/52). Thirty-three specimens were used to test species identification by sequencing the pan-Plasmodium 18S rRNA PCR product, with correct identification in all cases. The real-time PCR assay also identified two samples with mixed P. falciparum and P. ovale infection, which was confirmed by sequencing. The assay described here can be integrated into a malaria testing algorithm in low-prevalence areas, allowing definitive Plasmodium species identification shortly after malaria diagnosis by microscopy. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Comparison of nested-multiplex, Taqman & SYBR Green real-time PCR in diagnosis of amoebic liver abscess in a tertiary health care institute in India.

PubMed

Dinoop, K P; Parija, Subhash Chandra; Mandal, Jharna; Swaminathan, R P; Narayanan, P

2016-01-01

Amoebiasis is a common parasitic infection caused by Entamoeba histolytica and amoebic liver abscess (ALA) is the most common extraintestinal manifestation of amoebiasis. The aim of this study was to standardise real-time PCR assays (Taqman and SYBR Green) to detect E. histolytica from liver abscess pus and stool samples and compare its results with nested-multiplex PCR. Liver abscess pus specimens were subjected to DNA extraction. The extracted DNA samples were subjected to amplification by nested-multiplex PCR, Taqman (18S rRNA) and SYBR Green real-time PCR (16S-like rRNA assays to detect E. histolytica/E. dispar/E. moshkovskii). The amplification products were further confirmed by DNA sequence analysis. Receiver operator characteristic (ROC) curve analysis was done for nested-multiplex and SYBR Green real-time PCR and the area under the curve was calculated for evaluating the accuracy of the tests to dignose ALA. In all, 17, 19 and 25 liver abscess samples were positive for E. histolytica by nested-multiplex PCR, SYBR Green and Taqman real-time PCR assays, respectively. Significant differences in detection of E. histolytica were noted in the real-time PCR assays evaluated ( P<0.0001). The nested-multiplex PCR, SYBR Green real-time PCR and Taqman real-time PCR evaluated showed a positivity rate of 34, 38 and 50 per cent, respectively. Based on ROC curve analysis (considering Taqman real-time PCR as the gold standard), it was observed that SYBR Green real-time PCR was better than conventional nested-multiplex PCR for the diagnosis of ALA. Taqman real-time PCR targeting the 18S rRNA had the highest positivity rate evaluated in this study. Both nested multiplex and SYBR Green real-time PCR assays utilized were evaluated to give accurate results. Real-time PCR assays can be used as the gold standard in rapid and reliable diagnosis, and appropriate management of amoebiasis, replacing the conventional molecular methods.

Photobacterium damselae ssp. piscicida: detection by direct amplification of 16S rRNA gene sequences and genotypic variation as determined by amplified fragment length polymorphism (AFLP).

PubMed

Kvitt, H; Ucko, M; Colorni, A; Batargias, C; Zlotkin, A; Knibb, W

2002-04-05

A PCR protocol for the rapid diagnosis of fish 'pasteurellosis' based on 16S rRNA gene sequences was developed. The procedure combines low annealing temperature that detects low titers of Photobacterium damselae but also related species, and high annealing temperature for the specific identification of P. damselae directly from infected fish. The PCR protocol was validated on 19 piscine isolates of P. damselae ssp. piscicida from different geographic regions (Japan, Italy, Spain, Greece and Israel), on spontaneously infected sea bream Sparus aurata and sea bass Dicentrarchus labrax, and on closely related American Type Culture Collection (ATCC) reference strains. PCR using high annealing temperature (64 degrees C) discriminated between P. damselae and closely related reference strains, including P. histaminum. Sixteen isolates of P. damselae ssp. piscicida, 2 P. damselae ssp. piscicida reference strains and 1 P. damselae ssp. damselae reference strain were subjected to Amplified Fragment Length Polymorphism (AFLP) analysis, and a similarity matrix was produced. Accordingly, the Japanese isolates of P. damselae ssp. piscicida were distinguished from the Mediterranean/European isolates at a cut-off value of 83% similarity. A further subclustering at a cut-off value of 97% allowed discrimination between the Israeli P. damselae ssp. piscicida isolates and the other Mediterranean/European isolates. The combination of PCR direct amplification and AFLP provides a 2-step procedure, where P. damselae is rapidly identified at genus level on the basis of its 16S rRNA gene sequence and then grouped into distinct clusters on the basis of AFLP polymorphisms. The first step of direct amplification is highly sensitive and has immediate practical consequences, offering fish farmers a rapid diagnosis, while the AFLP is more specific and detects intraspecific variation which, in our study, also reflected geographic correspondence. Because of its superior discriminative properties, AFLP can be an important tool for epidemiological and taxonomic studies of this highly homogeneous genus.

Diversity in the 18S SSU rRNA V4 hyper-variable region of Theileria spp. in Cape buffalo (Syncerus caffer) and cattle from southern Africa.

PubMed

Mans, Ben J; Pienaar, Ronel; Latif, Abdalla A; Potgieter, Fred T

2011-05-01

Sequence variation within the 18S SSU rRNA V4 hyper-variable region can affect the accuracy of real-time hybridization probe-based diagnostics for the detection of Theileria spp. infections. This is relevant for assays that use non-specific primers, such as the real-time hybridization assay for T. parva (Sibeko et al. 2008). To assess the effect of sequence variation on this test, the Theileria 18S gene from 62 buffalo and 49 cattle samples was cloned and ∼1000 clones sequenced. Twenty-six genotypes were detected which included known and novel genotypes for the T. buffeli, T. mutans, T. taurotragi and T. velifera clades. A novel genotype related to T. sp. (sable) was also detected in 1 bovine sample. Theileria genotypic diversity was higher in buffalo compared to cattle. Polymorphism within the T. parva hyper-variable region was confirmed by aberrant real-time melting peaks and supported by sequencing of the S5 ribosomal gene. Analysis of the S5 gene suggests that this gene can be a marker for species differentiation. T. parva, T. sp. (buffalo) and T. sp. (bougasvlei) remain the only genotypes amplified by the primer set of the hybridization assay. Therefore, the 18S sequence diversity observed does not seem to affect the current real-time hybridization assay for T. parva.

High prevalence of small Babesia species in canines of Kerala, South India.

PubMed

Jain, Kollannur Jose; Lakshmanan, Bindu; Syamala, Karunakaran; Praveena, Jose E; Aravindakshan, Thazhathuveetil

2017-11-01

Canine babesiosis is an important vector-borne hemoparasitic disease caused by Babesia canis vogeli and Babesia gibsoni , in India. The communication places on record the salient findings of the study directed to detect and characterize the pathogenic B. gibsoni isolates of Kerala state. A total of 150 dogs were examined for the presence of hemoparasites by light microscopy as well as by PCR targeting the 18S rRNA gene of B. gibsoni . Hematological parameters were also analysed. Phylogenetic tree was constructed based on Tamura kei model adopting ML method. A sensitive and specific polymerase chain reaction assay was developed with newly designed primer pair BAGI-F/BAGI-R for the amplification of 488 bp fragment of 18S rRNA gene of B. gibsoni . Out of the 150 dogs examined, molecular evidence of B. gibsoni was recorded in 47.3% animals, while light microscopy detected the infection in 26.67% cases. The phylogenetic analyses revealed that B. gibsoni , Kerala, isolate was closest and occurred together with Bareilly isolate. Anemia and thrombocytopenia were the significant hematological alterations in chronic B. gibsoni infection. A high prevalence of natural infection of B. gibsoni was detected among the study population. The affected animals showed anaemia and thrombocytopenia. Phylogenetic analysis of this pathogenic isolate from south India revealed the closest similarity with Bareilly isolates.

High prevalence of small Babesia species in canines of Kerala, South India

PubMed Central

Jain, Kollannur Jose; Lakshmanan, Bindu; Syamala, Karunakaran; Praveena, Jose E; Aravindakshan, Thazhathuveetil

2017-01-01

Aim: Canine babesiosis is an important vector-borne hemoparasitic disease caused by Babesia canis vogeli and Babesia gibsoni, in India. The communication places on record the salient findings of the study directed to detect and characterize the pathogenic B. gibsoni isolates of Kerala state. Materials and Methods:: A total of 150 dogs were examined for the presence of hemoparasites by light microscopy as well as by PCR targeting the 18S rRNA gene of B. gibsoni. Hematological parameters were also analysed. Phylogenetic tree was constructed based on Tamura kei model adopting ML method. Results:: A sensitive and specific polymerase chain reaction assay was developed with newly designed primer pair BAGI-F/BAGI-R for the amplification of 488 bp fragment of 18S rRNA gene of B. gibsoni. Out of the 150 dogs examined, molecular evidence of B. gibsoni was recorded in 47.3% animals, while light microscopy detected the infection in 26.67% cases. The phylogenetic analyses revealed that B. gibsoni, Kerala, isolate was closest and occurred together with Bareilly isolate. Anemia and thrombocytopenia were the significant hematological alterations in chronic B. gibsoni infection. Conclusion:: A high prevalence of natural infection of B. gibsoni was detected among the study population. The affected animals showed anaemia and thrombocytopenia. Phylogenetic analysis of this pathogenic isolate from south India revealed the closest similarity with Bareilly isolates. PMID:29263592

Novel Molecular Method for Identification of Streptococcus pneumoniae Applicable to Clinical Microbiology and 16S rRNA Sequence-Based Microbiome Studies

PubMed Central

Scholz, Christian F. P.; Poulsen, Knud

2012-01-01

The close phylogenetic relationship of the important pathogen Streptococcus pneumoniae and several species of commensal streptococci, particularly Streptococcus mitis and Streptococcus pseudopneumoniae, and the recently demonstrated sharing of genes and phenotypic traits previously considered specific for S. pneumoniae hamper the exact identification of S. pneumoniae. Based on sequence analysis of 16S rRNA genes of a collection of 634 streptococcal strains, identified by multilocus sequence analysis, we detected a cytosine at position 203 present in all 440 strains of S. pneumoniae but replaced by an adenosine residue in all strains representing other species of mitis group streptococci. The S. pneumoniae-specific sequence signature could be demonstrated by sequence analysis or indirectly by restriction endonuclease digestion of a PCR amplicon covering the site. The S. pneumoniae-specific signature offers an inexpensive means for validation of the identity of clinical isolates and should be used as an integrated marker in the annotation procedure employed in 16S rRNA-based molecular studies of complex human microbiotas. This may avoid frequent misidentifications such as those we demonstrate to have occurred in previous reports and in reference sequence databases. PMID:22442329

5S ribosomal ribonucleic acid sequences in Bacteroides and Fusobacterium: evolutionary relationships within these genera and among eubacteria in general

NASA Technical Reports Server (NTRS)

Van den Eynde, H.; De Baere, R.; Shah, H. N.; Gharbia, S. E.; Fox, G. E.; Michalik, J.; Van de Peer, Y.; De Wachter, R.

1989-01-01

The 5S ribosomal ribonucleic acid (rRNA) sequences were determined for Bacteroides fragilis, Bacteroides thetaiotaomicron, Bacteroides capillosus, Bacteroides veroralis, Porphyromonas gingivalis, Anaerorhabdus furcosus, Fusobacterium nucleatum, Fusobacterium mortiferum, and Fusobacterium varium. A dendrogram constructed by a clustering algorithm from these sequences, which were aligned with all other hitherto known eubacterial 5S rRNA sequences, showed differences as well as similarities with respect to results derived from 16S rRNA analyses. In the 5S rRNA dendrogram, Bacteroides clustered together with Cytophaga and Fusobacterium, as in 16S rRNA analyses. Intraphylum relationships deduced from 5S rRNAs suggested that Bacteroides is specifically related to Cytophaga rather than to Fusobacterium, as was suggested by 16S rRNA analyses. Previous taxonomic considerations concerning the genus Bacteroides, based on biochemical and physiological data, were confirmed by the 5S rRNA sequence analysis.

Next-generation sequencing identification of pathogenic bacterial genes and their relationship with fecal indicator bacteria in different water sources in the Kathmandu Valley, Nepal.

PubMed

Ghaju Shrestha, Rajani; Tanaka, Yasuhiro; Malla, Bikash; Bhandari, Dinesh; Tandukar, Sarmila; Inoue, Daisuke; Sei, Kazunari; Sherchand, Jeevan B; Haramoto, Eiji

2017-12-01

Bacteriological analysis of drinking water leads to detection of only conventional fecal indicator bacteria. This study aimed to explore and characterize bacterial diversity, to understand the extent of pathogenic bacterial contamination, and to examine the relationship between pathogenic bacteria and fecal indicator bacteria in different water sources in the Kathmandu Valley, Nepal. Sixteen water samples were collected from shallow dug wells (n=12), a deep tube well (n=1), a spring (n=1), and rivers (n=2) in September 2014 for 16S rRNA gene next-generation sequencing. A total of 525 genera were identified, of which 81 genera were classified as possible pathogenic bacteria. Acinetobacter, Arcobacter, and Clostridium were detected with a relatively higher abundance (>0.1% of total bacterial genes) in 16, 13, and 5 of the 16 samples, respectively, and the highest abundance ratio of Acinetobacter (85.14%) was obtained in the deep tube well sample. Furthermore, the bla OXA23-like genes of Acinetobacter were detected using SYBR Green-based quantitative PCR in 13 (35%) of 37 water samples, including the 16 samples that were analyzed for next-generation sequencing, with concentrations ranging 5.3-7.5logcopies/100mL. There was no sufficient correlation found between fecal indicator bacteria, such as Escherichia coli and total coliforms, and potential pathogenic bacteria, as well as the bla OXA23-like gene of Acinetobacter. These results suggest the limitation of using conventional fecal indicator bacteria in evaluating the pathogenic bacteria contamination of different water sources in the Kathmandu Valley. Copyright © 2017 Elsevier B.V. All rights reserved.

Semen Bacterial Concentrations and HIV-1 RNA Shedding Among HIV-1-Seropositive Kenyan Men.

PubMed

Korhonen, Christine J; Srinivasan, Sujatha; Huang, Dandi; Ko, Daisy L; Sanders, Eduard J; Peshu, Norbert M; Krieger, John N; Muller, Charles H; Coombs, Robert W; Fredricks, David N; Graham, Susan M

2017-03-01

HIV-1 is transmitted through semen from men to their sexual partners. Genital infections can increase HIV-1 RNA shedding in semen, but shedding also occurs in the absence of typical pathogens. We hypothesized that higher bacterial concentrations in semen would be associated with higher HIV-1 RNA levels. We analyzed semen samples from 42 HIV-1-seropositive Kenyan men using quantitative polymerase chain reaction (PCR) to assess bacterial concentrations and real-time PCR to measure HIV-1 RNA levels. Generalized estimation equations were used to evaluate associations between these 2 measures. Broad-range 16S rRNA gene PCR with pyrosequencing was performed on a subset of 13 samples to assess bacterial community composition. Bacteria were detected in 96.6% of 88 samples by quantitative PCR. Semen bacterial concentration and HIV-1 RNA levels were correlated 0.30 (P = 0.01). The association between bacterial concentration and HIV-1 RNA detection was not significant after adjustment for antiretroviral therapy (ART) (adjusted odds ratio: 1.27, 95% CI: 0.84 to 1.91). Factors associated with semen bacterial concentration included insertive anal sex (adjusted beta 0.92, 95% CI: 0.12 to 1.73) and ART use (adjusted beta: -0.77, 95% CI: -1.50 to 0.04). Among 13 samples with pyrosequencing data, Corynebacterium spp., Staphylococcus spp., and Streptococcus spp. were most frequently detected. Most of these HIV-1-infected men had bacteria in their semen. ART use was associated with undetectable semen HIV-1 RNA and lower semen bacterial concentrations, whereas insertive anal sex was associated with higher bacterial concentrations. Additional studies evaluating the relationship between semen bacteria, inflammation, mucosal immunity, and HIV-1 shedding are needed to understand implications for HIV-1 transmission.

Semen Bacterial Concentrations and HIV-1 RNA Shedding Among HIV-1–Seropositive Kenyan Men

PubMed Central

Srinivasan, Sujatha; Huang, Dandi; Ko, Daisy L.; Sanders, Eduard J.; Peshu, Norbert M.; Krieger, John N.; Muller, Charles H.; Coombs, Robert W.; Fredricks, David N.; Graham, Susan M.

2017-01-01

Introduction: HIV-1 is transmitted through semen from men to their sexual partners. Genital infections can increase HIV-1 RNA shedding in semen, but shedding also occurs in the absence of typical pathogens. We hypothesized that higher bacterial concentrations in semen would be associated with higher HIV-1 RNA levels. Methods: We analyzed semen samples from 42 HIV-1–seropositive Kenyan men using quantitative polymerase chain reaction (PCR) to assess bacterial concentrations and real-time PCR to measure HIV-1 RNA levels. Generalized estimation equations were used to evaluate associations between these 2 measures. Broad-range 16S rRNA gene PCR with pyrosequencing was performed on a subset of 13 samples to assess bacterial community composition. Results: Bacteria were detected in 96.6% of 88 samples by quantitative PCR. Semen bacterial concentration and HIV-1 RNA levels were correlated 0.30 (P = 0.01). The association between bacterial concentration and HIV-1 RNA detection was not significant after adjustment for antiretroviral therapy (ART) (adjusted odds ratio: 1.27, 95% CI: 0.84 to 1.91). Factors associated with semen bacterial concentration included insertive anal sex (adjusted beta 0.92, 95% CI: 0.12 to 1.73) and ART use (adjusted beta: −0.77, 95% CI: −1.50 to 0.04). Among 13 samples with pyrosequencing data, Corynebacterium spp., Staphylococcus spp., and Streptococcus spp. were most frequently detected. Conclusion: Most of these HIV-1–infected men had bacteria in their semen. ART use was associated with undetectable semen HIV-1 RNA and lower semen bacterial concentrations, whereas insertive anal sex was associated with higher bacterial concentrations. Additional studies evaluating the relationship between semen bacteria, inflammation, mucosal immunity, and HIV-1 shedding are needed to understand implications for HIV-1 transmission. PMID:27861240

Quantitative secondary electron detection

DOE Office of Scientific and Technical Information (OSTI.GOV)

Agrawal, Jyoti; Joy, David C.; Nayak, Subuhadarshi

Quantitative Secondary Electron Detection (QSED) using the array of solid state devices (SSD) based electron-counters enable critical dimension metrology measurements in materials such as semiconductors, nanomaterials, and biological samples (FIG. 3). Methods and devices effect a quantitative detection of secondary electrons with the array of solid state detectors comprising a number of solid state detectors. An array senses the number of secondary electrons with a plurality of solid state detectors, counting the number of secondary electrons with a time to digital converter circuit in counter mode.

A guide to enterotypes across the human body: meta-analysis of microbial community structures in human microbiome datasets.

PubMed

Koren, Omry; Knights, Dan; Gonzalez, Antonio; Waldron, Levi; Segata, Nicola; Knight, Rob; Huttenhower, Curtis; Ley, Ruth E

2013-01-01

Recent analyses of human-associated bacterial diversity have categorized individuals into 'enterotypes' or clusters based on the abundances of key bacterial genera in the gut microbiota. There is a lack of consensus, however, on the analytical basis for enterotypes and on the interpretation of these results. We tested how the following factors influenced the detection of enterotypes: clustering methodology, distance metrics, OTU-picking approaches, sequencing depth, data type (whole genome shotgun (WGS) vs.16S rRNA gene sequence data), and 16S rRNA region. We included 16S rRNA gene sequences from the Human Microbiome Project (HMP) and from 16 additional studies and WGS sequences from the HMP and MetaHIT. In most body sites, we observed smooth abundance gradients of key genera without discrete clustering of samples. Some body habitats displayed bimodal (e.g., gut) or multimodal (e.g., vagina) distributions of sample abundances, but not all clustering methods and workflows accurately highlight such clusters. Because identifying enterotypes in datasets depends not only on the structure of the data but is also sensitive to the methods applied to identifying clustering strength, we recommend that multiple approaches be used and compared when testing for enterotypes.

A Guide to Enterotypes across the Human Body: Meta-Analysis of Microbial Community Structures in Human Microbiome Datasets

PubMed Central

Waldron, Levi; Segata, Nicola; Knight, Rob; Huttenhower, Curtis; Ley, Ruth E.

2013-01-01

Recent analyses of human-associated bacterial diversity have categorized individuals into ‘enterotypes’ or clusters based on the abundances of key bacterial genera in the gut microbiota. There is a lack of consensus, however, on the analytical basis for enterotypes and on the interpretation of these results. We tested how the following factors influenced the detection of enterotypes: clustering methodology, distance metrics, OTU-picking approaches, sequencing depth, data type (whole genome shotgun (WGS) vs.16S rRNA gene sequence data), and 16S rRNA region. We included 16S rRNA gene sequences from the Human Microbiome Project (HMP) and from 16 additional studies and WGS sequences from the HMP and MetaHIT. In most body sites, we observed smooth abundance gradients of key genera without discrete clustering of samples. Some body habitats displayed bimodal (e.g., gut) or multimodal (e.g., vagina) distributions of sample abundances, but not all clustering methods and workflows accurately highlight such clusters. Because identifying enterotypes in datasets depends not only on the structure of the data but is also sensitive to the methods applied to identifying clustering strength, we recommend that multiple approaches be used and compared when testing for enterotypes. PMID:23326225

Diversity of anaerobic microbes in spacecraft assembly clean rooms.

PubMed

Probst, Alexander; Vaishampayan, Parag; Osman, Shariff; Moissl-Eichinger, Christine; Andersen, Gary L; Venkateswaran, Kasthuri

2010-05-01

Although the cultivable and noncultivable microbial diversity of spacecraft assembly clean rooms has been previously documented using conventional and state-of-the-art molecular techniques, the occurrence of obligate anaerobes within these clean rooms is still uncertain. Therefore, anaerobic bacterial communities of three clean-room facilities were analyzed during assembly of the Mars Science Laboratory rover. Anaerobic bacteria were cultured on several media, and DNA was extracted from suitable anaerobic enrichments and examined with conventional 16S rRNA gene clone library, as well as high-density phylogenetic 16S rRNA gene microarray (PhyloChip) technologies. The culture-dependent analyses predominantly showed the presence of clostridial and propionibacterial strains. The 16S rRNA gene sequences retrieved from clone libraries revealed distinct microbial populations associated with each clean-room facility, clustered exclusively within gram-positive organisms. PhyloChip analysis detected a greater microbial diversity, spanning many phyla of bacteria, and provided a deeper insight into the microbial community structure of the clean-room facilities. This study presents an integrated approach for assessing the anaerobic microbial population within clean-room facilities, using both molecular and cultivation-based analyses. The results reveal that highly diverse anaerobic bacterial populations persist in the clean rooms even after the imposition of rigorous maintenance programs and will pose a challenge to planetary protection implementation activities.

The nucleotide sequence of the intergenic region between the 5.8S and 26S rRNA genes of the yeast ribosomal RNA operon. Possible implications for the interaction between 5.8S and 26S rRNA and the processing of the primary transcript.

PubMed Central

Veldman, G M; Klootwijk, J; van Heerikhuizen, H; Planta, R J

1981-01-01

We have determined the nucleotide sequence of part of a cloned yeast ribosomal RNA operon extending from the 5.8S RNA gene downstream into the 5' -terminal region of the 26S RNA gene. We mapped the pertinent processing sites, viz. the 5' end of 26S rRNA and the 3'ends of 5.8S rRNA and its immediate precursor, 7S RNA. At the 3' end of 7S RNA we find the sequence UCGUUU which is very similar to the type I consensus sequence UCAUUA/U present at the 3' ends of 17S, 5.8S and 26S rRNA as well as 18S precursor rRNA in yeast. At the 5' end of the 26S RNA gene we find a sequence of thirteen nucleotides which is homologous to the type II sequence present at the 5' termini of both the 17S and the 5.8S RNA gene. These findings further support the suggestion put forward earlier (G.M. Veldman et al. (1980) Nucl. Acids Res. 8, 2907-2920) that both consensus sequences are involved in the recognition of precursor rRNA by the processing nuclease(s). We discuss a model for the processing of yeast rRNA in which a processing enzyme sequentially recognizes several combinations of a type I and a type II consensus sequence. We also describe the existence of a significant base complementarity between sequences in the 5' -terminal region of 26S rRNA and the 3' -terminal region of 5.8S rRNA. We suggest that base pairing between these sequences contributes to the binding between 5.8S and 26S rRNA. Images PMID:7312619

Development of a polymerase chain reaction-based assay for the detection of Alternaria fungal contamination in food products.

PubMed

Zur, G; Hallerman, E M; Sharf, R; Kashi, Y

1999-10-01

Alternaria sp. are important fungal contaminants of vegetable, fruit, and grain products, including Alternaria alternata, a contaminant of tomato products. To date, the Howard method, based on microscopic observation of fungal filaments, has been the standard examination for inspection of tomato products. We report development of a polymerase chain reaction (PCR)-based method for detection of Alternaria DNA. PCR primers were designed to anneal to the internal transcribed regions ITS1 and ITS2 of the 5.8S rRNA gene of Alternaria but not to other microbial or tomato DNA. We demonstrate use of the PCR assay to detect Alternaria DNA in experimentally infested and commercially obtained tomato sauce and tomato powder. Use of the PCR method offers a rapid and sensitive assay for the presence of Alternaria DNA in tomato products. The apparent breakdown of DNA in tomato sauce may limit the utility of the assay to freshly prepared products. The assay for tomato powder is not affected by storage time.

Identification of Medically Important Yeasts Using PCR-Based Detection of DNA Sequence Polymorphisms in the Internal Transcribed Spacer 2 Region of the rRNA Genes

PubMed Central

Chen, Y. C.; Eisner, J. D.; Kattar, M. M.; Rassoulian-Barrett, S. L.; LaFe, K.; Yarfitz, S. L.; Limaye, A. P.; Cookson, B. T.

2000-01-01

Identification of medically relevant yeasts can be time-consuming and inaccurate with current methods. We evaluated PCR-based detection of sequence polymorphisms in the internal transcribed spacer 2 (ITS2) region of the rRNA genes as a means of fungal identification. Clinical isolates (401), reference strains (6), and type strains (27), representing 34 species of yeasts were examined. The length of PCR-amplified ITS2 region DNA was determined with single-base precision in less than 30 min by using automated capillary electrophoresis. Unique, species-specific PCR products ranging from 237 to 429 bp were obtained from 92% of the clinical isolates. The remaining 8%, divided into groups with ITS2 regions which differed by ≤2 bp in mean length, all contained species-specific DNA sequences easily distinguishable by restriction enzyme analysis. These data, and the specificity of length polymorphisms for identifying yeasts, were confirmed by DNA sequence analysis of the ITS2 region from 93 isolates. Phenotypic and ITS2-based identification was concordant for 427 of 434 yeast isolates examined using sequence identity of ≥99%. Seven clinical isolates contained ITS2 sequences that did not agree with their phenotypic identification, and ITS2-based phylogenetic analyses indicate the possibility of new or clinically unusual species in the Rhodotorula and Candida genera. This work establishes an initial database, validated with over 400 clinical isolates, of ITS2 length and sequence polymorphisms for 34 species of yeasts. We conclude that size and restriction analysis of PCR-amplified ITS2 region DNA is a rapid and reliable method to identify clinically significant yeasts, including potentially new or emerging pathogenic species. PMID:10834993

Planetary protection - assaying new methods

NASA Astrophysics Data System (ADS)

Nellen, J.; Rettberg, P.; Horneck, G.

Space age began in 1957 when the USSR launched the first satellite into earth orbit. In response to this new challenge the International Council for Science, formerly know as International Council of Scientific Unions (ICSU), established the Committee on Space Research (COSPAR) in 1958. The role of COSPAR was to channel the international scientific research in space and establish an international forum. Through COSPAR the scientific community agreed on the need for screening interplanetary probes for forward (contamination of foreign planets) and backward (contamination of earth by returned samples/probes) contamination. To prevent both forms of contamination a set of rules, as a guideline was established. Nowadays the standard implementation of the planetary protection rules is based on the experience gained during NASA's Viking project in 1975/76. Since then the evaluation-methods for microbial contamination of spacecrafts have been changed or updated just slowly. In this study the standard method of sample taking will be evaluated. New methods for examination of those samples, based on the identification of life on the molecular level, will be reviewed and checked for their feasibility as microbial detection systems. The methods will be examined for their qualitative (detection and verification of different organisms) and quantitative (detection limit and concentration verification) qualities. Amongst the methods analyzed will be i.e. real-time / PCR (poly-chain-reaction), using specific primer-sets for the amplification of highly conserved rRNA or DNA regions. Measurement of intrinsic fluorescence, i.e ATP using luciferin-luciferase reagents. The use of FAME (fatty acid methyl esters) and microchips for microbial identification purposes. The methods will be chosen to give a good overall coverage of different possible molecular markers and approaches. The most promising methods shall then be lab-tested and evaluated for their use under spacecraft assembly conditions. Since mars became one of the most sought-after planets in our solar system and will be visited by man-made probes quiet often in the near future, planetary protection is as important as never before.

Housekeeping while brain's storming Validation of normalizing factors for gene expression studies in a murine model of traumatic brain injury

PubMed Central

Rhinn, Hervé; Marchand-Leroux, Catherine; Croci, Nicole; Plotkine, Michel; Scherman, Daniel; Escriou, Virginie

2008-01-01

Background Traumatic brain injury models are widely studied, especially through gene expression, either to further understand implied biological mechanisms or to assess the efficiency of potential therapies. A large number of biological pathways are affected in brain trauma models, whose elucidation might greatly benefit from transcriptomic studies. However the suitability of reference genes needed for quantitative RT-PCR experiments is missing for these models. Results We have compared five potential reference genes as well as total cDNA level monitored using Oligreen reagent in order to determine the best normalizing factors for quantitative RT-PCR expression studies in the early phase (0–48 h post-trauma (PT)) of a murine model of diffuse brain injury. The levels of 18S rRNA, and of transcripts of β-actin, glyceraldehyde-3P-dehydrogenase (GAPDH), β-microtubulin and S100β were determined in the injured brain region of traumatized mice sacrificed at 30 min, 3 h, 6 h, 12 h, 24 h and 48 h post-trauma. The stability of the reference genes candidates and of total cDNA was evaluated by three different methods, leading to the following rankings as normalization factors, from the most suitable to the less: by using geNorm VBA applet, we obtained the following sequence: cDNA(Oligreen); GAPDH > 18S rRNA > S100β > β-microtubulin > β-actin; by using NormFinder Excel Spreadsheet, we obtained the following sequence: GAPDH > cDNA(Oligreen) > S100β > 18S rRNA > β-actin > β-microtubulin; by using a Confidence-Interval calculation, we obtained the following sequence: cDNA(Oligreen) > 18S rRNA; GAPDH > S100β > β-microtubulin > β-actin. Conclusion This work suggests that Oligreen cDNA measurements, 18S rRNA and GAPDH or a combination of them may be used to efficiently normalize qRT-PCR gene expression in mouse brain trauma injury, and that β-actin and β-microtubulin should be avoided. The potential of total cDNA as measured by Oligreen as a first-intention normalizing factor with a broad field of applications is highlighted. Pros and cons of the three methods of normalization factors selection are discussed. A generic time- and cost-effective procedure for normalization factor validation is proposed. PMID:18611280

5S rRNA and ribosome.

PubMed

Gongadze, G M

2011-12-01

5S rRNA is an integral component of the ribosome of all living organisms. It is known that the ribosome without 5S rRNA is functionally inactive. However, the question about the specific role of this RNA in functioning of the translation apparatus is still open. This review presents a brief history of the discovery of 5S rRNA and studies of its origin and localization in the ribosome. The previously expressed hypotheses about the role of this RNA in the functioning of the ribosome are discussed considering the unique location of 5S rRNA in the ribosome and its intermolecular contacts. Based on analysis of the current data on ribosome structure and its functional complexes, the role of 5S rRNA as an intermediary between ribosome functional domains is discussed.

Identification of vector-borne pathogens in dogs and cats from Southern Brazil.

PubMed

Malheiros, J; Costa, M M; do Amaral, R B; de Sousa, K C M; André, M R; Machado, R Z; Vieira, M I B

2016-07-01

Dogs and cats are often infected with vector-borne pathogens and play a crucial role as reservoirs and hosts in their life cycles. The aim of the present study was to investigate the occurrence of vector-borne pathogens among dogs and cats in the northwestern region of Rio Grande do Sul (RS) State, Brazil. One hundred and ten blood samples were collected from dogs (n=80) and cats (n=30). Laboratory analysis were carried out through stained blood smears, indirect enzyme-linked immunosorbent assay (ELISA) for Babesia vogeli and Ehrlichia canis (only for dogs) and polymerase chain reaction (PCR) aiming the detection of pathogens. The following pathogens were screened by PCR among dogs and cats: Babesia spp. and Hepatozoon spp. (18S rRNA gene), Anaplasma spp. (16S rRNA gene), and Ehrlichia spp. (dsb gene for dogs and 16S rRNA gene for cats) and Bartonella spp. (nuoG gene only for cats). Using blood smears structures morphologically compatible with piroplasms were found in 5.45% (6/110) of the samples. Anti-B. vogeli and anti-E. canis antibodies were detected in 91% (73/80) and 9% (7/80) of the dogs, respectively. All the seropositive dogs to E. canis were also to B. vogeli. Nineteen (17.3%) animals were positive to hemoparasites by PCR. After sequencing Rangelia vitalii 6/80 (7.5%), B. vogeli 3/80 (4%), Hepatozoon spp. 1/80 (1%), and Anaplasma spp. 1/80 (1%) were found in the dogs, and B. vogeli 2/30 (7%) and Bartonella spp. 6/30 (20%) were detected in the screened cats. No sample was positive for genes dsb and 16S rRNA of Ehrlichia spp. Only those animals which were positive for R. vitalii showed findings compatible with rangeliosis, such as anemia (100%), thrombocytopenia (67%), jaundice (50%), external bleeding (50%), and anorexia (50%). This is the first time that B. vogeli detected among cats in Southern Brazil. Copyright © 2016 Elsevier GmbH. All rights reserved.

Improving membrane based multiplex immunoassays for semi-quantitative detection of multiple cytokines in a single sample

PubMed Central

2014-01-01

Background Inflammatory mediators can serve as biomarkers for the monitoring of the disease progression or prognosis in many conditions. In the present study we introduce an adaptation of a membrane-based technique in which the level of up to 40 cytokines and chemokines can be determined in both human and rodent blood in a semi-quantitative way. The planar assay was modified using the LI-COR (R) detection system (fluorescence based) rather than chemiluminescence and semi-quantitative outcomes were achieved by normalizing the outcomes using the automated exposure settings of the Odyssey readout device. The results were compared to the gold standard assay, namely ELISA. Results The improved planar assay allowed the detection of a considerably higher number of analytes (n = 30 and n = 5 for fluorescent and chemiluminescent detection, respectively). The improved planar method showed high sensitivity up to 17 pg/ml and a linear correlation of the normalized fluorescence intensity with the results from the ELISA (r = 0.91). Conclusions The results show that the membrane-based technique is a semi-quantitative assay that correlates satisfactorily to the gold standard when enhanced by the use of fluorescence and subsequent semi-quantitative analysis. This promising technique can be used to investigate inflammatory profiles in multiple conditions, particularly in studies with constraints in sample sizes and/or budget. PMID:25022797

Reclassification of Theileria annae as Babesia vulpes sp. nov.

PubMed

Baneth, Gad; Florin-Christensen, Monica; Cardoso, Luís; Schnittger, Leonhard

2015-04-08

Theileria annae is a tick-transmitted small piroplasmid that infects dogs and foxes in North America and Europe. Due to disagreement on its placement in the Theileria or Babesia genera, several synonyms have been used for this parasite, including Babesia Spanish dog isolate, Babesia microti-like, Babesia (Theileria) annae, and Babesia cf. microti. Infections by this parasite cause anemia, thrombocytopenia, and azotemia in dogs but are mostly subclinical in red foxes (Vulpes vulpes). Furthermore, high infection rates have been detected among red fox populations in distant regions strongly suggesting that these canines act as the parasite's natural host. This study aims to reassess and harmonize the phylogenetic placement and binomen of T. annae within the order Piroplasmida. Four molecular phylogenetic trees were constructed using a maximum likelihood algorithm based on DNA alignments of: (i) near-complete 18S rRNA gene sequences (n = 76 and n = 93), (ii) near-complete and incomplete 18S rRNA gene sequences (n = 92), and (iii) tubulin-beta gene sequences (n = 32) from B. microti and B. microti-related parasites including those detected in dogs and foxes. All phylogenetic trees demonstrate that T. annae and its synonyms are not Theileria parasites but are most closely related with B. microti. The phylogenetic tree based on the 18S rRNA gene forms two separate branches with high bootstrap value, of which one branch corresponds to Babesia species infecting rodents, humans, and macaques, while the other corresponds to species exclusively infecting carnivores. Within the carnivore group, T. annae and its synonyms from distant regions segregate into a single clade with a highly significant bootstrap value corroborating their separate species identity. Phylogenetic analysis clearly shows that T. annae and its synonyms do not pertain to Theileria and can be clearly defined as a separate species. Based on the facts that T. annae and its synonyms have not been shown to have a leukocyte stage, as expected in Theileria, do not infect humans and rodents as B. microti, and cluster phylogenetically as a separate species, this study proposes to name this parasite Babesia vulpes sp. nov., after its natural host, the red fox V. vulpes.

Molecular genotyping of Giardia duodenalis in children from Behbahan, southwestern Iran.

PubMed

Kasaei, Raziyeh; Carmena, David; Jelowdar, Ali; Beiromvand, Molouk

2018-05-01

Giardia duodenalis is an intestinal flagellated protozoan that infects humans and several animal species. Giardiasis causing more than 200 million symptomatic infections globally is one of the most common causes of diarrhea in developing countries. Based on molecular studies mainly targeting the small-subunit (SSU) rRNA gene locus of the parasite, eight assemblages (A to H) have been identified in humans and other animal species. The aim of the current study was to evaluate the frequency and molecular diversity of G. duodenalis in children from rural and urban day care centers from Behbahan, southwestern Iran. This cross-sectional study was based on a concentration method for the microscopic detection of G. duodenalis in stool samples of 450 children, aged 1-7 years, in Behbahan, southwestern Iran. The survey was conducted from December 2015 to May 2016. PCR methods targeting the SSU rRNA and triose phosphate isomerase (TPI) genes of G. duodenalis were used for the identification and genotyping of the parasite isolates. Based on sucrose flotation and microscopy techniques, 2.7% (12/450) of children were infected with G. duodenalis, of which six (50.0%) were males and the other six (50.0%) were females. Overall, 91.7% (11/12) of the infections were detected in children from rural areas. The SSU rRNA and TPI genes were amplified successfully in nine and eight, respectively, of the Giardia-positive samples at microscopy. Among the eight TPI sequences, assemblage A, sub-assemblage AII, was identified in five of the isolates. The sequences of the three remaining samples were untypable. Although no significantly statistical difference between genotype and clinical symptoms was found, five out of the eight isolates identified as assemblage A were obtained in asymptomatic children. Giardia duodenalis infections were more prevalent in children from rural day care schools, and the predominant assemblage was A, sub-assemblage AII. The higher prevalence of giardiasis in rural areas might be related to differences in personal hygiene habits, parents' education level, source of drinking water, and inadequate hygienic toilet facilities in rural areas.

Application of TaqMan qPCR for the detection and monitoring of Naegleria species in reservoirs used as a source for drinking water.

PubMed

Kao, Po-Min; Hsu, Bing-Mu; Hsu, Tsui-Kang; Chiu, Yi-Chou; Chang, Chung-Liang; Ji, Wen-Tsai; Huang, Shih-Wei; Fan, Cheng-Wei

2014-10-01

Naegleria spp. can be found in the natural aquatic environments. Naegleria fowleri can cause fatal infections in the central nervous system in humans and animals, and the most important source of infection is through direct water contact. In this study, PCR of 5.8S ribosomal RNA (rRNA) gene and internal transcribed spacer (ITS) region was performed in order to identify Naegleria isolates and quantify the Naegleria spp. by TaqMan real-time quantitative PCR in reservoir water samples. The occurrence of Naegleria spp. was investigated in 57 water samples from reservoirs with culture and PCR positive in 2 of them (3.5%), respectively. The total detection rate was 7.0% (4/ 57) for Naegleria spp. The identified species included Naegleria spp., Naegleria canariensis, and Naegleria clarki. N. fowleri was not found in Taiwan's reservoirs used for drinking purposes. The concentrations of Naegleria spp. in detected positive reservoir water samples were in the range of 599 and 3.1 × 10(3) cells/L. The presence or absence of Naegleria spp. within the reservoir water samples showed significant difference with the levels of water temperature. The presence of Naegleria spp. in reservoirs considered a potential public health threat if pathogenic species exist in reservoirs.

A morphogenetic survey on ciliate plankton from a mountain lake pinpoints the necessity of lineage-specific barcode markers in microbial ecology.

PubMed

Stoeck, Thorsten; Breiner, Hans-Werner; Filker, Sabine; Ostermaier, Veronika; Kammerlander, Barbara; Sonntag, Bettina

2014-02-01

Analyses of high-throughput environmental sequencing data have become the 'gold-standard' to address fundamental questions of microbial diversity, ecology and biogeography. Findings that emerged from sequencing are, e.g. the discovery of the extensive 'rare microbial biosphere' and its potential function as a seed-bank. Even though applied since several years, results from high-throughput environmental sequencing have hardly been validated. We assessed how well pyrosequenced amplicons [the hypervariable eukaryotic V4 region of the small subunit ribosomal RNA (SSU rRNA) gene] reflected morphotype ciliate plankton. Moreover, we assessed if amplicon sequencing had the potential to detect the annual ciliate plankton stock. In both cases, we identified significant quantitative and qualitative differences. Our study makes evident that taxon abundance distributions inferred from amplicon data are highly biased and do not mirror actual morphotype abundances at all. Potential reasons included cell losses after fixation, cryptic morphotypes, resting stages, insufficient sequence data availability of morphologically described species and the unsatisfying resolution of the V4 SSU rRNA fragment for accurate taxonomic assignments. The latter two underline the necessity of barcoding initiatives for eukaryotic microbes to better and fully exploit environmental amplicon data sets, which then will also allow studying the potential of seed-bank taxa as a buffer for environmental changes. © 2013 The Authors. Environmental Microbiology published by Society for Applied Microbiology and John Wiley & Sons Ltd.

Polyphasic Analyses of Methanogenic Archaeal Communities in Agricultural Biogas Plants▿

PubMed Central

Nettmann, E.; Bergmann, I.; Pramschüfer, S.; Mundt, K.; Plogsties, V.; Herrmann, C.; Klocke, M.

2010-01-01

Knowledge of the microbial consortia participating in the generation of biogas, especially in methane formation, is still limited. To overcome this limitation, the methanogenic archaeal communities in six full-scale biogas plants supplied with different liquid manures and renewable raw materials as substrates were analyzed by a polyphasic approach. Fluorescence in situ hybridization (FISH) was carried out to quantify the methanogenic Archaea in the reactor samples. In addition, quantitative real-time PCR (Q-PCR) was used to support and complete the FISH analysis. Five of the six biogas reactors were dominated by hydrogenotrophic Methanomicrobiales. The average values were between 60 to 63% of archaeal cell counts (FISH) and 61 to 99% of archaeal 16S rRNA gene copies (Q-PCR). Within this order, Methanoculleus was found to be the predominant genus as determined by amplified rRNA gene restriction analysis. The aceticlastic family Methanosaetaceae was determined to be the dominant methanogenic group in only one biogas reactor, with average values for Q-PCR and FISH between 64% and 72%. Additionally, in three biogas reactors hitherto uncharacterized but potentially methanogenic species were detected. They showed closest accordance with nucleotide sequences of the hitherto unclassified CA-11 (85%) and ARC-I (98%) clusters. These results point to hydrogenotrophic methanogenesis as a predominant pathway for methane synthesis in five of the six analyzed biogas plants. In addition, a correlation between the absence of Methanosaetaceae in the biogas reactors and high concentrations of total ammonia (sum of NH3 and NH4+) was observed. PMID:20154117

New insight into stratification of anaerobic methanotrophs in cold seep sediments.

PubMed

Roalkvam, Irene; Jørgensen, Steffen Leth; Chen, Yifeng; Stokke, Runar; Dahle, Håkon; Hocking, William Peter; Lanzén, Anders; Haflidason, Haflidi; Steen, Ida Helene

2011-11-01

Methane seepages typically harbor communities of anaerobic methane oxidizers (ANME); however, knowledge about fine-scale vertical variation of ANME in response to geochemical gradients is limited. We investigated microbial communities in sediments below a white microbial mat in the G11 pockmark at Nyegga by 16S rRNA gene tag pyrosequencing and real-time quantitative PCR. A vertical stratification of dominating ANME communities was observed at 4 cmbsf (cm below seafloor) and below in the following order: ANME-2a/b, ANME-1 and ANME-2c. The ANME-1 community was most numerous and comprised single or chains of cells with typical rectangular morphology, accounting up to 89.2% of the retrieved 16S rRNA gene sequences. Detection rates for sulfate-reducing Deltaproteobacteria possibly involved in anaerobic oxidation of methane were low throughout the core. However, a correlation in the abundance of Candidate division JS-1 with ANME-2 was observed, indicating involvement in metabolisms occurring in ANME-2-dominated horizons. The white microbial mat and shallow sediments were dominated by organisms affiliated with Sulfurovum (Epsilonproteobacteria) and Methylococcales (Gammaproteobacteria), suggesting that aerobic oxidation of sulfur and methane is taking place. In intermediate horizons, typical microbial groups associated with methane seeps were recovered. The data are discussed with respect to co-occurring microbial assemblages and interspecies interactions. FEMS Microbiology Ecology © 2011 Federation of Microbiological Societies. Published by Blackwell Publishing Ltd. No claim to original Norwegian works.

A web-based quantitative signal detection system on adverse drug reaction in China.

PubMed

Li, Chanjuan; Xia, Jielai; Deng, Jianxiong; Chen, Wenge; Wang, Suzhen; Jiang, Jing; Chen, Guanquan

2009-07-01

To establish a web-based quantitative signal detection system for adverse drug reactions (ADRs) based on spontaneous reporting to the Guangdong province drug-monitoring database in China. Using Microsoft Visual Basic and Active Server Pages programming languages and SQL Server 2000, a web-based system with three software modules was programmed to perform data preparation and association detection, and to generate reports. Information component (IC), the internationally recognized measure of disproportionality for quantitative signal detection, was integrated into the system, and its capacity for signal detection was tested with ADR reports collected from 1 January 2002 to 30 June 2007 in Guangdong. A total of 2,496 associations including known signals were mined from the test database. Signals (e.g., cefradine-induced hematuria) were found early by using the IC analysis. In addition, 291 drug-ADR associations were alerted for the first time in the second quarter of 2007. The system can be used for the detection of significant associations from the Guangdong drug-monitoring database and could be an extremely useful adjunct to the expert assessment of very large numbers of spontaneously reported ADRs for the first time in China.

Psychrophile spoilers dominate the bacterial microbiome in musculature samples of slaughter pigs.

PubMed

Mann, Evelyne; Wetzels, Stefanie U; Pinior, Beate; Metzler-Zebeli, Barbara U; Wagner, Martin; Schmitz-Esser, Stephan

2016-07-01

The aim of this study was to disentangle the microbial diversity on porcine musculature. The hypervariable V1-V2 region of the 16S rRNA gene was amplified from DNA samples of clinically healthy slaughter pigs (n=8). Pyrosequencing yielded 37,000 quality-controlled reads and a diverse microbiome with 54-159 OTUs per sample was detected. Interestingly, 6 out of 8 samples were strongly dominated by 1-2 highly abundant OTUs (best hits of highly abundant OTUs: Serratia proteamaculans, Pseudomonas syringae, Aeromonas allosaccharophila, Brochothrix thermosphacta, Acidiphilium cryptum and Escherichia coli). In 1g musculature scraping, 3.20E+06 16S rRNA gene copies and 4.45E+01 Enterobacteriaceae rRNA gene copies were detected with qPCR. We conclude that i.) next-generation sequencing technologies help encompass the full content of complex, bacterial contamination, ii.) psychrophile spoilers dominated the microbiota and iii.) E. coli is an effective marker species for pork contamination, as it was one of very few abundant species being present in all samples. Copyright © 2016 Elsevier Ltd. All rights reserved.

Distribution of cultivated and uncultivated cyanobacteria and Chloroflexus-like bacteria in hot spring microbial mats

NASA Technical Reports Server (NTRS)

Ruff-Roberts, A. L.; Kuenen, J. G.; Ward, D. M.

1994-01-01

Oligodeoxynucleotide hybridization probes were developed to complement specific regions of the small subunit (SSU) rRNA sequences of cultivated and uncultivated cyanobacteria and Chloroflexus-like bacteria, which inhabit hot spring microbial mats. The probes were used to investigate the natural distribution of SSU rRNAs from these species in mats of Yellowstone hot springs of different temperatures and pHs as well as changes in SSU rRNA distribution resulting from 1-week in situ shifts in temperature, pH, and light intensity. Synechococcus lividus Y-7c-s SSU rRNA was detected only in the mat of a slightly acid spring, from which it may have been initially isolated, or when samples from a more alkaline spring were incubated in the more acid spring. Chloroflexus aurantiacus Y-400-fl SSU rRNA was detected only in a high-temperature mat sample from the alkaline Octopus Spring or when lower-temperature samples from this mat were incubated at the high-temperature site. SSU rRNAs of uncultivated species were more widely distributed. Temperature distributions and responses to in situ temperature shifts suggested that some of the uncultivated cyanobacteria might be adapted to high-, moderate-, and low-temperature ranges whereas an uncultivated Chloroflexus-like bacterium appears to have broad temperature tolerance. SSU rRNAs of all uncultivated species inhabiting a 48 to 51 degrees C Octopus Spring mat site were most abundant in the upper 1 mm and were not detected below a 2.5-to 3.5-mm depth, a finding consistent with their possible phototrophic nature. However, the effects of light intensity reduction on these SSU rRNAs were variable, indicating the difficulty of demonstrating a phototrophic phenotype in light reduction experiments.

Development and validation of a duplex real-time PCR assay for the diagnosis of equine piroplasmosis.

PubMed

Lobanov, Vladislav A; Peckle, Maristela; Massard, Carlos L; Brad Scandrett, W; Gajadhar, Alvin A

2018-03-02

Equine piroplasmosis (EP) is an economically significant infection of horses and other equine species caused by the tick-borne protozoa Theileria equi and Babesia caballi. The long-term carrier state in infected animals makes importation of such subclinical cases a major risk factor for the introduction of EP into non-enzootic areas. Regulatory testing for EP relies on screening of equines by serological methods. The definitive diagnosis of EP infection in individual animals will benefit from the availability of sensitive direct detection methods, for example, when used as confirmatory assays for non-negative serological test results. The objectives of this study were to develop a real-time quantitative polymerase chain reaction (qPCR) assay for simultaneous detection of both agents of EP, perform comprehensive evaluation of its performance and assess the assay's utility for regulatory testing. We developed a duplex qPCR targeting the ema-1 gene of T. equi and the 18S rRNA gene of B. caballi and demonstrated that the assay has high analytical sensitivities for both piroplasm species. Validation of the duplex qPCR on samples from 362 competitive enzyme-linked immunosorbent assay (cELISA)-negative horses from Canada and the United States yielded no false-positive reactions. The assay's performance was further evaluated using samples collected from 430 horses of unknown EP status from a highly endemic area in Brazil. This set of samples was also tested by a single-target 18S rRNA qPCR for T. equi developed at the OIE reference laboratory for EP in Japan, and a previously published single-target 18S rRNA qPCR for B. caballi whose oligonucleotides we adopted for use in the duplex qPCR. Matching serum samples were tested for antibodies to these parasites using cELISA. By the duplex qPCR, T. equi-specific 18S rRNA qPCR and cELISA, infections with T. equi were detected in 87.9% (95% confidence interval, CI: 84.5-90.7%), 90.5% (95% CI: 87.3-92.3%) and 87.4% (95% CI: 84.0-90.2%) of the horses, respectively. The B. caballi prevalence estimates were 9.3% (95% CI: 6.9-12.4%) by the duplex qPCR and 7.9% (95% CI: 5.7-10.9%) by the respective single-target qPCR assay. These values were markedly lower compared to the seroprevalence of 58.6% (95% CI: 53.9-63.2%) obtained by B. caballi-specific cELISA. The relative diagnostic sensitivity of the duplex qPCR for T. equi was 95.5%, as 359 of the 376 horses with exposure to T. equi confirmed by cELISA had parasitemia levels above the detection limit of the molecular assay. In contrast, only 39 (15.5%) of the 252 horses with detectable B. caballi-specific antibodies were positive for this piroplasm species by the duplex qPCR. The duplex qPCR described here performed comparably to the existing single-target qPCR assays for T. equi and B. caballi and will be more cost-effective in terms of results turnaround time and reagent costs when both pathogens are being targeted for disease control and epidemiological investigations. These validation data also support the reliability of the ema-1 gene-specific oligonucleotides developed in this study for confirmatory testing of non-negative serological test results for T. equi by qPCR. However, the B. caballi-specific qPCR cannot be similarly recommended as a confirmatory assay for routine regulatory testing due to the low level of agreement with serological test results demonstrated in this study. Further studies are needed to determine the transmission risk posed by PCR-negative equines with detectable antibodies to B. caballi.

Prevalence of Antibiotic Resistance Genes and Bacterial Community Composition in a River Influenced by a Wastewater Treatment Plant

PubMed Central

Marti, Elisabet; Jofre, Juan; Balcazar, Jose Luis

2013-01-01

Antibiotic resistance represents a global health problem, requiring better understanding of the ecology of antibiotic resistance genes (ARGs), their selection and their spread in the environment. Antibiotics are constantly released to the environment through wastewater treatment plant (WWTP) effluents. We investigated, therefore, the effect of these discharges on the prevalence of ARGs and bacterial community composition in biofilm and sediment samples of a receiving river. We used culture-independent approaches such as quantitative PCR to determine the prevalence of eleven ARGs and 16S rRNA gene-based pyrosequencing to examine the composition of bacterial communities. Concentration of antibiotics in WWTP influent and effluent were also determined. ARGs such as qnrS, bla TEM, bla CTX-M, bla SHV, erm(B), sul(I), sul(II), tet(O) and tet(W) were detected in all biofilm and sediment samples analyzed. Moreover, we observed a significant increase in the relative abundance of ARGs in biofilm samples collected downstream of the WWTP discharge. We also found significant differences with respect to community structure and composition between upstream and downstream samples. Therefore, our results indicate that WWTP discharges may contribute to the spread of ARGs into the environment and may also impact on the bacterial communities of the receiving river. PMID:24205347

Microbial diversity in a submarine carbonate edifice from the serpentinizing hydrothermal system of the Prony Bay (New Caledonia) over a 6-year period.

PubMed

Postec, Anne; Quéméneur, Marianne; Bes, Méline; Mei, Nan; Benaïssa, Fatma; Payri, Claude; Pelletier, Bernard; Monnin, Christophe; Guentas-Dombrowsky, Linda; Ollivier, Bernard; Gérard, Emmanuelle; Pisapia, Céline; Gérard, Martine; Ménez, Bénédicte; Erauso, Gaël

2015-01-01

Active carbonate chimneys from the shallow marine serpentinizing Prony Hydrothermal Field were sampled 3 times over a 6 years period at site ST09. Archaeal and bacterial communities composition was investigated using PCR-based methods (clone libraries, Denaturating Gel Gradient Electrophoresis, quantitative PCR) targeting 16S rRNA genes, methyl coenzyme M reductase A and dissimilatory sulfite reductase subunit B genes. Methanosarcinales (Euryarchaeota) and Thaumarchaea were the main archaeal members. The Methanosarcinales, also observed by epifluorescent microscopy and FISH, consisted of two phylotypes that were previously solely detected in two other serpentinitzing ecosystems (The Cedars and Lost City Hydrothermal Field). Surprisingly, members of the hyperthermophilic order Thermococcales were also found which may indicate the presence of a hot subsurface biosphere. The bacterial community mainly consisted of Firmicutes, Chloroflexi, Alpha-, Gamma-, Beta-, and Delta-proteobacteria and of the candidate division NPL-UPA2. Members of these taxa were consistently found each year and may therefore represent a stable core of the indigenous bacterial community of the PHF chimneys. Firmicutes isolates representing new bacterial taxa were obtained by cultivation under anaerobic conditions. Our study revealed diverse microbial communities in PHF ST09 related to methane and sulfur compounds that share common populations with other terrestrial or submarine serpentinizing ecosystems.

Molecular characterization of Hepatozoon species in reptiles from the Seychelles.

PubMed

Harris, D James; Maia, João P M C; Perera, Ana

2011-02-01

Hepatozoon parasites were examined for the first time in reptiles from the Seychelles Islands. Although both prevalence and intensity were low, Hepatozoon species were detected in individuals from 2 endemic species, the lizard Mabuya wrightii and the snake Lycognathophis seychellensis. This was confirmed using visual identification and through sequencing part of the 18s rRNA gene. Phylogenetic analysis indicates that the Hepatozoon on the Seychelles form a monophyletic lineage, although more data are clearly needed to stabilize estimates of relationships based on this marker.

Isolation and partial characterization of halotolerant lactic acid bacteria from two Mexican cheeses.

PubMed

Morales, Fredy; Morales, Jesús I; Hernández, César H; Hernández-Sánchez, Humberto

2011-07-01

Isolated strains of halotolerant or halophilic lactic acid bacteria (HALAB) from Cotija and doble crema cheeses were identified and partially characterized by phenotypic and genotypic methods, and their technological abilities were studied in order to test their potential use as dairy starter components. Humidity, a(w), pH, and salt concentration of cheeses were determined. Genotypic diversity was evaluated by randomly amplified polymorphic DNA-polymerase chain reaction. Molecular identification and phylogenetic reconstructions based on 16S rRNA gene sequences were performed. Additional technological abilities such as salt tolerance, acidifying, and proteolytic and lipolytic activities were also investigated. The differences among strains reflected the biodiversity of HALAB in both types of cheeses. Lactobacillus acidipiscis, Tetragenococcus halophilus, Weissella thailandensis, and Lactobacillus pentosus from Cotija cheese, and L. acidipiscis, Enterococcus faecium, Lactobacillus plantarum, Lactobacillus farciminis, and Lactobacillus rhamnosus from doble crema cheese were identified based on 16S rRNA. Quantitative and qualitative assessments showed strains of T. halophilus and L. plantarum to be proteolytic, along with E. faecium, L. farciminis, and L. pentosus to a lesser extent. Lipolytic activity could be demonstrated in strains of E. faecium, L. pentosus, L. plantarum, and T. halophilus. Strains belonging to the species L. pentosus, L. plantarum, and E. faecium were able to acidify the milk media. This study evidences the presence of HALAB that may play a role in the ripening of cheeses.

Diversity, Biogeography, and Biodegradation Potential of Actinobacteria in the Deep-Sea Sediments along the Southwest Indian Ridge

PubMed Central

Chen, Ping; Zhang, Limin; Guo, Xiaoxuan; Dai, Xin; Liu, Li; Xi, Lijun; Wang, Jian; Song, Lei; Wang, Yuezhu; Zhu, Yaxin; Huang, Li; Huang, Ying

2016-01-01

The phylum Actinobacteria has been reported to be common or even abundant in deep marine sediments, however, knowledge about the diversity, distribution, and function of actinobacteria is limited. In this study, actinobacterial diversity in the deep sea along the Southwest Indian Ridge (SWIR) was investigated using both 16S rRNA gene pyrosequencing and culture-based methods. The samples were collected at depths of 1662–4000 m below water surface. Actinobacterial sequences represented 1.2–9.1% of all microbial 16S rRNA gene amplicon sequences in each sample. A total of 5 actinobacterial classes, 17 orders, 28 families, and 52 genera were detected by pyrosequencing, dominated by the classes Acidimicrobiia and Actinobacteria. Differences in actinobacterial community compositions were found among the samples. The community structure showed significant correlations to geochemical factors, notably pH, calcium, total organic carbon, total phosphorus, and total nitrogen, rather than to spatial distance at the scale of the investigation. In addition, 176 strains of the Actinobacteria class, belonging to 9 known orders, 18 families, and 29 genera, were isolated. Among these cultivated taxa, 8 orders, 13 families, and 15 genera were also recovered by pyrosequencing. At a 97% 16S rRNA gene sequence similarity, the pyrosequencing data encompassed 77.3% of the isolates but the isolates represented only 10.3% of the actinobacterial reads. Phylogenetic analysis of all the representative actinobacterial sequences and isolates indicated that at least four new orders within the phylum Actinobacteria were detected by pyrosequencing. More than half of the isolates spanning 23 genera and all samples demonstrated activity in the degradation of refractory organics, including polycyclic aromatic hydrocarbons and polysaccharides, suggesting their potential ecological functions and biotechnological applications for carbon recycling. PMID:27621725

Antimicrobial susceptibility and molecular characterization of macrolide resistance of Mycoplasma bovis isolates from multiple provinces in China

PubMed Central

KONG, Ling-Cong; GAO, Duo; JIA, Bo-Yan; WANG, Zi; GAO, Yun-Hang; PEI, Zhi-Hua; LIU, Shu-Ming; XIN, Jiu-Qing; MA, Hong-Xia

2015-01-01

Mycoplasma bovis has spread widely throughout the world via animal movement and has become an important pathogen of bovine respiratory disease. However, the minimum inhibitory concentrations of antimicrobials for Mycoplasma bovis have not been studied in China. The objective of this study was to determine the prevalence and antibiotic resistance of Mycoplasma bovis isolated from young cattle with respiratory infection in China. Mycoplasma bovis was detected in 32/45 bovine respiratory infection outbreaks at beef farms in 8 provinces in China. The isolates were susceptible or had medium sensitivity to ciprofloxacin, enrofloxacin and doxycycline, but were frequently resistant to macrolides (13/32, 41%). An A2058G (Escherichia coli Numbering) mutation located in the rrnA operon in domain V of 23S rRNA was observed in strains that were resistant to macrolides. This single mutations at the rrnA operon in domain V of 23S rRNA may play an important role in the resistance of Mycoplasma bovis strains to macrolides. PMID:26346744

Identification and molecular survey of Borrelia burgdorferi sensu lato in sika deer (Cervus nippon) from Jilin Province, north-eastern China.

PubMed

Zhai, Bintao; Niu, Qingli; Yang, Jifei; Liu, Zhijie; Liu, Junlong; Yin, Hong; Zeng, Qiaoying

2017-02-01

Lyme disease caused by Borrelia burgdorferi sensu lato (s.l.) is a common disease of domestic animals and wildlife worldwide. Sika deer is first-grade state-protected wildlife animals in China and have economic consequences for humans. It is reported that sika deer may serve as an important reservoir host for several species of B. burgdorferi s.l. and may transmit these species to humans and animals. However, little is known about the presence of Borrelia pathogens in sika deer in China. In this study, the existence and prevalence of Borrelia sp. in sika deer from four regions of Jilin Province in China was assessed. Seventy-one blood samples of sika deer were collected and tested by nested-PCRs based on 16S ribosomal RNA (16S rRNA), outer surface protein A (OspA), flagenllin (fla), and 5S-23S rRNA intergenic spacer (5S-23S rRNA) genes of B. burgdorferi s.l. Six (8.45%) samples were positive for Borrelia sp. based on sequences of 4 genes. The positive samples were detected 18 for 16S rRNA, 10 for OspA, 16 for fla and 6 for 5S-23S, with the positive rates 25.35% (95% CI=3.8-35.6), 14.08% (95% CI=3.0-21.6), 22.54% (95% CI=4.3-36.9) and 8.45% (95% CI=1.7-22.9), respectively. Sequence analysis of the positive PCR products revealed that the partial 4 genes sequences in this study were all most similar to the sequences of B. garinii and B. burgdorferi sensu stricto (s.s.), no other Borrelia genospecies were found. This is the first report of Borrelia pathogens in sika deer in China. The findings in this study indicated that sika deer as potential natural host and may spread Lyme disease pathogen to animals, ticks, and even humans. Copyright © 2016. Published by Elsevier B.V.

How many novel eukaryotic 'kingdoms'? Pitfalls and limitations of environmental DNA surveys

PubMed Central

Berney, Cédric; Fahrni, José; Pawlowski, Jan

2004-01-01

Background Over the past few years, the use of molecular techniques to detect cultivation-independent, eukaryotic diversity has proven to be a powerful approach. Based on small-subunit ribosomal RNA (SSU rRNA) gene analyses, these studies have revealed the existence of an unexpected variety of new phylotypes. Some of them represent novel diversity in known eukaryotic groups, mainly stramenopiles and alveolates. Others do not seem to be related to any molecularly described lineage, and have been proposed to represent novel eukaryotic kingdoms. In order to review the evolutionary importance of this novel high-level eukaryotic diversity critically, and to test the potential technical and analytical pitfalls and limitations of eukaryotic environmental DNA surveys (EES), we analysed 484 environmental SSU rRNA gene sequences, including 81 new sequences from sediments of the small river, the Seymaz (Geneva, Switzerland). Results Based on a detailed screening of an exhaustive alignment of eukaryotic SSU rRNA gene sequences and the phylogenetic re-analysis of previously published environmental sequences using Bayesian methods, our results suggest that the number of novel higher-level taxa revealed by previously published EES was overestimated. Three main sources of errors are responsible for this situation: (1) the presence of undetected chimeric sequences; (2) the misplacement of several fast-evolving sequences; and (3) the incomplete sampling of described, but yet unsequenced eukaryotes. Additionally, EES give a biased view of the diversity present in a given biotope because of the difficult amplification of SSU rRNA genes in some taxonomic groups. Conclusions Environmental DNA surveys undoubtedly contribute to reveal many novel eukaryotic lineages, but there is no clear evidence for a spectacular increase of the diversity at the kingdom level. After re-analysis of previously published data, we found only five candidate lineages of possible novel high-level eukaryotic taxa, two of which comprise several phylotypes that were found independently in different studies. To ascertain their taxonomic status, however, the organisms themselves have now to be identified. PMID:15176975

Application of a unique server-based oligonucleotide probe selection tool toward a novel biosensor for the detection of Streptococcus pyogenes.

PubMed

Nugen, Sam R; Leonard, Barbara; Baeumner, Antje J

2007-05-15

We developed a software program for the rapid selection of detection probes to be used in nucleic acid-based assays. In comparison to commercially available software packages, our program allows the addition of oligotags as required by nucleic acid sequence-based amplification (NASBA) as well as automatic BLAST searches for all probe/primer pairs. We then demonstrated the usefulness of the program by designing a novel lateral flow biosensor for Streptococcus pyogenes that does not rely on amplification methods such as the polymerase chain reaction (PCR) or NASBA to obtain low limits of detection, but instead uses multiple reporter and capture probes per target sequence and an instantaneous amplification via dye-encapsulating liposomes. These assays will decrease the detection time to just a 20 min hybridization reaction and avoid costly enzymatic gene amplification reactions. The lateral flow assay was developed quantifying the 16S rRNA from S. pyogenes by designing reporter and capture probes that specifically hybridize with the RNA and form a sandwich. DNA reporter probes were tagged with dye-encapsulating liposomes, biotinylated DNA oligonucleotides were used as capture probes. From the initial number of capture and reporter probes chosen, a combination of two capture and three reporter probes were found to provide optimal signal generation and significant enhancement over single capture/reporter probe combinations. The selectivity of the biosensor was proven by analyzing organisms closely related to S. pyogenes, such as other Streptococcus and Enterococcus species. All probes had been selected by the software program within minutes and no iterative optimization and re-design of the oligonucleotides was required which enabled a very rapid biosensor prototyping. While the sensitivity obtained with the biosensor was only 135 ng, future experiments will decrease this significantly by the addition of more reporter and capture probes for either the same rRNA or a different nucleic acid target molecule. This will lead to the possibility of detecting S. pyogenes with a rugged assay that does not require a cell culturing or gene amplification step and will therefore enable rapid, specific and sensitive onsite testing.

Altered gravity influences rDNA and NopA100 localization in nucleoli

NASA Astrophysics Data System (ADS)

Sobol, M. A.; Kordyum, E. L.

Fundamental discovery of gravisensitivity of cells no specified to gravity perception focused increasing attention on an elucidation of the mechanisms involved in altered gravity effects at the cellular and subcellular levels. The nucleolus is the transcription site of rRNA genes as well as the site of processing and initial packaging of their transcripts with ribosomal and nonribosomal proteins. The mechanisms inducing the changes in the subcomponents of the nucleolus that is morphologically defined yet highly dynamic structure are still unknown in detail. To understand the functional organization of the nucleolus as in the control as under altered gravity conditions it is essential to determine both the precise location of rDNA and the proteins playing the key role in rRNA processing. Lepidium sativum seeds were germinated in 1% agar medium on the slow horizontal clinostat (2 rpm) and in the stationary conditions. We investigated the root meristematic cells dissected from the seedlings grown in darkness for two days. The investigations were carried out with anti-DNA and anti-NopA100 antibodies labeling as well as with TdT procedure, and immunogold electron microscopy. In the stationary growth conditions, the anti-DNA antibody as well TdT procedure were capable of detecting fibrillar centers (FCs) and the dense fibrillar component (DFC) in the nucleolus. In FCs, gold particles were revealed on the condensed chromatin inclusions, internal fibrils of decondensed rDNA and the transition zone FC-DFC. Quantitatively, FCs appeared 1,5 times more densely labeled than DFC. NopA100 was localized in FCs and in DFC. In FCs, the most of protein was revealed in the transition zone FC-DFC. After a quantitative study, FCs and the transition zone FC-DFC appeared to contain NopA100 1,7 times more than DFC. Under the conditions of altered gravity, quantitative data clearly showed a redistribution of nucleolar DNA and NopA100 between FCs and DFC in comparison with the control. In labeling both with anti-DNA antibody and by TdT method, 1,5 times more gold particles were localized on FCs, and 1,5 times less in DFC. Unlike the control, condensed r-chromatin blocks and inner rDNA were labeled much more than the transition zone FC-DFC in fibrillar centers. The content of NopA100 in FCs and the transition zone FC-DFC was 2,4 times more than in the control. Twice less quantity of the protein was revealed in DFC as compared to the control. In fibrillar centers, the majority of NopA100 was localized in the inner space of FCs than in the transition zone FC-DFC. Re-localization of rDNA and NopA100 in the nucleolar subcomponents indicates lowering the level of rDNA transcription as well as middle and late processing of rRNA that let us to propose lowering the functional activity of the nucleolus under the influence of altered gravity.

MIPE: A metagenome-based community structure explorer and SSU primer evaluation tool

PubMed Central

Zhou, Quan

2017-01-01

An understanding of microbial community structure is an important issue in the field of molecular ecology. The traditional molecular method involves amplification of small subunit ribosomal RNA (SSU rRNA) genes by polymerase chain reaction (PCR). However, PCR-based amplicon approaches are affected by primer bias and chimeras. With the development of high-throughput sequencing technology, unbiased SSU rRNA gene sequences can be mined from shotgun sequencing-based metagenomic or metatranscriptomic datasets to obtain a reflection of the microbial community structure in specific types of environment and to evaluate SSU primers. However, the use of short reads obtained through next-generation sequencing for primer evaluation has not been well resolved. The software MIPE (MIcrobiota metagenome Primer Explorer) was developed to adapt numerous short reads from metagenomes and metatranscriptomes. Using metagenomic or metatranscriptomic datasets as input, MIPE extracts and aligns rRNA to reveal detailed information on microbial composition and evaluate SSU rRNA primers. A mock dataset, a real Metagenomics Rapid Annotation using Subsystem Technology (MG-RAST) test dataset, two PrimerProspector test datasets and a real metatranscriptomic dataset were used to validate MIPE. The software calls Mothur (v1.33.3) and the SILVA database (v119) for the alignment and classification of rRNA genes from a metagenome or metatranscriptome. MIPE can effectively extract shotgun rRNA reads from a metagenome or metatranscriptome and is capable of classifying these sequences and exhibiting sensitivity to different SSU rRNA PCR primers. Therefore, MIPE can be used to guide primer design for specific environmental samples. PMID:28350876

First Report of the 23S rRNA Gene A2058G Point Mutation Associated With Macrolide Resistance in Treponema pallidum From Syphilis Patients in Cuba.

PubMed

Noda, Angel A; Matos, Nelvis; Blanco, Orestes; Rodríguez, Islay; Stamm, Lola Virginia

2016-05-01

This study aimed to assess the presence of macrolide-resistant Treponema pallidum subtypes in Havana, Cuba. Samples from 41 syphilis patients were tested for T. pallidum 23S rRNA gene mutations. Twenty-five patients (61%) harbored T. pallidum with the A2058G mutation, which was present in all 8 subtypes that were identified. The A2059G mutation was not detected.

Catalyzed Reporter Deposition-Fluorescence In Situ Hybridization Allows for Enrichment-Independent Detection of Microcolony-Forming Soil Bacteria

PubMed Central

Ferrari, Belinda C.; Tujula, Niina; Stoner, Kate; Kjelleberg, Staffan

2006-01-01

Advances in the growth of hitherto unculturable soil bacteria have emphasized the requirement for rapid bacterial identification methods. Due to the slow-growing strategy of microcolony-forming soil bacteria, successful fluorescence in situ hybridization (FISH) requires an rRNA enrichment step for visualization. In this study, catalyzed reporter deposition (CARD)-FISH was employed as an alternative method to rRNA enhancement and was found to be superior to conventional FISH for the detection of microcolonies that are cultivated by using the soil substrate membrane system. CARD-FISH enabled real-time identification of oligophilic microcolony-forming soil bacteria without the requirement for enrichment on complex media and the associated shifts in community composition. PMID:16391135

QUANTITATIVE DETECTION OF ENVIRONMENTALLY IMPORTANT DYES USING DIODE LASER/FIBER-OPTIC RAMAN

EPA Science Inventory

A compact diode laser/fiber-optic Raman spectrometer is used for quantitative detection of environmentally important dyes. This system is based on diode laser excitation at 782 mm, fiber optic probe technology, an imaging spectrometer, and state-of-the-art scientific CCD camera. ...

Location of rRNA transcription to the nucleolar components: disappearance of the fibrillar centers in nucleoli of regenerating rat hepatocytes.

PubMed

Montanaro, Lorenzo; Govoni, Marzia; Orrico, Catia; Treré, Davide; Derenzini, Massimo

2011-01-01

The precise location of rDNA transcription to the components of mammalian cell nucleolus is still debated. This was due to the fact that all the molecules necessary for rRNA synthesis are located in two of the three components, the fibrillar centers (FCs) and the dense fibrillar component (DFC), which together with the granular component (GC) are considered to be constantly present in mammalian cell nucleoli. In the present study we demonstrated that in nucleoli of many regenerating rat hepatocytes at 15 h after partial hepatectomy the FCs were no longer present, only the DFC and the GC being detected. At this time of regeneration the rRNA transcriptional activity was three fold that of resting hepatocytes, while the synthesis of DNA was not yet significantly increased, indicating that these nucleolar changes were due to the rRNA synthesis up-regulation. The DFC appeared to be organized in numerous, small, roundish tufts of fibrils. The silver staining procedure for AgNOR proteins, which are associated with the ribosomal genes, selectively and homogeneously stained these fibrillar tufts. Immuno-gold visualization of the Upstream Binding Factor (UBF), which is associated with the promoter region and the transcribed portion of the rRNA 45S gene, demonstrated that UBF was selectively located in the fibrillar tufts. We concluded that in proliferating rat hepatocytes the increased synthesis of rRNA induced an activation of the rRNA transcription machinery located in the fibrillar centers which, by becoming associated with the ribonucleoprotein transcripts, assumed the morphological pattern of the DFC.

Towards high-throughput molecular detection of Plasmodium: new approaches and molecular markers

PubMed Central

Steenkeste, Nicolas; Incardona, Sandra; Chy, Sophy; Duval, Linda; Ekala, Marie-Thérèse; Lim, Pharath; Hewitt, Sean; Sochantha, Tho; Socheat, Doung; Rogier, Christophe; Mercereau-Puijalon, Odile; Fandeur, Thierry; Ariey, Frédéric

2009-01-01

Background Several strategies are currently deployed in many countries in the tropics to strengthen malaria control toward malaria elimination. To measure the impact of any intervention, there is a need to detect malaria properly. Mostly, decisions still rely on microscopy diagnosis. But sensitive diagnosis tools enabling to deal with a large number of samples are needed. The molecular detection approach offers a much higher sensitivity, and the flexibility to be automated and upgraded. Methods Two new molecular methods were developed: dot18S, a Plasmodium-specific nested PCR based on the 18S rRNA gene followed by dot-blot detection of species by using species-specific probes and CYTB, a Plasmodium-specific nested PCR based on cytochrome b gene followed by species detection using SNP analysis. The results were compared to those obtained with microscopic examination and the "standard" 18S rRNA gene based nested PCR using species specific primers. 337 samples were diagnosed. Results Compared to the microscopy the three molecular methods were more sensitive, greatly increasing the estimated prevalence of Plasmodium infection, including P. malariae and P. ovale. A high rate of mixed infections was uncovered with about one third of the villagers infected with more than one malaria parasite species. Dot18S and CYTB sensitivity outranged the "standard" nested PCR method, CYTB being the most sensitive. As a consequence, compared to the "standard" nested PCR method for the detection of Plasmodium spp., the sensitivity of dot18S and CYTB was respectively 95.3% and 97.3%. Consistent detection of Plasmodium spp. by the three molecular methods was obtained for 83% of tested isolates. Contradictory results were mostly related to detection of Plasmodium malariae and Plasmodium ovale in mixed infections, due to an "all-or-none" detection effect at low-level parasitaemia. Conclusion A large reservoir of asymptomatic infections was uncovered using the molecular methods. Dot18S and CYTB, the new methods reported herein are highly sensitive, allow parasite DNA extraction as well as genus- and species-specific diagnosis of several hundreds of samples, and are amenable to high-throughput scaling up for larger sample sizes. Such methods provide novel information on malaria prevalence and epidemiology and are suited for active malaria detection. The usefulness of such sensitive malaria diagnosis tools, especially in low endemic areas where eradication plans are now on-going, is discussed in this paper. PMID:19402894

Isolation and characterization of Acidithiobacillus caldus from a sulfur-oxidizing bacterial biosensor and its role in detection of toxic chemicals.

PubMed

Hassan, Sedky H A; Van Ginkel, Steven W; Kim, Sung-Min; Yoon, Sung-Hwan; Joo, Jin-Ho; Shin, Beom-Soo; Jeon, Byong-Hun; Bae, Wookeun; Oh, Sang-Eun

2010-08-01

A novel toxicity detection methodology based on sulfur-oxidizing bacteria (SOB) has been developed for the rapid and reliable detection of toxic chemicals in water. The methodology exploits the ability of SOB to oxidize sulfur particles in the presence of oxygen to produce sulfuric acid. The reaction results in an increase in electrical conductivity (EC) and a decrease in pH. The assay is based on the inhibition of SOB in the presence of toxic chemicals by measuring changes in EC and pH. We found that SOB biosensor can detect toxic chemicals, such as heavy metals and CN-, in the 5-2000ppb range. One bacterium was isolated from an SOB biosensor and the 16S rRNA gene of the bacterial strain has 99% and 96% sequence similarity to Acidithiobacillus sp. ORCS6 and Acidithiobacillus caldus DSM 8584, respectively. The isolate was identified as A. caldus SMK. The SOB biosensor is ideally suited for monitoring toxic chemicals in water having the advantages of high sensitivity and quick detection.

Single Fluorescence Channel-based Multiplex Detection of Avian Influenza Virus by Quantitative PCR with Intercalating Dye

PubMed Central

Ahberg, Christian D.; Manz, Andreas; Neuzil, Pavel

2015-01-01

Since its invention in 1985 the polymerase chain reaction (PCR) has become a well-established method for amplification and detection of segments of double-stranded DNA. Incorporation of fluorogenic probe or DNA intercalating dyes (such as SYBR Green) into the PCR mixture allowed real-time reaction monitoring and extraction of quantitative information (qPCR). Probes with different excitation spectra enable multiplex qPCR of several DNA segments using multi-channel optical detection systems. Here we show multiplex qPCR using an economical EvaGreen-based system with single optical channel detection. Previously reported non quantitative multiplex real-time PCR techniques based on intercalating dyes were conducted once the PCR is completed by performing melting curve analysis (MCA). The technique presented in this paper is both qualitative and quantitative as it provides information about the presence of multiple DNA strands as well as the number of starting copies in the tested sample. Besides important internal control, multiplex qPCR also allows detecting concentrations of more than one DNA strand within the same sample. Detection of the avian influenza virus H7N9 by PCR is a well established method. Multiplex qPCR greatly enhances its specificity as it is capable of distinguishing both haemagglutinin (HA) and neuraminidase (NA) genes as well as their ratio. PMID:26088868

A Novel Pretreatment-Free Duplex Chamber Digital PCR Detection System for the Absolute Quantitation of GMO Samples.

PubMed

Zhu, Pengyu; Wang, Chenguang; Huang, Kunlun; Luo, Yunbo; Xu, Wentao

2016-03-18

Digital polymerase chain reaction (PCR) has developed rapidly since it was first reported in the 1990s. However, pretreatments are often required during preparation for digital PCR, which can increase operation error. The single-plex amplification of both the target and reference genes may cause uncertainties due to the different reaction volumes and the matrix effect. In the current study, a quantitative detection system based on the pretreatment-free duplex chamber digital PCR was developed. The dynamic range, limit of quantitation (LOQ), sensitivity and specificity were evaluated taking the GA21 event as the experimental object. Moreover, to determine the factors that may influence the stability of the duplex system, we evaluated whether the pretreatments, the primary and secondary structures of the probes and the SNP effect influence the detection. The results showed that the LOQ was 0.5% and the sensitivity was 0.1%. We also found that genome digestion and single nucleotide polymorphism (SNP) sites affect the detection results, whereas the unspecific hybridization within different probes had little side effect. This indicated that the detection system was suited for both chamber-based and droplet-based digital PCR. In conclusion, we have provided a simple and flexible way of achieving absolute quantitation for genetically modified organism (GMO) genome samples using commercial digital PCR detection systems.

A Novel Pretreatment-Free Duplex Chamber Digital PCR Detection System for the Absolute Quantitation of GMO Samples

PubMed Central

Zhu, Pengyu; Wang, Chenguang; Huang, Kunlun; Luo, Yunbo; Xu, Wentao

2016-01-01

Digital polymerase chain reaction (PCR) has developed rapidly since it was first reported in the 1990s. However, pretreatments are often required during preparation for digital PCR, which can increase operation error. The single-plex amplification of both the target and reference genes may cause uncertainties due to the different reaction volumes and the matrix effect. In the current study, a quantitative detection system based on the pretreatment-free duplex chamber digital PCR was developed. The dynamic range, limit of quantitation (LOQ), sensitivity and specificity were evaluated taking the GA21 event as the experimental object. Moreover, to determine the factors that may influence the stability of the duplex system, we evaluated whether the pretreatments, the primary and secondary structures of the probes and the SNP effect influence the detection. The results showed that the LOQ was 0.5% and the sensitivity was 0.1%. We also found that genome digestion and single nucleotide polymorphism (SNP) sites affect the detection results, whereas the unspecific hybridization within different probes had little side effect. This indicated that the detection system was suited for both chamber-based and droplet-based digital PCR. In conclusion, we have provided a simple and flexible way of achieving absolute quantitation for genetically modified organism (GMO) genome samples using commercial digital PCR detection systems. PMID:26999129

Thermus and the Pink Discoloration Defect in Cheese

PubMed Central

Quigley, Lisa; O’Sullivan, Daniel J.; Daly, David; O’Sullivan, Orla; Burdikova, Zuzana; Vana, Rostislav; Beresford, Tom P.; Ross, R. Paul; Fitzgerald, Gerald F.; McSweeney, Paul L. H.; Giblin, Linda

2016-01-01

ABSTRACT A DNA sequencing-based strategy was applied to study the microbiology of Continental-type cheeses with a pink discoloration defect. The basis for this phenomenon has remained elusive, despite decades of research. The bacterial composition of cheese containing the defect was compared to that of control cheese using 16S rRNA gene and shotgun metagenomic sequencing as well as quantitative PCR (qPCR). Throughout, it was apparent that Thermus, a carotenoid-producing genus, was present at higher levels in defect-associated cheeses than in control cheeses. Prompted by this finding and data confirming the pink discoloration to be associated with the presence of a carotenoid, a culture-based approach was employed, and Thermus thermophilus was successfully cultured from defect-containing cheeses. The link between Thermus and the pinking phenomenon was then established through the cheese defect equivalent of Koch’s postulates when the defect was recreated by the reintroduction of a T. thermophilus isolate to a test cheese during the manufacturing process. IMPORTANCE Pink discoloration in cheese is a defect affecting many cheeses throughout the world, leading to significant financial loss for the dairy industry. Despite decades of research, the cause of this defect has remained elusive. The advent of high-throughput, next-generation sequencing has revolutionized the field of food microbiology and, with respect to this study, provided a means of testing a possible microbial basis for this defect. In this study, a combined 16S rRNA, whole-genome sequencing, and quantitative PCR approach was taken. This resulted in the identification of Thermus, a carotenoid-producing thermophile, in defect-associated cheeses and the recreation of the problem in cheeses to which Thermus was added. This finding has the potential to lead to new strategies to eliminate this defect, and our method represents an approach that can be employed to investigate the role of microbes in other food defects of unknown origin. PMID:27822529

First molecular detection and characterization of Hepatozoon and Sarcocystis spp. in field mice and voles from Japan.

PubMed

Moustafa, Mohamed Abdallah Mohamed; Shimozuru, Michito; Mohamed, Wessam; Taylor, Kyle Rueben; Nakao, Ryo; Sashika, Mariko; Tsubota, Toshio

2017-08-01

Sarcocystis and Hepatozoon species are protozoan parasites that are frequently detected in domestic and wild animals. Rodents are considered common intermediate and paratenic hosts for several Sarcocystis and Hepatozoon species. Here, blood DNA samples from a total of six rodents, including one Myodes rutilus, one Myodes rufocanus, and four Apodemus speciosus, collected from Hokkaido, Japan, were shown by conventional PCR of the 18S ribosomal RNA (rRNA) gene to contain Sarcocystis and Hepatozoon DNA. Sequencing of the DNA detected one Sarcocystis sp. in the M. rufocanus sample and two different Hepatozoon spp. in the M. rutilus and A. speciosus samples. Phylogenetic analysis showed that the detected Sarcocystis sp. sequence grouped with GenBank Sarcocystis sequences from rodents, snakes, and raccoons from Japan and China. The 18S rRNA partial gene sequences of both detected Hepatozoon spp. clustered with GenBank Hepatozoon sequences from snakes, geckos and voles in Europe, Africa, and Asia. This study provides evidence that wild rodents have a role in the maintenance of Sarcocystis and Hepatozoon species on the island of Hokkaido.

Control of rRNA and tRNA syntheses in Escherichia coli by guanosine tetraphosphate.

PubMed Central

Ryals, J; Little, R; Bremer, H

1982-01-01

The expression of stable RNA (rRNA and tRNA) genes and the concentration of guanosine tetraphosphate (ppGpp) were measured in an isogenic pair of relA+ and relA derivatives of Escherichia coli B/r. The cells were either growing exponentially at different rates or subject to amino acid starvation when they were measured. The specific stable RNA gene activity (rs/rt, the rate of rRNA and tRNA synthesis relative to the total instantaneous rate of RNA synthesis) was found to decrease from 1.0 at a ppGpp concentration of 0 (extrapolated value) to 0.24 at saturating concentrations of ppGpp (above 100 pmoles per optical density at 460 nm unit of cell mass). The same relationship between the rs/rt ratio and ppGpp concentration was obtained independent of the physiological state of the bacteria (i.e., independent of the growth rate or of amino acid starvation) and independent of the relA allele. It can be concluded that ppGpp is an effector for stable RNA gene control and that stable RNA genes are not controlled by factors other than the ppGpp-mediated system. The results were shown to be qualitatively and quantitatively consistent with data on in vitro rRNA gene control by ppGpp, and they were interpreted in the light of reported ideas derived from those in vitro experiments. PMID:6179924

High-resolution phylogenetic microbial community profiling

DOE Office of Scientific and Technical Information (OSTI.GOV)

Singer, Esther; Bushnell, Brian; Coleman-Derr, Devin

Over the past decade, high-throughput short-read 16S rRNA gene amplicon sequencing has eclipsed clone-dependent long-read Sanger sequencing for microbial community profiling. The transition to new technologies has provided more quantitative information at the expense of taxonomic resolution with implications for inferring metabolic traits in various ecosystems. We applied single-molecule real-time sequencing for microbial community profiling, generating full-length 16S rRNA gene sequences at high throughput, which we propose to name PhyloTags. We benchmarked and validated this approach using a defined microbial community. When further applied to samples from the water column of meromictic Sakinaw Lake, we show that while community structuresmore » at the phylum level are comparable between PhyloTags and Illumina V4 16S rRNA gene sequences (iTags), variance increases with community complexity at greater water depths. PhyloTags moreover allowed less ambiguous classification. Last, a platform-independent comparison of PhyloTags and in silico generated partial 16S rRNA gene sequences demonstrated significant differences in community structure and phylogenetic resolution across multiple taxonomic levels, including a severe underestimation in the abundance of specific microbial genera involved in nitrogen and methane cycling across the Lake's water column. Thus, PhyloTags provide a reliable adjunct or alternative to cost-effective iTags, enabling more accurate phylogenetic resolution of microbial communities and predictions on their metabolic potential.« less

High-resolution phylogenetic microbial community profiling

DOE PAGES

Singer, Esther; Bushnell, Brian; Coleman-Derr, Devin; ...

2016-02-09

Over the past decade, high-throughput short-read 16S rRNA gene amplicon sequencing has eclipsed clone-dependent long-read Sanger sequencing for microbial community profiling. The transition to new technologies has provided more quantitative information at the expense of taxonomic resolution with implications for inferring metabolic traits in various ecosystems. We applied single-molecule real-time sequencing for microbial community profiling, generating full-length 16S rRNA gene sequences at high throughput, which we propose to name PhyloTags. We benchmarked and validated this approach using a defined microbial community. When further applied to samples from the water column of meromictic Sakinaw Lake, we show that while community structuresmore » at the phylum level are comparable between PhyloTags and Illumina V4 16S rRNA gene sequences (iTags), variance increases with community complexity at greater water depths. PhyloTags moreover allowed less ambiguous classification. Last, a platform-independent comparison of PhyloTags and in silico generated partial 16S rRNA gene sequences demonstrated significant differences in community structure and phylogenetic resolution across multiple taxonomic levels, including a severe underestimation in the abundance of specific microbial genera involved in nitrogen and methane cycling across the Lake's water column. Thus, PhyloTags provide a reliable adjunct or alternative to cost-effective iTags, enabling more accurate phylogenetic resolution of microbial communities and predictions on their metabolic potential.« less

The subgingival microbiota of Papillon-Lefèvre syndrome.

PubMed

Albandar, Jasim M; Khattab, Razan; Monem, Fawza; Barbuto, Sara M; Paster, Bruce J

2012-07-01

There is little information about the microbiologic profiles of periodontal lesions in Papillon-Lefèvre syndrome (PLS) and the significance of bacteria in the pathogenesis of periodontitis in these patients. This comprehensive analysis of the subgingival microbiota in patients with PLS used 16S ribosomal RNA (rRNA) clonal analysis and the 16S rRNA-based Human Oral Microbe Identification Microarray (HOMIM). Thirteen patients with PLS from seven unrelated families volunteered for this microbiologic study. Subgingival plaque was collected with sterile paper points from multiple sites with ≥5 mm probing depth, and whole genomic DNA was extracted. The 16S rRNA genes were amplified, cloned, and sequenced. The samples were then probed for ≈300 predominant oral bacterial species using the HOMIM. The most commonly detected phylotypes in the clonal analysis were Gemella morbillorum, Gemella haemolysans, Granulicatella adiacens, Lachnospiraceae OT 100 (EI074), Parvimonas micra, Selenomonas noxia, and Veillonella parvula. As a group, streptococci were commonly detected in these individuals. In the HOMIM analysis, a total of 170 bacterial species/phylotypes were detected, with a range of 40 to 80 species per patient with PLS. Of these, 12 bacterial species were detected in medium to high levels in ≥50% of the individuals. The high-frequency strains were clustered into eight groups: Aggregatibacter actinomycetemcomitans, Campylobacter spp., Capnocytophaga granulosa, G. morbillorum, P. micra, Porphyromonas endodontalis, Streptococcus spp., and Tannerella forsythia. The subgingival microbiota in PLS is diverse. Periodontal pathogens commonly associated with chronic and aggressive periodontitis and opportunistic pathogens may be associated with the development of severe periodontitis in patients with PLS.

Quantitative and Sensitive Detection of Chloramphenicol by Surface-Enhanced Raman Scattering

PubMed Central

Ding, Yufeng; Yin, Hongjun; Meng, Qingyun; Zhao, Yongmei; Liu, Luo; Wu, Zhenglong; Xu, Haijun

2017-01-01

We used surface-enhanced Raman scattering (SERS) for the quantitative and sensitive detection of chloramphenicol (CAP). Using 30 nm colloidal Au nanoparticles (NPs), a low detection limit for CAP of 10−8 M was obtained. The characteristic Raman peak of CAP centered at 1344 cm−1 was used for the rapid quantitative detection of CAP in three different types of CAP eye drops, and the accuracy of the measurement result was verified by high-performance liquid chromatography (HPLC). The experimental results reveal that the SERS technique based on colloidal Au NPs is accurate and sensitive, and can be used for the rapid detection of various antibiotics. PMID:29261161

Facile and quantitative electrochemical detection of yeast cell apoptosis

NASA Astrophysics Data System (ADS)

Yue, Qiulin; Xiong, Shiquan; Cai, Dongqing; Wu, Zhengyan; Zhang, Xin

2014-03-01

An electrochemical method based on square wave anodic stripping voltammetry (SWASV) was developed to detect the apoptosis of yeast cells conveniently and quantitatively through the high affinity between Cu2+ and phosphatidylserine (PS) translocated from the inner to the outer plasma membrane of the apoptotic cells. The combination of negatively charged PS and Cu2+ could decrease the electrochemical response of Cu2+ on the electrode. The results showed that the apoptotic rates of cells could be detected quantitatively through the variations of peak currents of Cu2+ by SWASV, and agreed well with those obtained through traditional flow cytometry detection. This work thus may provide a novel, simple, immediate and accurate detection method for cell apoptosis.

A HIGHLY SELECTIVE PCR PROTOCOL FOR DETECTING 16S RRNA GENES OF THE GENUS PSEUDOMONAS (SENSU STRICTO) IN ENVIRONMENTAL SAMPLES

EPA Science Inventory

Pseudomonas species are plant, animal, and human pathogens; exhibit plant pathogen-suppressing properties useful in biological control; or express metabolic versatilities valued in biotechnology and bioremediation. Specific detection of Pseudomonas species in the environment may ...

Redescriptions of three trachelocercid ciliates (Protista, Ciliophora, Karyorelictea), with notes on their phylogeny based on small subunit rRNA gene sequences.

PubMed

Yan, Ying; Xu, Yuan; Yi, Zhenzhen; Warren, Alan

2013-09-01

Three trachelocercid ciliates, Kovalevaia sulcata (Kovaleva, 1966) Foissner, 1997, Trachelocerca sagitta (Müller, 1786) Ehrenberg, 1840 and Trachelocerca ditis (Wright, 1982) Foissner, 1996, isolated from two coastal habitats at Qingdao, China, were investigated using live observation and silver impregnation methods. Data on their infraciliature and morphology are supplied. The small subunit rRNA (SSU rRNA) genes of K. sulcata and Trachelocerca sagitta were sequenced for the first time. Phylogenetic analyses based on SSU rRNA gene sequence data indicate that both organisms, and the previously sequenced Trachelocerca ditis, are located within the trachelocercid assemblage and that K. sulcata is sister to an unidentified taxon forming a clade that is basal to the core trachelocercids.

Development of a Prokaryotic Universal Primer for Simultaneous Analysis of Bacteria and Archaea Using Next-Generation Sequencing

PubMed Central

Takahashi, Shunsuke; Tomita, Junko; Nishioka, Kaori; Hisada, Takayoshi; Nishijima, Miyuki

2014-01-01

For the analysis of microbial community structure based on 16S rDNA sequence diversity, sensitive and robust PCR amplification of 16S rDNA is a critical step. To obtain accurate microbial composition data, PCR amplification must be free of bias; however, amplifying all 16S rDNA species with equal efficiency from a sample containing a large variety of microorganisms remains challenging. Here, we designed a universal primer based on the V3-V4 hypervariable region of prokaryotic 16S rDNA for the simultaneous detection of Bacteria and Archaea in fecal samples from crossbred pigs (Landrace×Large white×Duroc) using an Illumina MiSeq next-generation sequencer. In-silico analysis showed that the newly designed universal prokaryotic primers matched approximately 98.0% of Bacteria and 94.6% of Archaea rRNA gene sequences in the Ribosomal Database Project database. For each sequencing reaction performed with the prokaryotic universal primer, an average of 69,330 (±20,482) reads were obtained, of which archaeal rRNA genes comprised approximately 1.2% to 3.2% of all prokaryotic reads. In addition, the detection frequency of Bacteria belonging to the phylum Verrucomicrobia, including members of the classes Verrucomicrobiae and Opitutae, was higher in the NGS analysis using the prokaryotic universal primer than that performed with the bacterial universal primer. Importantly, this new prokaryotic universal primer set had markedly lower bias than that of most previously designed universal primers. Our findings demonstrate that the prokaryotic universal primer set designed in the present study will permit the simultaneous detection of Bacteria and Archaea, and will therefore allow for a more comprehensive understanding of microbial community structures in environmental samples. PMID:25144201

Raman spectroscopy-based detection of chemical contaminants in food powders

USDA-ARS?s Scientific Manuscript database

Raman spectroscopy technique has proven to be a reliable method for qualitative detection of chemical contaminants in food ingredients and products. For quantitative imaging-based detection, each contaminant particle in a food sample must be detected and it is important to determine the necessary sp...

Watershed scale fungal community characterization along a pH gradient in a subsurface environment co-contaminated with uranium and nitrate

DOE Office of Scientific and Technical Information (OSTI.GOV)

Jasrotia, Puja; Green, Stefan; Canion, Andy

2014-01-01

The objective of this study was to characterize fungal communities in a subsurface environment co-contaminated with uranium and nitrate at the watershed scale, and to determine the potential contribution of fungi to contaminant transformation (nitrate attenuation). The abundance, distribution and diversity of fungi in subsurface groundwater samples were determined using quantitative and semi-quantitative molecular techniques, including quantitative PCR of eukaryotic SSU rRNA genes and pyrosequencing of fungal internal transcribed spacer (ITS) regions. Potential bacterial and fungal denitrification was assessed in sediment-groundwater slurries amended with antimicrobial compounds and in fungal pure cultures isolated from subsurface. Our results demonstrate that subsurface fungalmore » communities are dominated by members of the phylum Ascomycota, and a pronounced shift in fungal community composition occurs across the groundwater pH gradient at the field site, with lower diversity observed under acidic (pH < 4.5) conditions. Fungal isolates recovered from subsurface sediments were shown to reduce nitrate to nitrous oxide, including cultures of the genus Coniochaeta that were detected in abundance in pyrosequence libraries of site groundwater samples. Denitrifying fungal isolates recovered from the site were classified, and found to be distributed broadly within the phylum Ascomycota, and within a single genus within the Basidiomycota. Potential denitrification rate assays with sediment-groundwater slurries showed the potential for subsurface fungi to reduce nitrate to nitrous oxide under in situ acidic pH conditions.« less

The Functional Implications of Bottom Up and Top Down Controls on Marine Bacteria in Arthur Harbor, a Highly Productive Coastal Setting on the West Antarctic Peninsula

NASA Astrophysics Data System (ADS)

Bowman, J. S.; Amaral-Zettler, L. A.; Rich, J. J.; Luria, C.; Ducklow, H. W.

2016-02-01

Marine bacteria can be broadly classified into two groups based on their ecology; slow growing oligotrophic specialists and fast growing copiotrophs. These ecological strategies are associated with specific taxonomic and functional groups, making it possible to use 16S rRNA gene amplicon and shotgun metagenomic data to qualitatively, and possibly quantitatively, identify the contribution of each strategy to marine biogeochemical cycles. We leveraged a 5-year (2009 to 2014) time series of 16S rRNA gene amplicon data for Arthur Harbor, located near Palmer Station, Antarctica, to identify trends in the abundance of taxa associated with each ecological strategy. Using emergent self-organizing maps, we identified four recurring "modes" in bacterial community structure based on the relative abundance of the ubiquitous SAR11 clade. A different bacterial genus was dominant in each mode; Pelagibacter, Polaribacter, Roseobacter, and Colwellia. To explore the functional implications of these different modes we applied shotgun metagenomics and functional predictions using the newly available tool PAPRICA, in combination with flow cytometry and estimates of bacterial production. Our annotation and assembly of binned contigs corresponding to the dominant genera illuminate the succession of metabolic functions across the 2013-2014 austral summer and inform the timing of autotrophic and mixotrophic (putatively bacterivorous) phytoplankton blooms. Surprisingly, while the abundance of Pelagibacter 16S rRNA gene reads was negatively correlated with the concentration of chlorophyll a, the ratio of Pelagibacter to Polaribacter and Roseobacter was poorly correlated with the ratio of high nucleic acid (HNA) to low nucleic acid (LNA) bacteria as determined by flow cytometry, and the relative size of the HNA population at times contrasted sharply with chlorophyll a. These findings suggest that the physiological state of bacterial cells and top down controls play a strong role in HNA: LNA dynamics.

Molecular characterization of Hepatozoon felis in Rhipicephalus sanguineus ticks infested on captive lions (Panthera leo).

PubMed

Bhusri, Benjaporn; Sariya, Ladawan; Mongkolphan, Chalisa; Suksai, Parut; Kaewchot, Supakarn; Changbunjong, Tanasak

2017-09-01

Hepatozoon spp. are protozoan parasites that infect a wide range of domestic and wild animals. The infection occurs by ingestion of an infected tick. This study was carried out to detect and characterize Hepatozoon spp. in ticks collected from captive lions ( Panthera leo ) in Thailand based on the partial 18S rRNA gene sequence. A total of 30 ticks were collected and identified as Rhipicephalus sanguineus . The collected ticks were separated into 10 tick pools by sex and life stages. Of the 10 tick pools examined, only one (10%) was found to be infected with the Hepatozoon species. Sequencing and phylogenetic analysis showed a clustering of the partial 18S rRNA gene sequence like that of H. felis from the GenBank database. This is the first report of H. felis in R. sanguineus ticks collected from captive lions in Thailand. Our results indicated that R. sanguineus may be a possible vector of feline Hepatozoon in Thailand.

Microelectrode-based technology for the detection of low levels of bacteria

NASA Technical Reports Server (NTRS)

Rogers, Tom D.; Hitchens, G. D.; Mishra, S. K.; Pierson, D. L.

1992-01-01

A microelectrode-based electrochemical detection method was used for quantitation of bacteria in water samples. The redox mediator, benzoquinone, was used to accept electrons from the bacterial metabolic pathway to create a flow of electrons by reducing the mediator. Electrochemical monitoring electrodes detected the reduced mediator as it diffused out of the cells and produced a small electrical current. By using a combination of microelectrodes and monitoring instrumentation, the cumulative current generated by a particular bacterial population could be monitored. Using commercially available components, an electrochemical detection system was assembled and tested to evaluate its potential as an emerging technology for rapid detection and quantitation of bacteria in water samples.

Co-generating synthetic parts toward a self-replicating system

DOE Office of Scientific and Technical Information (OSTI.GOV)

Li, Jun; Haas, Wilhelm; Jackson, Kirsten

To build replicating systems with new functions, the engineering of existing biological machineries requires a sensible strategy. Protein synthesis Using Recombinant Elements (PURE) system consists of the desired components for transcription, translation, aminoacylation and energy regeneration. PURE, might be the basis for a radically alterable, lifelike system after optimization. Here, we regenerated 54 E. coli ribosomal (r-) proteins individually from DNA templates in the PURE system. We show that using stable isotope labeling with amino acids, mass spectrometry based quantitative proteomics could detect 26 of the 33 50S and 20 of the 21 30S subunit r-proteins when co-expressed in batchmore » format PURE system. By optimizing DNA template concentrations and adapting a miniaturized Fluid Array Device with optimized feeding solution, we were able to cogenerate and detect at least 29 of the 33 50S and all of the 21 30S subunit r-proteins in one pot. The boost on yield of a single r-protein in co-expression pool varied from ~1.5 to 5-fold compared to the batch mode, with up to ~ 2.4 µM yield for a single r-protein. Reconstituted ribosomes under physiological condition from PURE system synthesized 30S r-proteins and native 16S rRNA showed ~13% activity of native 70S ribosomes, which increased to 21% when supplemented with GroEL/ES. As a result, this work also points to what is still needed to obtain self-replicating synthetic ribosomes in-situ in the PURE system.« less

Co-generating synthetic parts toward a self-replicating system

DOE PAGES

Li, Jun; Haas, Wilhelm; Jackson, Kirsten; ...

2017-03-23

To build replicating systems with new functions, the engineering of existing biological machineries requires a sensible strategy. Protein synthesis Using Recombinant Elements (PURE) system consists of the desired components for transcription, translation, aminoacylation and energy regeneration. PURE, might be the basis for a radically alterable, lifelike system after optimization. Here, we regenerated 54 E. coli ribosomal (r-) proteins individually from DNA templates in the PURE system. We show that using stable isotope labeling with amino acids, mass spectrometry based quantitative proteomics could detect 26 of the 33 50S and 20 of the 21 30S subunit r-proteins when co-expressed in batchmore » format PURE system. By optimizing DNA template concentrations and adapting a miniaturized Fluid Array Device with optimized feeding solution, we were able to cogenerate and detect at least 29 of the 33 50S and all of the 21 30S subunit r-proteins in one pot. The boost on yield of a single r-protein in co-expression pool varied from ~1.5 to 5-fold compared to the batch mode, with up to ~ 2.4 µM yield for a single r-protein. Reconstituted ribosomes under physiological condition from PURE system synthesized 30S r-proteins and native 16S rRNA showed ~13% activity of native 70S ribosomes, which increased to 21% when supplemented with GroEL/ES. As a result, this work also points to what is still needed to obtain self-replicating synthetic ribosomes in-situ in the PURE system.« less

Culture-dependent and independent studies of microbial diversity in highly copper-contaminated Chilean marine sediments.

PubMed

Besaury, Ludovic; Marty, Florence; Buquet, Sylvaine; Mesnage, Valérie; Muyzer, Gerard; Quillet, Laurent

2013-02-01

Cultivation and molecular-based approaches were used to study microbial diversity in two Chilean marine sediments contaminated with high (835 ppm) and very high concentrations of copper (1,533 ppm). The diversity of cultivable bacteria resistant to copper was studied at oxic and anoxic conditions, focusing on sulfate-, thiosulfate-, and iron-reducing bacteria. For both sediments, the cultivable bacteria isolated at oxic conditions were mostly affiliated to the genus Bacillus, while at anoxic conditions the majority of the cultivable bacteria found were closely related to members of the genera Desulfovibrio, Sphingomonas, and Virgibacillus. Copper resistance was between 100 and 400 ppm, with the exception of a strain affiliated to members of the genus Desulfuromonas, which was resistant up to 1,000 ppm of copper. In parallel, cloning and sequencing of 16S rRNA was performed to study the total bacterial diversity in the sediments. A weak correlation was observed between the isolated strains and the 16S rRNA operational taxonomic units detected. The presence of copper resistance genes (copA, cusA, and pcoA) was tested for all the strains isolated; only copA was detected in a few isolates, suggesting that other copper resistance mechanisms could be used by the bacteria in those highly copper-contaminated sediments.

Prevalence of Corynebacterial 16S rRNA Sequences in Patients with Bacterial and “Nonbacterial” Prostatitis

PubMed Central

Tanner, Michael A.; Shoskes, Daniel; Shahed, Asha; Pace, Norman R.

1999-01-01

The etiology of chronic prostatitis syndromes in men is controversial, particularly when positive cultures for established uropathogens are lacking. Although identification of bacteria in prostatic fluid has relied on cultivation and microscopy, most microorganisms in the environment, including some human pathogens, are resistant to cultivation. We report here on an rRNA-based molecular phylogenetic approach to the identification of bacteria in prostate fluid from prostatitis patients. Positive bacterial signals were seen for 65% of patients with chronic prostatitis overall. Seven of 11 patients with bacterial signals but none of 6 patients without bacterial signals were cured with antibiotic-based therapy. Results indicate the occurrence in the prostate fluid of a wide spectrum of bacterial species representing several genera. Most rRNA genes were closely related to those of species belonging to the genera Corynebacterium, Staphylococcus, Peptostreptococcus, Streptococcus, and Escherichia. Unexpectedly, a wide diversity of Corynebacterium species was found in high proportion compared to the proportions of other bacterial species found. A subset of these 16S rRNA sequences represent those of undescribed species on the basis of their positions in phylogenetic trees. These uncharacterized organisms were not detected in control samples, suggesting that the organisms have a role in the disease or are the consequence of the disease. These studies show that microorganisms associated with prostatitis generally occur as complex microbial communities that differ between patients. The results also indicate that microbial communities distinct from those associated with prostatitis may occur at low levels in normal prostatic fluid. PMID:10325338

Analysis of the 16S–23S rRNA Gene Internal Transcribed Spacer Region in Klebsiella Species▿

PubMed Central

Wang, Min; Cao, Boyang; Yu, Qunfang; Liu, Lei; Gao, Qili; Wang, Lei; Feng, Lu

2008-01-01

The 16S-23S rRNA gene internal transcribed spacer (ITS) regions of Klebsiella spp., including Klebsiella pneumoniae subsp. pneumoniae, Klebsiella pneumoniae subsp. ozaenae, Klebsiella pneumoniae subsp. rhinoscleromatis, Klebsiella oxytoca, Klebsiella planticola, Klebsiella terrigena, and Klebsiella ornithinolytica, were characterized, and the feasibility of using ITS sequences to discriminate Klebsiella species and subspecies was explored. A total of 336 ITS sequences from 21 representative strains and 11 clinical isolates of Klebsiella were sequenced and analyzed. Three distinct ITS types—ITSnone (without tRNA genes), ITSglu [with a tRNAGlu (UUC) gene], and ITSile+ala [with tRNAIle (GAU) and tRNAAla (UGC) genes]—were detected in all species except for K. pneumoniae subsp. rhinoscleromatis, which has only ITSglu and ITSile+ala. The presence of ITSnone in Enterobacteriaceae had never been reported before. Both the length and the sequence of each ITS type are highly conserved within the species, with identity levels from 0.961 to 1.000 for ITSnone, from 0.967 to 1.000 for ITSglu, and from 0.968 to 1.000 for ITSile+ala. Interspecies sequence identities range from 0.775 to 0.989 for ITSnone, from 0.798 to 0.997 for ITSglu, and from 0.712 to 0.985 for ITSile+ala. Regions with significant interspecies variations but low intraspecies polymorphisms were identified; these may be targeted in the design of probes for the identification of Klebsiella to the species level. Phylogenetic analysis based on ITS regions reveals the relationships among Klebsiella species similarly to that based on 16S rRNA genes. PMID:18753345

Ultratrace level determination and quantitative analysis of kidney injury biomarkers in patient samples attained by zinc oxide nanorods

NASA Astrophysics Data System (ADS)

Singh, Manpreet; Alabanza, Anginelle; Gonzalez, Lorelis E.; Wang, Weiwei; Reeves, W. Brian; Hahm, Jong-In

2016-02-01

Determining ultratrace amounts of protein biomarkers in patient samples in a straightforward and quantitative manner is extremely important for early disease diagnosis and treatment. Here, we successfully demonstrate the novel use of zinc oxide nanorods (ZnO NRs) in the ultrasensitive and quantitative detection of two acute kidney injury (AKI)-related protein biomarkers, tumor necrosis factor (TNF)-α and interleukin (IL)-8, directly from patient samples. We first validate the ZnO NRs-based IL-8 results via comparison with those obtained from using a conventional enzyme-linked immunosorbent method in samples from 38 individuals. We further assess the full detection capability of the ZnO NRs-based technique by quantifying TNF-α, whose levels in human urine are often below the detection limits of conventional methods. Using the ZnO NR platforms, we determine the TNF-α concentrations of all 46 patient samples tested, down to the fg per mL level. Subsequently, we screen for TNF-α levels in approximately 50 additional samples collected from different patient groups in order to demonstrate a potential use of the ZnO NRs-based assay in assessing cytokine levels useful for further clinical monitoring. Our research efforts demonstrate that ZnO NRs can be straightforwardly employed in the rapid, ultrasensitive, quantitative, and simultaneous detection of multiple AKI-related biomarkers directly in patient urine samples, providing an unparalleled detection capability beyond those of conventional analysis methods. Additional key advantages of the ZnO NRs-based approach include a fast detection speed, low-volume assay condition, multiplexing ability, and easy automation/integration capability to existing fluorescence instrumentation. Therefore, we anticipate that our ZnO NRs-based detection method will be highly beneficial for overcoming the frequent challenges in early biomarker development and treatment assessment, pertaining to the facile and ultrasensitive quantification of hard-to-trace biomolecules.Determining ultratrace amounts of protein biomarkers in patient samples in a straightforward and quantitative manner is extremely important for early disease diagnosis and treatment. Here, we successfully demonstrate the novel use of zinc oxide nanorods (ZnO NRs) in the ultrasensitive and quantitative detection of two acute kidney injury (AKI)-related protein biomarkers, tumor necrosis factor (TNF)-α and interleukin (IL)-8, directly from patient samples. We first validate the ZnO NRs-based IL-8 results via comparison with those obtained from using a conventional enzyme-linked immunosorbent method in samples from 38 individuals. We further assess the full detection capability of the ZnO NRs-based technique by quantifying TNF-α, whose levels in human urine are often below the detection limits of conventional methods. Using the ZnO NR platforms, we determine the TNF-α concentrations of all 46 patient samples tested, down to the fg per mL level. Subsequently, we screen for TNF-α levels in approximately 50 additional samples collected from different patient groups in order to demonstrate a potential use of the ZnO NRs-based assay in assessing cytokine levels useful for further clinical monitoring. Our research efforts demonstrate that ZnO NRs can be straightforwardly employed in the rapid, ultrasensitive, quantitative, and simultaneous detection of multiple AKI-related biomarkers directly in patient urine samples, providing an unparalleled detection capability beyond those of conventional analysis methods. Additional key advantages of the ZnO NRs-based approach include a fast detection speed, low-volume assay condition, multiplexing ability, and easy automation/integration capability to existing fluorescence instrumentation. Therefore, we anticipate that our ZnO NRs-based detection method will be highly beneficial for overcoming the frequent challenges in early biomarker development and treatment assessment, pertaining to the facile and ultrasensitive quantification of hard-to-trace biomolecules. Electronic supplementary information (ESI) available: Typical SEM images of the ZnO NRs used in the biomarker assays are provided in Fig. S1. See DOI: 10.1039/c5nr08706f

Nearly complete rRNA genes assembled from across the metazoan animals: effects of more taxa, a structure-based alignment, and paired-sites evolutionary models on phylogeny reconstruction.

PubMed

Mallatt, Jon; Craig, Catherine Waggoner; Yoder, Matthew J

2010-04-01

This study (1) uses nearly complete rRNA-gene sequences from across Metazoa (197 taxa) to reconstruct animal phylogeny; (2) presents a highly annotated, manual alignment of these sequences with special reference to rRNA features including paired sites (http://purl.oclc.org/NET/rRNA/Metazoan_alignment) and (3) tests, after eliminating as few disruptive, rogue sequences as possible, if a likelihood framework can recover the main metazoan clades. We found that systematic elimination of approximately 6% of the sequences, including the divergent or unstably placed sequences of cephalopods, arrowworm, symphylan and pauropod myriapods, and of myzostomid and nemertodermatid worms, led to a tree that supported Ecdysozoa, Lophotrochozoa, Protostomia, and Bilateria. Deuterostomia, however, was never recovered, because the rRNA of urochordates goes (nonsignificantly) near the base of the Bilateria. Counterintuitively, when we modeled the evolution of the paired sites, phylogenetic resolution was not increased over traditional tree-building models that assume all sites in rRNA evolve independently. The rRNA genes of non-bilaterians contain a higher % AT than do those of most bilaterians. The rRNA genes of Acoela and Myzostomida were found to be secondarily shortened, AT-enriched, and highly modified, throwing some doubt on the location of these worms at the base of Bilateria in the rRNA tree--especially myzostomids, which other evidence suggests are annelids instead. Other findings are marsupial-with-placental mammals, arrowworms in Ecdysozoa (well supported here but contradicted by morphology), and Placozoa as sister to Cnidaria. Finally, despite the difficulties, the rRNA-gene trees are in strong concordance with trees derived from multiple protein-coding genes in supporting the new animal phylogeny. (c) 2009 Elsevier Inc. All rights reserved.

Biodiversity within hot spring microbial mat communities: molecular monitoring of enrichment cultures

NASA Technical Reports Server (NTRS)

Ward, D. M.; Santegoeds, C. M.; Nold, S. C.; Ramsing, N. B.; Ferris, M. J.; Bateson, M. M.

1997-01-01

We have begun to examine the basis for incongruence between hot spring microbial mat populations detected by cultivation or by 16S rRNA methods. We used denaturing gradient gel electrophoresis (DGGE) to monitor enrichments and isolates plated therefrom. At near extincting inoculum dilutions we observed Chloroflexus-like and cyanobacterial populations whose 16S rRNA sequences have been detected in the 'New Pit' Spring Chloroflexus mat and the Octopus Spring cyanobacterial mat. Cyanobacterial populations enriched from 44 to 54 degrees C and 56 to 63 degrees C samples at near habitat temperatures were similar to those previously detected in mat samples of comparable temperatures. However, a lower temperature enrichment from the higher temperature sample selected for the populations found in the lower temperature sample. Three Thermus populations detected by both DGGE and isolation exemplify even more how enrichment may bias our view of community structure. The most abundant population was adapted to the habitat temperature (50 degrees C), while populations adapted to 65 degrees C and 70 degrees C were 10(2)- and 10(4)-fold less abundant, respectively. However, enrichment at 70 degrees C favored the least abundant strain. Inoculum dilution and incubation at the habitat temperature favored the more numerically relevant populations. We enriched many other aerobic chemoorganotrophic populations at various inoculum dilutions and substrate concentrations, most of whose 16S rRNA sequences have not been detected in mats. A common feature of numerically relevant cyanobacterial, Chloroflexus-like and aerobic chemorganotrophic populations, is that they grow poorly and resist cultivation on solidified medium, suggesting plating bias, and that the medium composition and incubation conditions may not reflect the natural microenvironments these populations inhabit.

Seasonal variation in detection of bacterial DNA in arthritic stifle joints of dogs with cranial cruciate ligament rupture using PCR amplification of the 16S rRNA gene.

PubMed

Muir, Peter; Fox, Robin; Wu, Qiang; Baker, Theresa A; Zitzer, Nina C; Hudson, Alan P; Manley, Paul A; Schaefer, Susan L; Hao, Zhengling

2010-02-24

An underappreciated cause and effect relationship between environmental bacteria and arthritis may exist. Previously, we found that stifle arthritis in dogs was associated with the presence of environmental bacteria within synovium. Cranial cruciate ligament rupture (CCLR) is often associated with stifle arthritis in dogs. We now wished to determine whether seasonal variation in detection of bacterial material may exist in affected dogs, and to also conduct analyses of both synovium and synovial fluid. We also wished to analyze a larger clone library of the 16S rRNA gene to further understanding of the microbial population in the canine stifle. Synovial biopsies were obtained from 117 affected dogs from January to December 2006. Using PCR, synovium and synovial fluid were tested for Borrelia burgdorferi and Stenotrophomonas maltophilia DNA. Broad-ranging 16S rRNA primers were also used and PCR products were cloned and sequenced for bacterial identification. Overall, 41% of arthritic canine stifle joints contained bacterial DNA. Detection of bacterial DNA in synovial fluid samples was increased, when compared with synovium (p<0.01). Detection rates were highest in the winter and spring and lowest in the summer period, suggesting environmental factors influence the risk of translocation to the stifle. Organisms detected were predominately Gram's negative Proteobacteria, particularly the orders Rhizobiales (32.8% of clones) and Burkholderiales (20.0% of clones), usually as part of a polymicrobial population. PCR-positivity was inversely correlated with severity of arthritis assessed radiographically and with dog age. Bacterial translocation to the canine stifle may be associated with changes to the indoor environment. Copyright 2009 Elsevier B.V. All rights reserved.

Characterization of a Newly Discovered Symbiont of the Whitefly Bemisia tabaci (Hemiptera: Aleyrodidae)

PubMed Central

Bing, Xiao-Li; Yang, Jiao; Zchori-Fein, Einat; Wang, Xiao-Wei

2013-01-01

Bemisia tabaci (Hemiptera: Aleyrodidae) is a species complex containing >28 cryptic species, some of which are important crop pests worldwide. Like many other sap-sucking insects, whiteflies harbor an obligatory symbiont, “Candidatus Portiera aleyrodidarum,” and a number of secondary symbionts. So far, six genera of secondary symbionts have been identified in B. tabaci. In this study, we report and describe the finding of an additional bacterium in the indigenous B. tabaci cryptic species China 1 (formerly known as B. tabaci biotype ZHJ3). Phylogenetic analysis based on the 16S rRNA and gltA genes showed that the bacterium belongs to the Alphaproteobacteria subdivision of the Proteobacteria and has a close relationship with human pathogens of the genus Orientia. Consequently, we temporarily named it Orientia-like organism (OLO). OLO was found in six of eight wild populations of B. tabaci China 1, with the infection rate ranging from 46.2% to 76.8%. Fluorescence in situ hybridization (FISH) of B. tabaci China 1 in nymphs and adults revealed that OLOs are confined to the bacteriome and co-occur with “Ca. Portiera aleyrodidarum.” The vertical transmission of OLO was demonstrated by detection of OLO at the anterior pole end of the oocytes through FISH. Quantitative PCR analysis of population dynamics suggested a complex interaction between “Ca. Portiera aleyrodidarum” and OLO. Based on these results, we propose “Candidatus Hemipteriphilus asiaticus” for the classification of this symbiont from B. tabaci. PMID:23144129

Combined quantification of pulmonary Pneumocystis jirovecii DNA and serum (1->3)-β-D-glucan for differential diagnosis of pneumocystis pneumonia and Pneumocystis colonization.

PubMed

Damiani, Céline; Le Gal, Solène; Da Costa, Cécilia; Virmaux, Michèle; Nevez, Gilles; Totet, Anne

2013-10-01

This study assessed a quantitative PCR (qPCR) assay for Pneumocystis jirovecii quantification in bronchoalveolar lavage (BAL) fluid samples combined with serum (1→3)-β-d-glucan (BG) level detection to distinguish Pneumocystis pneumonia (PCP) from pulmonary colonization with P. jirovecii. Forty-six patients for whom P. jirovecii was initially detected in BAL fluid samples were retrospectively enrolled. Based on clinical data and results of P. jirovecii detection, 17 and 29 patients were diagnosed with PCP and colonization, respectively. BAL fluid samples were reassayed using a qPCR assay targeting the mitochondrial large subunit rRNA gene. qPCR results and serum BG levels (from a Fungitell kit) were analyzed conjointly. P. jirovecii DNA copy numbers were significantly higher in the PCP group than in the colonization group (1.3 × 10(7) versus 3.4 × 10(3) copies/μl, P < 0.05). A lower cutoff value (1.6 × 10(3) copies/μl) achieving 100% sensitivity for PCP diagnosis and an upper cutoff value (2 × 10(4) copies/μl) achieving 100% specificity were determined. Applying these two values, 13/17 PCP patients and 19/29 colonized patients were correctly assigned to their patient groups. For the remaining 14 patients with P. jirovecii DNA copy numbers between the cutoff values, PCP and colonization could not be distinguished on the basis of qPCR results. Four of these patients who were initially assigned to the PCP group presented BG levels of ≥100 pg/ml. The other 10 patients, who were initially assigned to the colonization group, presented BG levels of <100 pg/ml. These results suggest that the combination of the qPCR assay, applying cutoff values of 1.6 × 10(3) and 2 × 10(4) copies/μl, and serum BG detection, applying a 100 pg/ml threshold, can differentiate PCP and colonization diagnoses.

Combined Quantification of Pulmonary Pneumocystis jirovecii DNA and Serum (1→3)-β-d-Glucan for Differential Diagnosis of Pneumocystis Pneumonia and Pneumocystis Colonization

PubMed Central

Le Gal, Solène; Da Costa, Cécilia; Virmaux, Michèle; Nevez, Gilles; Totet, Anne

2013-01-01

This study assessed a quantitative PCR (qPCR) assay for Pneumocystis jirovecii quantification in bronchoalveolar lavage (BAL) fluid samples combined with serum (1→3)-β-d-glucan (BG) level detection to distinguish Pneumocystis pneumonia (PCP) from pulmonary colonization with P. jirovecii. Forty-six patients for whom P. jirovecii was initially detected in BAL fluid samples were retrospectively enrolled. Based on clinical data and results of P. jirovecii detection, 17 and 29 patients were diagnosed with PCP and colonization, respectively. BAL fluid samples were reassayed using a qPCR assay targeting the mitochondrial large subunit rRNA gene. qPCR results and serum BG levels (from a Fungitell kit) were analyzed conjointly. P. jirovecii DNA copy numbers were significantly higher in the PCP group than in the colonization group (1.3 × 107 versus 3.4 × 103 copies/μl, P < 0.05). A lower cutoff value (1.6 × 103 copies/μl) achieving 100% sensitivity for PCP diagnosis and an upper cutoff value (2 × 104 copies/μl) achieving 100% specificity were determined. Applying these two values, 13/17 PCP patients and 19/29 colonized patients were correctly assigned to their patient groups. For the remaining 14 patients with P. jirovecii DNA copy numbers between the cutoff values, PCP and colonization could not be distinguished on the basis of qPCR results. Four of these patients who were initially assigned to the PCP group presented BG levels of ≥100 pg/ml. The other 10 patients, who were initially assigned to the colonization group, presented BG levels of <100 pg/ml. These results suggest that the combination of the qPCR assay, applying cutoff values of 1.6 × 103 and 2 × 104 copies/μl, and serum BG detection, applying a 100 pg/ml threshold, can differentiate PCP and colonization diagnoses. PMID:23903553

Mitochondrial DNA Targets Increase Sensitivity of Malaria Detection Using Loop-Mediated Isothermal Amplification ▿

PubMed Central

Polley, Spencer D.; Mori, Yasuyoshi; Watson, Julie; Perkins, Mark D.; González, Iveth J.; Notomi, Tsugunori; Chiodini, Peter L.; Sutherland, Colin J.

2010-01-01

Loop-mediated isothermal amplification (LAMP) of DNA offers the ability to detect very small quantities of pathogen DNA following minimal tissue sample processing and is thus an attractive methodology for point-of-care diagnostics. Previous attempts to diagnose malaria by the use of blood samples and LAMP have targeted the parasite small-subunit rRNA gene, with a resultant sensitivity for Plasmodium falciparum of around 100 parasites per μl. Here we describe the use of mitochondrial targets for LAMP-based detection of any Plasmodium genus parasite and of P. falciparum specifically. These new targets allow routine amplification from samples containing as few as five parasites per μl of blood. Amplification is complete within 30 to 40 min and is assessed by real-time turbidimetry, thereby offering rapid diagnosis with greater sensitivity than is achieved by the most skilled microscopist or antigen detection using lateral flow immunoassays. PMID:20554824

Optimization of nested polymerase chain reaction assays for identification of Aeromonas salmonicida, Yersinia ruckeri and Flavobacterium psychrophilum

USGS Publications Warehouse

Taylor, P.W.; Winton, J.R.

2002-01-01

Nested polymerase chain reaction (PCR) assays were developed using first-round primers complementary to highly conserved regions within the bacterial 16S ribosomal RNA (rRNA) gene (universal eubacterial primers) and second-round primers specific for sequences within the 16S rRNA genes of Aeromonas salmonicida, Yersinia ruckeri, andFlavobacterium psychrophilum. Following optimization of the MgCl2 concentration and primer annealing temperature, PCR employing the universal eubacterial primers was used to amplify a 1,500-base-pair (bp) product visible in agarose gels stained with ethidium bromide. The calculated detection limit of this single-round assay was less than 1.4 × 104 colony-forming units (CFU) per reaction for all bacterial species tested. Single-round PCR using primer sets specific for A. salmonicida, Y. ruckeri, and F. psychrophilumamplified bands of 271, 575, and 1,100 bp, respectively, with detection limits of less than 1.4 × 104, 1.4 × 105, and 1.4 × 105 CFU per reaction. Using the universal eubacterial primers in the first round and the species-specific primer sets in the second round of nested PCR assays improved the detection ability by approximately four orders of magnitude to fewer than 14 CFU per sample for each of the three bacterial species. Such nested assays could be adapted to a wide variety of bacterial fish pathogens for which 16S sequences are available.

A Natural View of Microbial Biodiversity within Hot Spring Cyanobacterial Mat Communities

PubMed Central

Ward, David M.; Ferris, Michael J.; Nold, Stephen C.; Bateson, Mary M.

1998-01-01

This review summarizes a decade of research in which we have used molecular methods, in conjunction with more traditional approaches, to study hot spring cyanobacterial mats as models for understanding principles of microbial community ecology. Molecular methods reveal that the composition of these communities is grossly oversimplified by microscopic and cultivation methods. For example, none of 31 unique 16S rRNA sequences detected in the Octopus Spring mat, Yellowstone National Park, matches that of any prokaryote previously cultivated from geothermal systems; 11 are contributed by genetically diverse cyanobacteria, even though a single cyanobacterial species was suspected based on morphologic and culture analysis. By studying the basis for the incongruity between culture and molecular samplings of community composition, we are beginning to cultivate isolates whose 16S rRNA sequences are readily detected. By placing the genetic diversity detected in context with the well-defined natural environmental gradients typical of hot spring mat systems, the relationship between gene and species diversity is clarified and ecological patterns of species occurrence emerge. By combining these ecological patterns with the evolutionary patterns inherently revealed by phylogenetic analysis of gene sequence data, we find that it may be possible to understand microbial biodiversity within these systems by using principles similar to those developed by evolutionary ecologists to understand biodiversity of larger species. We hope that such an approach guides microbial ecologists to a more realistic and predictive understanding of microbial species occurrence and responsiveness in both natural and disturbed habitats. PMID:9841675

A natural view of microbial biodiversity within hot spring cyanobacterial mat communities

NASA Technical Reports Server (NTRS)

Ward, D. M.; Ferris, M. J.; Nold, S. C.; Bateson, M. M.

1998-01-01

This review summarizes a decade of research in which we have used molecular methods, in conjunction with more traditional approaches, to study hot spring cyanobacterial mats as models for understanding principles of microbial community ecology. Molecular methods reveal that the composition of these communities is grossly oversimplified by microscopic and cultivation methods. For example, none of 31 unique 16S rRNA sequences detected in the Octopus Spring mat, Yellowstone National Park, matches that of any prokaryote previously cultivated from geothermal systems; 11 are contributed by genetically diverse cyanobacteria, even though a single cyanobacterial species was suspected based on morphologic and culture analysis. By studying the basis for the incongruity between culture and molecular samplings of community composition, we are beginning to cultivate isolates whose 16S rRNA sequences are readily detected. By placing the genetic diversity detected in context with the well-defined natural environmental gradients typical of hot spring mat systems, the relationship between gene and species diversity is clarified and ecological patterns of species occurrence emerge. By combining these ecological patterns with the evolutionary patterns inherently revealed by phylogenetic analysis of gene sequence data, we find that it may be possible to understand microbial biodiversity within these systems by using principles similar to those developed by evolutionary ecologists to understand biodiversity of larger species. We hope that such an approach guides microbial ecologists to a more realistic and predictive understanding of microbial species occurrence and responsiveness in both natural and disturbed habitats.

Bacterial consortia at different wine fermentation phases of two typical Central European grape varieties: Blaufränkisch (Frankovka modrá) and Grüner Veltliner (Veltlínske zelené).

PubMed

Godálová, Zuzana; Kraková, Lucia; Puškárová, Andrea; Bučková, Mária; Kuchta, Tomáš; Piknová, Ľubica; Pangallo, Domenico

2016-01-18

This is the first study focused to bacterial diversity and dynamic during the vinification of two important Central Europe grape vines: Blaufränkisch and Grüner Veltliner. The investigation strategy included culture-dependent and culture-independent approaches. Four different agar media were utilized for the isolation of various bacteria occurring in several fermentation stages. The isolates were clustered by fluorescent-ITS PCR and, one or more representatives of each cluster, were identified by 16 rRNA gene sequencing. The culture-independent approach, based on 16S rRNA gene amplification, combined the denaturing gradient gel electrophoresis (DGGE) method and the construction of bacterial clone library for each wine fermentation step. A complex bacterial community was identified, comprising different lactic acid bacteria and acetic acid bacteria, such as Leuconostoc spp., Lactobacillus spp. and Gluconobacter spp. Other OTUs and bacterial isolates embraced the Actinobacteria, Bacilli, Alpha-, Beta- and Gamma-proteobacteria classes. Different taxa already detected by recent studies, such as Sphingomonas, Variovorax, Pantoea, Enterobacter and Tatumella, were detected confirming the continuous occurrence of these kinds of bacteria in wine environment. Moreover, novel genera (Amycolatopsis, Hydrogenophilus, Snodgrassella, Telluria, Gilliamella, Lelliottia, and Lonsdale quercina) never detected before were recognized, too. The role of these, until now anonymous, bacteria during vinification deserves investigation, which could open a new research field in wine technology. Copyright © 2015 Elsevier B.V. All rights reserved.

Development and Evaluation of a Quantitative PCR Assay Targeting Sandhill Crane (Grus canadensis) Fecal Pollution

PubMed Central

Ryu, Hodon; Lu, Jingrang; Vogel, Jason; Elk, Michael; Chávez-Ramírez, Felipe; Ashbolt, Nicholas

2012-01-01

While the microbial water quality in the Platte River is seasonally impacted by excreta from migrating cranes, there are no methods available to study crane fecal contamination. Here we characterized microbial populations in crane feces using phylogenetic analysis of 16S rRNA gene fecal clone libraries. Using these sequences, a novel crane quantitative PCR (Crane1) assay was developed, and its applicability as a microbial source tracking (MST) assay was evaluated by determining its host specificity and detection ability in environmental waters. Bacteria from crane excreta were dominated by bacilli and proteobacteria, with a notable paucity of sequences homologous to Bacteroidetes and Clostridia. The Crane1 marker targeted a dominant clade of unclassified Lactobacillales sequences closely related to Catellicoccus marimammalium. The host distribution of the Crane1 marker was relatively high, being positive for 69% (66/96) of the crane excreta samples tested. The assay also showed high host specificity, with 95% of the nontarget fecal samples (i.e., n = 553; 20 different free-range hosts) being negative. Of the presumed crane-impacted water samples (n = 16), 88% were positive for the Crane1 assay, whereas none of the water samples not impacted by cranes were positive (n = 165). Bayesian statistical models of the Crane1 MST marker demonstrated high confidence in detecting true-positive signals and a low probability of false-negative signals from environmental water samples. Altogether, these data suggest that the newly developed marker could be used in environmental monitoring studies to study crane fecal pollution dynamics. PMID:22492437

Antibiotic Resistance Gene Detection in the Microbiome Context.

PubMed

Do, Thi Thuy; Tamames, Javier; Stedtfeld, Robert D; Guo, Xueping; Murphy, Sinead; Tiedje, James M; Walsh, Fiona

2018-06-01

Within the past decade, microbiologists have moved from detecting single antibiotic resistance genes (ARGs) to detecting all known resistance genes within a sample due to advances in next generation sequencing. This has provided a wealth of data on the variation and relative abundances of ARGs present in a total bacterial population. However, to use these data in terms of therapy or risk to patients, they must be analyzed in the context of the background microbiome. Using a quantitative PCR ARG chip and 16S rRNA amplicon sequencing, we have sought to identify the ARGs and bacteria present in a fecal sample of a healthy adult using genomic tools. Of the 42 ARGs detected, 12 fitted into the ResCon1 category of ARGs: cfxA, cphA, bacA, sul3, aadE, bla TEM , aphA1, aphA3, aph(2')-Id, aacA/aphd, catA1, and vanC. Therefore, we describe these 12 genes as the core resistome of this person's fecal microbiome and the remaining 30 ARGs as descriptors of the microbial population within the fecal microbiome. The dominant phyla and genera agree with those previously detected in the greatest abundances in fecal samples of healthy humans. The majority of the ARGs detected were associated with the presence of specific bacterial taxa, which were confirmed using microbiome analysis. We acknowledge the limitations of the data in the context of the limited sample set. However, the principle of combining qPCR and microbiome analysis was shown to be helpful to identify the association of the ARGs with specific taxa.

Identification of a novel acetate-utilizing bacterium belonging to Synergistes group 4 in anaerobic digester sludge.

PubMed

Ito, Tsukasa; Yoshiguchi, Kazumi; Ariesyady, Herto Dwi; Okabe, Satoshi

2011-12-01

Major acetate-utilizing bacterial and archaeal populations in methanogenic anaerobic digester sludge were identified and quantified by radioisotope- and stable-isotope-based functional analyses, microautoradiography-fluorescence in situ hybridization (MAR-FISH) and stable-isotope probing of 16S rRNA (RNA-SIP) that can directly link 16S rRNA phylogeny with in situ metabolic function. First, MAR-FISH with (14)C-acetate indicated the significant utilization of acetate by only two major groups, unidentified bacterial cells and Methanosaeta-like filamentous archaeal cells, in the digester sludge. To identify the acetate-utilizing unidentified bacteria, RNA-SIP was conducted with (13)C(6)-glucose and (13)C(3)-propionate as sole carbon source, which were followed by phylogenetic analysis of 16S rRNA. We found that bacteria belonging to Synergistes group 4 were commonly detected in both 16S rRNA clone libraries derived from the sludge incubated with (13)C-glucose and (13)C-propionate. To confirm that this bacterial group can utilize acetate, specific FISH probe targeting for Synergistes group 4 was newly designed and applied to the sludge incubated with (14)C-acetate for MAR-FISH. The MAR-FISH result showed that bacteria belonging to Synergistes group 4 significantly took up acetate and their active population size was comparable to that of Methanosaeta in this sludge. In addition, as bacteria belonging to Synergistes group 4 had high K(m) for acetate and maximum utilization rate, they are more competitive for acetate over Methanosaeta at high acetate concentrations (2.5-10  mM). To our knowledge, it is the first time to report the acetate-utilizing activity of uncultured bacteria belonging to Synergistes group 4 and its competitive significance to acetoclastic methanogen, Methanosaeta.

Identification of characteristic oligonucleotides in the bacterial 16S ribosomal RNA sequence dataset

NASA Technical Reports Server (NTRS)

Zhang, Zhengdong; Willson, Richard C.; Fox, George E.

2002-01-01

MOTIVATION: The phylogenetic structure of the bacterial world has been intensively studied by comparing sequences of 16S ribosomal RNA (16S rRNA). This database of sequences is now widely used to design probes for the detection of specific bacteria or groups of bacteria one at a time. The success of such methods reflects the fact that there are local sequence segments that are highly characteristic of particular organisms or groups of organisms. It is not clear, however, the extent to which such signature sequences exist in the 16S rRNA dataset. A better understanding of the numbers and distribution of highly informative oligonucleotide sequences may facilitate the design of hybridization arrays that can characterize the phylogenetic position of an unknown organism or serve as the basis for the development of novel approaches for use in bacterial identification. RESULTS: A computer-based algorithm that characterizes the extent to which any individual oligonucleotide sequence in 16S rRNA is characteristic of any particular bacterial grouping was developed. A measure of signature quality, Q(s), was formulated and subsequently calculated for every individual oligonucleotide sequence in the size range of 5-11 nucleotides and for 15mers with reference to each cluster and subcluster in a 929 organism representative phylogenetic tree. Subsequently, the perfect signature sequences were compared to the full set of 7322 sequences to see how common false positives were. The work completed here establishes beyond any doubt that highly characteristic oligonucleotides exist in the bacterial 16S rRNA sequence dataset in large numbers. Over 16,000 15mers were identified that might be useful as signatures. Signature oligonucleotides are available for over 80% of the nodes in the representative tree.

Group 16SrXI phytoplasma strains, including subgroup 16SrXI-B and a new subgroup, 16SrXI-D, are associated with sugar cane white leaf.

PubMed

Zhang, Rong-Yue; Li, Wen-Feng; Huang, Ying-Kun; Wang, Xiao-Yan; Shan, Hong-Li; Luo, Zhi-Ming; Yin, Jiong

2016-01-01

Sugar cane white leaf (SCWL) is a serious disease caused by phytoplasmas. In this study, we performed nested PCR with phytoplasma universal primer pairs (P1/P7 and R16F2n/R16R2) for the 16S rRNA gene to detect SCWL phytoplasmas in 31 SCWL samples collected from Baoshan and Lincang, Yunnan, China. We cloned and sequenced the nested PCR products, revealing that the 16S rRNA gene sequences from 31 SCWL samples were all 1247 bp in length and shared more than 99 % nucleotide sequence similarity with the 16S rRNA gene sequences of SCWL phytoplasmas from various countries. Based on the reported 16S rRNA gene sequence data from SCWL isolates of various countries, we conducted phylogenetic and virtual RFLP analysis. In the resulting phylogenetic tree, all SCWL isolates clustered into two branches, with the Lincang and Baoshan SCWL phytoplasma isolates belonging to different branches. The virtual RFLP patterns show that phytoplasmas of the Lincang branch belong to subgroup 16SrXI-B. However, the virtual RFLP patterns revealed by HaeIII digestion of phytoplasmas of the Baoshan branch differed from those of subgroup 16SrXI-B. According to the results of phylogenetic and virtual RFLP analysis, we propose that the phytoplasmas of the Baoshan branch represent a new subgroup, 16SrXI-D. These findings suggest that SCWL is caused by phytoplasmas from group 16SrXI, including subgroup 16SrXI-B and a new subgroup, 16SrXI-D.

Identification of a novel acetate-utilizing bacterium belonging to Synergistes group 4 in anaerobic digester sludge

PubMed Central

Ito, Tsukasa; Yoshiguchi, Kazumi; Ariesyady, Herto Dwi; Okabe, Satoshi

2011-01-01

Major acetate-utilizing bacterial and archaeal populations in methanogenic anaerobic digester sludge were identified and quantified by radioisotope- and stable-isotope-based functional analyses, microautoradiography-fluorescence in situ hybridization (MAR-FISH) and stable-isotope probing of 16S rRNA (RNA-SIP) that can directly link 16S rRNA phylogeny with in situ metabolic function. First, MAR-FISH with 14C-acetate indicated the significant utilization of acetate by only two major groups, unidentified bacterial cells and Methanosaeta-like filamentous archaeal cells, in the digester sludge. To identify the acetate-utilizing unidentified bacteria, RNA-SIP was conducted with 13C6-glucose and 13C3-propionate as sole carbon source, which were followed by phylogenetic analysis of 16S rRNA. We found that bacteria belonging to Synergistes group 4 were commonly detected in both 16S rRNA clone libraries derived from the sludge incubated with 13C-glucose and 13C-propionate. To confirm that this bacterial group can utilize acetate, specific FISH probe targeting for Synergistes group 4 was newly designed and applied to the sludge incubated with 14C-acetate for MAR-FISH. The MAR-FISH result showed that bacteria belonging to Synergistes group 4 significantly took up acetate and their active population size was comparable to that of Methanosaeta in this sludge. In addition, as bacteria belonging to Synergistes group 4 had high Km for acetate and maximum utilization rate, they are more competitive for acetate over Methanosaeta at high acetate concentrations (2.5–10 m). To our knowledge, it is the first time to report the acetate-utilizing activity of uncultured bacteria belonging to Synergistes group 4 and its competitive significance to acetoclastic methanogen, Methanosaeta. PMID:21562600

Comparative evaluation of PCR amplification of RLEP, 16S rRNA, rpoT and Sod A gene targets for detection of M. leprae DNA from clinical and environmental samples.

PubMed

Turankar, Ravindra P; Pandey, Shradha; Lavania, Mallika; Singh, Itu; Nigam, Astha; Darlong, Joydeepa; Darlong, Fam; Sengupta, Utpal

2015-03-01

PCR assay is a highly sensitive, specific and reliable diagnostic tool for the identification of pathogens in many infectious diseases. Genome sequencing Mycobacterium leprae revealed several gene targets that could be used for the detection of DNA from clinical and environmental samples. The PCR sensitivity of particular gene targets for specific clinical and environmental isolates has not yet been established. The present study was conducted to compare the sensitivity of RLEP, rpoT, Sod A and 16S rRNA gene targets in the detection of M. leprae in slit skin smear (SSS), blood, soil samples of leprosy patients and their surroundings. Leprosy patients were classified into Paucibacillary (PB) and Multibacillary (MB) types. Ziehl-Neelsen (ZN) staining method for all the SSS samples and Bacteriological Index (BI) was calculated for all patients. Standard laboratory protocol was used for DNA extraction from SSS, blood and soil samples. PCR technique was performed for the detection of M. leprae DNA from all the above-mentioned samples. RLEP gene target was able to detect the presence of M. leprae in 83% of SSS, 100% of blood samples and in 36% of soil samples and was noted to be the best out of all other gene targets (rpoT, Sod A and 16S rRNA). It was noted that the RLEP gene target was able to detect the highest number (53%) of BI-negative leprosy patients amongst all the gene targets used in this study. Amongst all the gene targets used in this study, PCR positivity using RLEP gene target was the highest in all the clinical and environmental samples. Further, the RLEP gene target was able to detect 53% of blood samples as positive in BI-negative leprosy cases indicating its future standardization and use for diagnostic purposes. Copyright © 2015 Asian African Society for Mycobacteriology. Published by Elsevier Ltd. All rights reserved.

PCR diagnostics underestimate the prevalence of avian malaria (Plasmodium relictum) in experimentally-infected passerines

USGS Publications Warehouse

Jarvi, Susan I.; Schultz, Jeffrey J.; Atkinson, Carter T.

2002-01-01

Several polymerase chain reaction (PCR)-based methods have recently been developed for diagnosing malarial infections in both birds and reptiles, but a critical evaluation of their sensitivity in experimentally-infected hosts has not been done. This study compares the sensitivity of several PCR-based methods for diagnosing avian malaria (Plasmodium relictum) in captive Hawaiian honeycreepers using microscopy and a recently developed immunoblotting technique. Sequential blood samples were collected over periods of up to 4.4 yr after experimental infection and rechallenge to determine both the duration and detectability of chronic infections. Two new nested PCR approaches for detecting circulating parasites based on P. relictum 18S rRNA genes and the thrombospondin-related anonymous protein (TRAP) gene are described. The blood smear and the PCR tests were less sensitive than serological methods for detecting chronic malarial infections. Individually, none of the diagnostic methods was 100% accurate in detecting subpatent infections, although serological methods were significantly more sensitive (97%) than either nested PCR (61–84%) or microscopy (27%). Circulating parasites in chronically infected birds either disappear completely from circulation or to drop to intensities below detectability by nested PCR. Thus, the use of PCR as a sole means of detection of circulating parasites may significantly underestimate true prevalence.

Community structure and function of planktonic Crenarchaeota: changes with depth in the South China Sea.

PubMed

Hu, Anyi; Jiao, Nianzhi; Zhang, Chuanlun L

2011-10-01

Marine Crenarchaeota represent a widespread and abundant microbial group in marine ecosystems. Here, we investigated the abundance, diversity, and distribution of planktonic Crenarchaeota in the epi-, meso-, and bathypelagic zones at three stations in the South China Sea (SCS) by analysis of crenarchaeal 16S rRNA gene, ammonia monooxygenase gene amoA involved in ammonia oxidation, and biotin carboxylase gene accA putatively involved in archaeal CO(2) fixation. Quantitative PCR analyses indicated that crenarchaeal amoA and accA gene abundances varied similarly with archaeal and crenarchaeal 16S rRNA gene abundances at all stations, except that crenarchaeal accA genes were almost absent in the epipelagic zone. Ratios of the crenarchaeal amoA gene to 16S rRNA gene abundances decreased ~2.6 times from the epi- to bathypelagic zones, whereas the ratios of crenarchaeal accA gene to marine group I crenarchaeal 16S rRNA gene or to crenarchaeal amoA gene abundances increased with depth, suggesting that the metabolism of Crenarchaeota may change from the epi- to meso- or bathypelagic zones. Denaturing gradient gel electrophoresis profiling of the 16S rRNA genes revealed depth partitioning in archaeal community structures. Clone libraries of crenarchaeal amoA and accA genes showed two clusters: the "shallow" cluster was exclusively derived from epipelagic water and the "deep" cluster was from meso- and/or bathypelagic waters, suggesting that niche partitioning may take place between the shallow and deep marine Crenarchaeota. Overall, our results show strong depth partitioning of crenarchaeal populations in the SCS and suggest a shift in their community structure and ecological function with increasing depth.

Stable Isotope Quantitative N-Glycan Analysis by Liquid Separation Techniques and Mass Spectrometry.

PubMed

Mittermayr, Stefan; Albrecht, Simone; Váradi, Csaba; Millán-Martín, Silvia; Bones, Jonathan

2017-01-01

Liquid phase separation analysis and subsequent quantitation remains a challenging task for protein-derived oligosaccharides due to their inherent structural complexity and diversity. Incomplete resolution or co-detection of multiple glycan species complicates peak area-based quantitation and associated statistical analysis when optical detection methods are used. The approach outlined herein describes the utilization of stable isotope variants of commonly used fluorescent tags that allow for mass-based glycan identification and relative quantitation following separation by liquid chromatography (LC) or capillary electrophoresis (CE). Comparability assessment of glycoprotein-derived oligosaccharides is performed by derivatization with commercially available isotope variants of 2-aminobenzoic acid or aniline and analysis by LC- and CE-mass spectrometry. Quantitative information is attained from the extracted ion chromatogram/electropherogram ratios generated from the light and heavy isotope clusters.

A novel multi-walled carbon nanotube-based antibody conjugate for quantitative and semi-quantitative lateral flow assays.

PubMed

Sun, Wenjuan; Hu, Xiaolong; Liu, Jia; Zhang, Yurong; Lu, Jianzhong; Zeng, Libo

2017-10-01

In this study, the multi-walled carbon nanotubes (MWCNTs) were applied in lateral flow strips (LFS) for semi-quantitative and quantitative assays. Firstly, the solubility of MWCNTs was improved using various surfactants to enhance their biocompatibility for practical application. The dispersed MWCNTs were conjugated with the methamphetamine (MET) antibody in a non-covalent manner and then manufactured into the LFS for the quantitative detection of MET. The MWCNTs-based lateral flow assay (MWCNTs-LFA) exhibited an excellent linear relationship between the values of test line and MET when its concentration ranges from 62.5 to 1500 ng/mL. The sensitivity of the LFS was evaluated by conjugating MWCNTs with HCG antibody and the MWCNTs conjugated method is 10 times more sensitive than the one conjugated with classical colloidal gold nanoparticles. Taken together, our data demonstrate that MWCNTs-LFA is a more sensitive and reliable assay for semi-quantitative and quantitative detection which can be used in forensic analysis.

Cultivation of a highly enriched ammonia-oxidizing archaeon of thaumarchaeotal group I.1b from an agricultural soil.

PubMed

Kim, Jong-Geol; Jung, Man-Young; Park, Soo-Je; Rijpstra, W Irene C; Sinninghe Damsté, Jaap S; Madsen, Eugene L; Min, Deullae; Kim, Jin-Seog; Kim, Geun-Joong; Rhee, Sung-Keun

2012-06-01

Nitrification of excess ammonia in soil causes eutrophication of water resources and emission of atmospheric N(2) O gas. The first step of nitrification, ammonia oxidation, is mediated by Archaea as well as Bacteria. The physiological reactions mediated by ammonia-oxidizing archaea (AOA) and their contribution to soil nitrification are still unclear. Results of non-culture-based studies have shown the thaumarchaeotal group I.1b lineage of AOA to be dominant over both AOA of group I.1a and ammonia-oxidizing bacteria in various soils. We obtained from an agricultural soil a highly enriched ammonia-oxidizing culture dominated by a single archaeal population [c. 90% of total cells, as determined microscopically (by fluorescence in situ hybridization) and by quantitative PCR of its 16S rRNA gene]. The archaeon (termed 'strain JG1') fell within thaumarchaeotal group I.1b and was related to the moderately thermophilic archaeon, Candidatus Nitrososphaera gargensis, and the mesophilic archaeon, Ca. Nitrososphaera viennensis with 97.0% and 99.1% 16S rRNA gene sequence similarity respectively. Strain JG1 was neutrophilic (growth range pH 6.0-8.0) and mesophilic (growth range temperature 25-40°C). The optimum temperature of strain JG1 (35-40°C) is > 10°C higher than that of ammonia-oxidizing bacteria (AOB). Membrane analysis showed that strain JG1 contained a glycerol dialkyl glycerol tetraether, GDGT-4, and its regioisomer as major core lipids; this crenarchaeol regioisomer was previously detected in similar abundance in the thermophile, Ca. N. gargensis and has been frequently observed in tropical soils. Substrate uptake assays showed that the affinity of strain JG1 for ammonia and oxygen was much higher than those of AOB. These traits may give a competitive advantage to AOA related to strain JG1 in oligotrophic environments. (13) C-bicarbonate incorporation into archaeal lipids of strain JG1 established its ability to grow autotrophically. Strain JG1 produced a significant amount of N(2) O gas - implicating AOA as a possible source of N(2) O emission from soils. Sequences of archaeal amoA and 16S rRNA genes closely related to those of strain JG1 have been retrieved from various terrestrial environments in which lineage of strain JG1 is likely engaged in autotrophic nitrification. © 2012 Society for Applied Microbiology and Blackwell Publishing Ltd.

Guanosine 3'-diphosphate 5'-diphosphate is not required for growth rate-dependent control of rRNA synthesis in Escherichia coli.

PubMed Central

Gaal, T; Gourse, R L

1990-01-01

rRNA synthesis in Escherichia coli is subject to at least two regulation systems, growth rate-dependent control and stringent control. The inverse correlation between rRNA synthesis rates and guanosine 3'-diphosphate 5'-diphosphate (ppGpp) levels under various physiological conditions has led to the supposition that ppGpp is the mediator of both control mechanisms by inhibiting transcription from rrn P1 promoters. Recently, relA- spoT- strains have been constructed in which both ppGpp synthesis pathways most likely have been removed (M. Cashel, personal communication). We have confirmed that such strains produce no detectable ppGpp and therefore offer a direct means for testing the involvement of ppGpp in the regulation of rRNA synthesis in vivo. Stringent control was determined by measurement of rRNA synthesis after amino acid starvation, while growth rate control was determined by measurement of rRNA synthesis under different nutritional conditions. As expected, the relA- spoT- strain is relaxed for stringent control. However, growth rate-dependent regulation is unimpaired. These results indicate that growth rate regulation can occur in the absence of ppGpp and imply that ppGpp is not the mediator, or at least is not the sole mediator, of growth rate-dependent control. Therefore, growth rate-dependent control and stringent control may utilize different mechanisms for regulating stable RNA synthesis. PMID:2196571

Cytochemical features common to nucleoli and cytoplasmic nucleoloids of Olea europaea meiocytes: detection of rRNA by in situ hybridization.

PubMed

Alché, J D; Fernández, M C; Rodríguez-García, M I

1994-02-01

We used light and electron microscopic techniques to study the composition of cytoplasmic nucleoloids during meiotic division in Olea europaea. Nucleoloids were found in two clearly distinguishable morphological varieties: one similar in morphology to the nucleolus, and composed mainly of dense fibrillar component, and another surrounded by many ribosome-like particles. Cytochemical and immunocytochemical techniques showed similar reactivities in nucleoloids and the nucleolus: both are ribonucleoproteic in nature, and possess argyrophillic, argentaffinic and highly phosphorylated proteins. Immunohistochemical techniques failed to detect DNA in either structure. In situ hybridization to a 18 S rRNA probe demonstrated the presence of ribosomal transcripts in both the nucleolus and nucleoloids. These similarities in morphology and composition may reflect similar functionalities.

Molecular and Stable Isotope Investigation of Nitrite Respiring Bacterial Communities Capable of Anaerobic Ammonium Oxidation (ANAMMOX) and Denitrifying Anaerobic Methane Oxidation (DAMO) in Nitrogen Contaminated Groundwater

NASA Astrophysics Data System (ADS)

Song, B.; Hirsch, M.; Taylor, J.; Smith, R. L.; Repert, D.; Tobias, C. R.

2010-12-01

Anaerobic ammonium oxidation (ANAMMOX) and denitrifying anaerobic methane oxidation (DAMO) are two recently discovered N2 production pathways in the microbial nitrogen cycle. ANAMMOX has been relatively well investigated in various aquatic ecosystems, while DAMO has been examined only in freshwater wetlands. However, neither ANAMMOX nor DAMO have been studied in groundwater ecosystems as microbial N removal processes where they could compliment or compete with denitrification to remediate N contaminated aquifers. Thus, we conducted molecular and stable isotope analyses to detect and measure ANAMMOX and DAMO in a nitrogen contaminated aquifer on Cape Cod, Massachusetts. The study site has a plume of nitrogen contaminated groundwater as a result of continuous discharge of treated wastewater over 60 years. Groundwater was collected from multiport sampling devices installed at two sites, near the waste-water disposal location (A) and more than 3 km down gradient (B) along the contamination plume. Biomass was collected from water samples for DNA extraction and 15N tracer incubation experiments. PCR with specific 16S rRNA gene primers detected the presence of ANAMMOX and DAMO bacteria at both sites. Phylogenetic analysis of 16S rRNA genes revealed that the ANAMMOX community at site A was most associated with Kuenenia spp. while site B had a community more closely related to Brocadia spp. The DAMO communities at the two sites were quite different based on 16S rRNA gene analysis. The communities at site B are closely associated with Candidatus “Methylomirabilis oxyfera”, which is the first enriched DAMO culture. Most of the 16S rRNA sequences detected in site A were related to those found in other DAMO enrichment cultures established from a eutrophic ditch sediment. In order to determine active members of ANAMMOX communities, the transcriptional expression of hydrazine oxidase (hzo) and hydrazine hydrolase (hh) genes was examined at both sites. In addition, 15N tracer incubation experiments were used to measure the rates of ANAMMOX and denitrification. ANAMMOX was found to be higher than denitrification at site A where ANAMMOX accounted for 60% of the 15N2 production. In contrast, denitrification was higher than ANAMMOX at site B where Methylomirabilis spp. were found. Thus, this study clearly demonstrates the potential importance of ANAMMOX and DAMO in the nitrogen removal from groundwater and suggests that detailed characterization of the processes under in situ subsurface conditions could provide new information regarding the ecology of these microbes.

Culture-free, highly sensitive, quantitative detection of bacteria from minimally processed samples using fluorescence imaging by smartphone.

PubMed

Shrivastava, Sajal; Lee, Won-Il; Lee, Nae-Eung

2018-06-30

A critical unmet need in the diagnosis of bacterial infections, which remain a major cause of human morbidity and mortality, is the detection of scarce bacterial pathogens in a variety of samples in a rapid and quantitative manner. Herein, we demonstrate smartphone-based detection of Staphylococcus aureus in a culture-free, rapid, quantitative manner from minimally processed liquid samples using aptamer-functionalized fluorescent magnetic nanoparticles. The tagged S. aureus cells were magnetically captured in a detection cassette, and then fluorescence was imaged using a smartphone camera with a light-emitting diode as the excitation source. Our results showed quantitative detection capability with a minimum detectable concentration as low as 10 cfu/ml by counting individual bacteria cells, efficiently capturing S. aureus cells directly from a peanut milk sample within 10 min. When the selectivity of detection was investigated using samples spiked with other pathogenic bacteria, no significant non-specific detection occurred. Furthermore, strains of S. aureus from various origins showed comparable results, ensuring that the approach can be widely adopted. Therefore, the quantitative fluorescence imaging platform on a smartphone could allow on-site detection of bacteria, providing great potential assistance during major infectious disease outbreaks in remote and resource-limited settings. Copyright © 2018 Elsevier B.V. All rights reserved.

Estimating biodiversity of fungi in activated sludge communities using culture-independent methods.

PubMed

Evans, Tegan N; Seviour, Robert J

2012-05-01

Fungal diversity of communities in several activated sludge plants treating different influent wastes was determined by comparative sequence analyses of their 18S rRNA genes. Methods for DNA extraction and choice of primers for PCR amplification were both optimised using denaturing gradient gel electrophoresis profile patterns. Phylogenetic analysis revealed that the levels of fungal biodiversity in some communities, like those treating paper pulp wastes, were low, and most of the fungi detected in all communities examined were novel uncultured representatives of the major fungal subdivisions, in particular, the newly described clade Cryptomycota. The fungal populations in activated sludge revealed by these culture-independent methods were markedly different to those based on culture-dependent data. Members of the genera Penicillium, Cladosporium, Aspergillus and Mucor, which have been commonly identified in mixed liquor, were not identified in any of these plant communities. Non-fungal eukaryotic 18S rRNA genes were also amplified with the primer sets used. This is the first report where culture-independent methods have been applied to flocculated activated sludge biomass samples to estimate fungal community composition and, as expected, the data obtained gave a markedly different view of their population biodiversity compared to that based on culture-dependent methods.

Rapid and Quantitative Detection of Vibrio parahemolyticus by the Mixed-Dye-Based Loop-Mediated Isothermal Amplification Assay on a Self-Priming Compartmentalization Microfluidic Chip.

PubMed

Pang, Bo; Ding, Xiong; Wang, Guoping; Zhao, Chao; Xu, Yanan; Fu, Kaiyue; Sun, Jingjing; Song, Xiuling; Wu, Wenshuai; Liu, Yushen; Song, Qi; Hu, Jiumei; Li, Juan; Mu, Ying

2017-12-27

Vibrio parahemolyticus (VP) mostly isolated from aquatic products is one of the major causes of bacterial food-poisoning events worldwide, which could be reduced using a promising on-site detection method. Herein, a rapid and quantitative method for VP detection was developed by applying a mixed-dye-loaded loop-mediated isothermal amplification (LAMP) assay on a self-priming compartmentalization (SPC) microfluidic chip, termed on-chip mixed-dye-based LAMP (CMD-LAMP). In comparison to conventional approaches, CMD-LAMP was advantageous on the limit of detection, which reached down to 1 × 10 3 CFU/mL in food-contaminated samples without the pre-enrichment of bacteria. Additionally, as a result of the use of a mixed dye and SPC chip, the quantitative result could be easily acquired, avoiding the requirement of sophisticated instruments and tedious operation. Also, CMD-LAMP was rapid and cost-effective. Conclusively, CMD-LAMP has great potential in realizing the on-site quantitative analysis of VP for food safety.

Detection and differentiation of early acute and following age stages of myocardial infarction with quantitative post-mortem cardiac 1.5T MR.

PubMed

Schwendener, Nicole; Jackowski, Christian; Persson, Anders; Warntjes, Marcel J; Schuster, Frederick; Riva, Fabiano; Zech, Wolf-Dieter

2017-01-01

Recently, quantitative MR sequences have started being used in post-mortem imaging. The goal of the present study was to evaluate if early acute and following age stages of myocardial infarction can be detected and discerned by quantitative 1.5T post-mortem cardiac magnetic resonance (PMCMR) based on quantitative T1, T2 and PD values. In 80 deceased individuals (25 female, 55 male), a cardiac MR quantification sequence was performed prior to cardiac dissection at autopsy in a prospective study. Focal myocardial signal alterations detected in synthetically generated MR images were MR quantified for their T1, T2 and PD values. The locations of signal alteration measurements in PMCMR were targeted at autopsy heart dissection and cardiac tissue specimens were taken for histologic examinations. Quantified signal alterations in PMCMR were correlated to their according histologic age stage of myocardial infarction. In PMCMR seventy-three focal myocardial signal alterations were detected in 49 of 80 investigated hearts. These signal alterations were diagnosed histologically as early acute (n=39), acute (n=14), subacute (n=10) and chronic (n=10) age stages of myocardial infarction. Statistical analysis revealed that based on their quantitative T1, T2 and PD values, a significant difference between all defined age groups of myocardial infarction can be determined. It can be concluded that quantitative 1.5T PMCMR quantification based on quantitative T1, T2 and PD values is feasible for characterization and differentiation of early acute and following age stages of myocardial infarction. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.