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Sample records for 16s rdna pcr

  1. Genotypic Characterization of Bradyrhizobium Strains Nodulating Endemic Woody Legumes of the Canary Islands by PCR-Restriction Fragment Length Polymorphism Analysis of Genes Encoding 16S rRNA (16S rDNA) and 16S-23S rDNA Intergenic Spacers, Repetitive Extragenic Palindromic PCR Genomic Fingerprinting, and Partial 16S rDNA Sequencing

    PubMed Central

    Vinuesa, Pablo; Rademaker, Jan L. W.; de Bruijn, Frans J.; Werner, Dietrich

    1998-01-01

    We present a phylogenetic analysis of nine strains of symbiotic nitrogen-fixing bacteria isolated from nodules of tagasaste (Chamaecytisus proliferus) and other endemic woody legumes of the Canary Islands, Spain. These and several reference strains were characterized genotypically at different levels of taxonomic resolution by computer-assisted analysis of 16S ribosomal DNA (rDNA) PCR-restriction fragment length polymorphisms (PCR-RFLPs), 16S-23S rDNA intergenic spacer (IGS) RFLPs, and repetitive extragenic palindromic PCR (rep-PCR) genomic fingerprints with BOX, ERIC, and REP primers. Cluster analysis of 16S rDNA restriction patterns with four tetrameric endonucleases grouped the Canarian isolates with the two reference strains, Bradyrhizobium japonicum USDA 110spc4 and Bradyrhizobium sp. strain (Centrosema) CIAT 3101, resolving three genotypes within these bradyrhizobia. In the analysis of IGS RFLPs with three enzymes, six groups were found, whereas rep-PCR fingerprinting revealed an even greater genotypic diversity, with only two of the Canarian strains having similar fingerprints. Furthermore, we show that IGS RFLPs and even very dissimilar rep-PCR fingerprints can be clustered into phylogenetically sound groupings by combining them with 16S rDNA RFLPs in computer-assisted cluster analysis of electrophoretic patterns. The DNA sequence analysis of a highly variable 264-bp segment of the 16S rRNA genes of these strains was found to be consistent with the fingerprint-based classification. Three different DNA sequences were obtained, one of which was not previously described, and all belonged to the B. japonicum/Rhodopseudomonas rDNA cluster. Nodulation assays revealed that none of the Canarian isolates nodulated Glycine max or Leucaena leucocephala, but all nodulated Acacia pendula, C. proliferus, Macroptilium atropurpureum, and Vigna unguiculata. PMID:9603820

  2. [Rapid detection of Pseudomonas aeruginosa by the fluorescence quantitative PCR assay targeting 16S rDNA].

    PubMed

    Xue, Li-Jun; Wang, Yong-Zhi; Ren, Hao; Tong, Yi-Min; Zhao, Ping; Zhu, Shi-Ying; Qi, Zhong-Tian

    2006-09-01

    The 16S rDNA specific primers were designed for rapid detection of Pseudomonas aeruginosa (PA) by the fluorescence quantitative PCR (FQ-PCR) assay, based upon multiple sequence alignment and phylogenetic tree analysis of the 16S rDNAs of over 20 bacteria. After extraction of PA genomic DNA, the target 16S rDNA fragment was amplified by PCR with specific primers, and used to construct recombinant pMDT-Pfr plasmid, the dilution gradients of which were subjected to the standard quantitation curve in FQ-PCR assay. Different concentrations of PA genomic DNA were detected by FQ-PCR in a 20microL of reaction system with SYBR Green I. At the same time, various genomic DNAs of Staphylococcus aureus, Salmonella typhi, Shigella flexneri, Proteus vulgaris, Staphylococcus epidermidis, Escherichia coli, and Mycobacterium tuberculosis were used as negative controls to confirm specificity of the FQ-PCR detection assay. Results demonstrated that the predicted amplified product of designed primers was of high homology only with PA 16S rDNA, and that sensitivity of the FQ-PCR assay was of 3.6pg/microL of bacterial DNA or (2.1 x 10(3) +/- 3.1 x 10(2)) copies/microL of 16S rDNA, accompanied with high specificity, and that the whole detection process including DNA extraction could be completed in about two hours. In contrast to traditional culture method, the FQ-PCR assay targeting 16S rDNA gene can be used to detect PA rapidly, which exhibits perfect application prospect in future. PMID:17037203

  3. ASSESSMENT OF FECAL POLLUTION SOURCES IN PLUM CREEK WATERSHED USING PCR AND PHYLOGENETIC ANALYSES OF BACTEROIDETES 16S RDNA

    EPA Science Inventory

    Traditional methods for assessing fecal pollution in environmental systems, such as monitoring for fecal coliforms are not capable of discriminating between different sources fecal pollution. Recently, 16S rDNA Bacteroidetes-targeted PCR assays were developed to discriminate betw...

  4. PCR amplification of 16S rDNA from lyophilized cell cultures facilitates studies in molecular systematics

    NASA Technical Reports Server (NTRS)

    Wisotzkey, J. D.; Jurtshuk, P. Jr; Fox, G. E.

    1990-01-01

    The sequence of the major portion of a Bacillus cycloheptanicus strain SCH(T) 16S rRNA gene is reported. This sequence suggests that B. cycloheptanicus is genetically quite distinct from traditional Bacillus strains (e.g., B. subtilis) and may be properly regarded as belonging to a different genus. The sequence was determined from DNA that was produced by direct amplification of ribosomal DNA from a lyophilized cell pellet with straightforward polymerase chain reaction (PCR) procedures. By obviating the need to revive cell cultures from the lyophile pellet, this approach facilitates rapid 16S rDNA sequencing and thereby advances studies in molecular systematics.

  5. Genus-specific profile of acetic acid bacteria by 16S rDNA PCR-DGGE.

    PubMed

    De Vero, Luciana; Giudici, Paolo

    2008-06-30

    An effective method for grouping acetic acid bacteria (AAB) genera was defined and evaluated as a tool for preliminary screening of the major AAB species involved in vinegar production. Acetobacter, Gluconobacter, Gluconacetobacter, Asaia, Neoasaia, Saccharibacter, Frateuria and Kozakia AAB strains were screened on the basis of the 16S rDNA sequences using polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) technique. The DGGE profile of all the strains tested, consisted of one single band of approximately 330 bp for each strain and allowed their clustering. The results obtained clearly reflected in silico phylogenetic analysis of the AAB species used in this study, in fact, the species with a higher 16S rDNA sequence homology showed a similar electrophoretic profile. In particular almost all the species belonging to the genus Gluconacetobacter showed a DGGE pattern nearly identical and well distinct from all the other AAB genera. Furthermore by PCR-DGGE it was possible to clearly group the species more frequently recovered from vinegar fermentation which are mainly distributed in the genera Acetobacter, Gluconobacter and Gluconacetobacter. PMID:17919758

  6. Development of a PCR assay based on the 16S-23S rDNA internal transcribed spacer for identification of strictly anaerobic bacterium Zymophilus.

    PubMed

    Felsberg, Jurgen; Jelínková, Markéta; Kubizniaková, Petra; Matoulková, Dagmar

    2015-06-01

    PCR-primers were designed for identification of strictly anaerobic bacteria of the genus Zymophilus based on genus-specific sequences of the 16S-23S rDNA internal transcribed spacer region. The specificity of the primers was tested against 37 brewery-related non-target microorganisms that could potentially occur in the same brewery specimens. None DNA was amplified from any of the non-Zymophilus strains tested including genera from the same family (Pectinatus, Megasphaera, Selenomonas), showing thus 100% specificity. PCR assay developed in this study allows an extension of the spectra of detected beer spoilage microorganisms in brewery laboratories. PMID:25725268

  7. Genomic-Based Restriction Enzyme Selection for Specific Detection of Piscirickettsia salmonis by 16S rDNA PCR-RFLP

    PubMed Central

    Mandakovic, Dinka; Glasner, Benjamín; Maldonado, Jonathan; Aravena, Pamela; González, Mauricio; Cambiazo, Verónica; Pulgar, Rodrigo

    2016-01-01

    The gram negative facultative bacterium P. salmonis is the etiological agent of Salmonid Rickettsial Septicaemia (SRS), a severe disease that causes important economic losses in the global salmon farmer industry. Despite efforts to control this disease, the high frequency of new epizootic events indicate that the vaccine and antibiotics treatments have limited effectiveness, therefore the preventive and diagnostic approaches must be improved. A comparison of several methodologies for SRS diagnostic indicate differences in their specificity and its capacity to detect other bacteria coexisting with P. salmonis in culture media (contamination) and fish samples (coinfection), aspects relevant for research, vaccine development and clinical diagnostic. By computer-simulation analyses, we identified a group of restriction enzymes that generate unique P. salmonis 16S rDNA band patterns, distinguishable from all other bacteria. From this information, we designed and developed a PCR-RFLP (Polymerase Chain Reaction—Restriction Fragment Length Polymorphism) assay, which was validated using 16S rDNA universal primers and restriction enzyme PmaCI for the amplification and digestion, respectively. Experimental validation was performed by comparing the restriction pattern of P. salmonis with the restriction patterns generated by bacteria that cohabit with P. salmonis (fish bacterial isolates and culture media contaminants). Our results indicate that the restriction enzyme selection pipeline was suitable to design a more specific, sensible, faster and cheaper assay than the currently used P. salmonis detection methodologies. PMID:27242682

  8. Adaptation of a membrane bioreactor to 1,2-dichloroethane revealed by 16S rDNA pyrosequencing and dhlA qPCR.

    PubMed

    Munro, Jacob E; Liew, Elissa F; Coleman, Nicholas V

    2013-01-01

    A pilot-scale membrane bioreactor (MBR) was tested for bioremediation of 1,2-dichloroethane (DCA) in groundwater. Pyrosequencing of 16S rDNA was used to study changes in the microbiology of the MBR over 137 days, including a 67 day initial adaptation phase of increasing DCA concentration. The bacterial community in the MBR was distinct from those in soil and groundwater at the same site, and was dominated by alpha- and beta- proteobacteria, including Rhodobacter, Methylibium, Rhodopseudomonas, Methyloversatilis, Caldilinea, Thiobacillus, Azoarcus, Hyphomicrobium, and Leptothrix. Biodegradation of DCA in the MBR began after 26 days, and was sustained for the remainder of the experiment. A quantitative PCR (qPCR) assay for the dehalogenase gene dhlA was developed to monitor DCA-degrading bacteria in the MBR, and a positive correlation was seen between dhlA gene abundance and the cumulative amount of DCA that had entered the MBR. Genera previously associated with aerobic DCA biodegradation (Xanthobacter, Ancylobacter, Azoarcus) were present in the MBR, and the abundance of Azoarcus correlated well with dhlA gene abundance. This study shows that MBRs can be an effective method for removal of DCA from groundwater, and that the dhlA qPCR is a rapid and sensitive method for detection of DCA-degrading bacteria. PMID:24175727

  9. Food Targeting: A Real-Time PCR Assay Targeting 16S rDNA for Direct Quantification of Alicyclobacillus spp. Spores after Aptamer-Based Enrichment.

    PubMed

    Hünniger, Tim; Felbinger, Christine; Wessels, Hauke; Mast, Sophia; Hoffmann, Antonia; Schefer, Anna; Märtlbauer, Erwin; Paschke-Kratzin, Angelika; Fischer, Markus

    2015-05-01

    Spore-forming Alicyclobacillus spp. are able to form metabolites that induce even in small amounts an antiseptical or medicinal off-flavor in fruit juices. Microbial contaminations could occur by endospores, which overcame the pasteurization process. The current detection method for Alicyclobacillus spp. can take up to 1 week because of microbiological enrichment. In a previous study, DNA aptamers were selected and characterized for an aptamer-driven rapid enrichment of Alicyclobacillus spp. spores from orange juice by magnetic separation. In the present work, a direct quantification assay for Alicyclobacillus spp. spores was developed to complete the two-step approach of enrichment and detection. After mechanical treatment of the spores, the isolated DNA was quantified in a real-time PCR-assay targeting 16S rDNA. The assay was evaluated by the performance requirements of the European Network of Genetically Modified Organisms Laboratories (ENGL). Hence, the presented method is applicable for direct spore detection from orange juice in connection with an enrichment step. PMID:25880790

  10. Development of a real-time PCR method for the detection of fossil 16S rDNA fragments of phototrophic sulfur bacteria in the sediments of Lake Cadagno.

    PubMed

    Ravasi, D F; Peduzzi, S; Guidi, V; Peduzzi, R; Wirth, S B; Gilli, A; Tonolla, M

    2012-05-01

    Lake Cadagno is a crenogenic meromictic lake situated in the southern range of the Swiss Alps characterized by a compact chemocline that has been the object of many ecological studies. The population dynamics of phototrophic sulfur bacteria in the chemocline has been monitored since 1994 with molecular methods such as 16S rRNA gene clone library analysis. To reconstruct paleo-microbial community dynamics, we developed a quantitative real-time PCR methodology for specific detection of 16S rRNA gene sequences of purple and green sulfur bacteria populations from sediment samples. We detected fossil 16S rDNA of nine populations of phototrophic sulfur bacteria down to 9-m sediment depth, corresponding to about 9500 years of the lake's biogeological history. These results provide the first evidence for the presence of 16S rDNA of anoxygenic phototrophic bacteria in Holocene sediments of an alpine meromictic lake and indicate that the water column stratification and the bacterial plume were already present in Lake Cadagno thousands of years ago. The finding of Chlorobium clathratiforme remains in all the samples analyzed shows that this population, identified in the water column only in 2001, was already a part of the lake's biota in the past. PMID:22433067

  11. Identification of Lactobacillus strains of goose origin using MALDI-TOF mass spectrometry and 16S-23S rDNA intergenic spacer PCR analysis.

    PubMed

    Dec, Marta; Urban-Chmiel, Renata; Gnat, Sebastian; Puchalski, Andrzej; Wernicki, Andrzej

    2014-04-01

    The objective of our study was to identify Lactobacillus sp. strains of goose origin using MALDI-TOF mass spectrometry, ITS-PCR and ITS-PCR/RFLP. All three techniques proved to be valuable tools for identification of avian lactobacilli and produced comparable classification results. Lactobacillus strains were isolated from 100% of geese aged 3 weeks to 4 years, but from only 25% of chicks aged 1-10 days. Among the 104 strains isolated, we distinguished 14 Lactobacillus species. The dominant species was Lactobacillus salivarius (35.6%), followed by Lactobacillus johnsonii (18.3%), Lactobacillus ingluviei (11.5%) and Lactobacillus agilis (7.7%). The intact-cell MALDI-TOF mass spectrometry enabled rapid species identification of the lactobacilli with minimal pretreatment. However, it produced more than one identification result for 11.5% examined strains (mainly of the species L. johnsonii). ITS-PCR distinguished 12 genotypes among the isolates, but was not able to differentiate closely related strains, i.e. between Lactobacillus amylovorus and Lactobacillus kitasatonis and between Lactobacillus paracasei, Lactobacillus rhamnosus and Lactobacillus zeae. These species were differentiated by ITS-PCR/RFLP using the restriction enzymes TaqI and MseI. The results obtained indicate that ITS-PCR and ITS-PCR/RFLP assays could be used not only for interspecific, but also for intraspecific, typing. PMID:24607713

  12. Microbial community analysis in the denitrification process of saline-wastewater by denaturing gradient gel electrophoresis of PCR-amplified 16S rDNA and the cultivation method.

    PubMed

    Yoshie, S; Noda, N; Miyano, T; Tsuneda, S; Hirata, A; Inamori, Y

    2001-01-01

    The metallurgic wastewater generated from the processes of recovering precious metals from industrial wastes contains high concentrations of nitrogen compounds and salts. Biological nitrogen removal from this wastewater was attempted using a circulating bioreactor system equipped with an anaerobic packed bed or an anaerobic fluidized bed. The denitrification capability of the system with the anaerobic packed bed was more stable than that of the system with the anaerobic fluidized bed. The NOx removal rate of the anaerobic packed bed was as high as 97%. Microbial community analysis by denaturing gradient gel electrophoresis (DGGE) of PCR-amplified 16S ribosomal DNA (rDNA) fragments and the cultivation method revealed that the community diversity varied in accordance with wastewater composition such as the level of salinity and so on. Phylogenetic analysis suggested that the taxonomic affiliation of the dominant species in the anaerobic reactors was to the gamma-Proteobacteria including Halomonadaceae species. The PCR-DGGE method as a non-cultivation method was found to be a powerful tool for analysis of the microbial community, because the cultivation method could detect only a fraction of the microbial species present in these systems. The genetic diversity of the isolated bacteria belonging to the gamma-Proteobacteria which reduced both nitrate and nitrite in the anaerobic packed bed was higher than that of the bacteria in the anaerobic fluidized bed. This suggested that a genetically diverse microbial community stabilized the denitrifying performance in the anaerobic packed bed. PMID:16233109

  13. Using an intervening sequence of Faecalibacterium 16S rDNA to identify poultry feces.

    PubMed

    Shen, Zhenyu; Duan, Chuanren; Zhang, Chao; Carson, Andrew; Xu, Dong; Zheng, Guolu

    2013-10-15

    This study was designed to identify poultry feces-specific marker(s) within sequences of Faecalibacterium 16S rDNA for detecting poultry fecal pollution in water. Bioinformatics tools were used in the comparative analysis of 7,458 sequences of Faecalibacterium 16S rDNA, reportedly associated with various poultry (chicken and turkey) and animal species. One intervening sequence (IVS) within between the hypervariable region 1 and the conserved region 2, designated as IVS-p, was found to be unique to poultry feces. Based on this sequence, a PCR assay (PCR-p) was developed. The PCR-p produced an amplicon of 132 bp only in the test when fecal or wastewater samples from poultry were used, but not when using fecal or wastewater samples from other sources. The non-poultry sources included feces of beef or dairy cattle, dog, horse, human, domestic or wild geese, seagull, sheep, swine, and wild turkey. These data indicate that IVS-p may prove to be a useful genetic marker for the specific identification of poultry fecal pollution in environmental waterways. Furthermore, results of data mining and PCR assay indicate that the IVS-p may have a broad geographic distribution. This report represents initial evidence of the potential utility of ribosomal intervening sequences as genetic markers for tracking host sources of fecal pollution in waterways. PMID:24011842

  14. ASSESSMENT OF FECAL POLLUTION SOURCES IN PLUM CREEK WATERSHED USING BACTEROIDETES 16S RDNA-BASED ASSAYS

    EPA Science Inventory

    Recently, 16S rDNA Bacteroidetes-targeted PCR assays were developed to discriminate between ruminant and human fecal pollution. These assays are rapid and relatively inexpensive but have been used in a limited number of studies. In this study, we evaluated the efficacy o...

  15. Combining denaturing gradient gel electrophoresis of 16S rDNA V3 region and 16S-23S rDNA spacer region polymorphism analyses for the identification of staphylococci from Italian fermented sausages.

    PubMed

    Blaiotta, Giuseppe; Pennacchia, Carmelina; Ercolini, Danilo; Moschetti, Giancarlo; Villani, Francesco

    2003-09-01

    Separation of amplified V3 region from 16S rDNA by denaturing gradient gel electrophoresis (PCR-DGGE) and 16S-23S rDNA intergenic spacer region polymorphism (ISR-PCR) analyses were tested as tool for differentiation of staphylococcal strains commonly isolated from fermented sausages. Variable V3 regions of 25 staphylococcal reference strains and 96 wild strains of species belonging to the genera Staphylococcus, Micrococcus and Kocuria were analyzed. PCR-DGGE profiles obtained were species-specific for S. sciuri, S. haemolyticus, S. hominis, S. auricularis, S. condimenti, S. kloosi, S. vitulus, S. succinus, S. pasteuri, S. capitis and S. (Macrococcus) caseolyticus. Moreover, 7 groups could be distinguished gathering the remaining species as result of the separation of the V3 rDNA amplicons in DGGE. Furthermore, the combination of the results obtained by PCR-DGGE and ISR-PCR analyses allowed a clear differentiation of all the staphylococcal species analysed, with exception of the pairs S. equorum-S. cohnii and S. carnosus-S. schleiferi. The suitability of both molecular techniques and of the combination their results for the identification of staphylococci was validated analysing partial nucleotide sequence of the 16S rDNA of a representative number of wild strains. PMID:14529185

  16. PCR Conditions for 16S Primers for Analysis of Microbes in the Colon of Rats

    PubMed Central

    Camacho, H.; Tuero, A. D.; Bacardí, D.; Palenzuela, D. O.; Aguilera, A.; Silva, J. A.; Estrada, R.; Gell, O.; Suárez, J.; Ancizar, J.; Brown, E.; Colarte, A. B.; Castro, J.; Novoa, L. I.

    2016-01-01

    The study of the composition of the intestinal flora is important to the health of the host, playing a key role in maintaining intestinal homeostasis and the evolution of the immune system. For these studies, various universal primers of the 16S rDNA gene are used in microbial taxonomy. Here, we report an evaluation of 5 universal primers to explore the presence of microbial DNA in colon biopsies preserved in RNAlater solution. The DNA extracted was used for the amplification of PCR products containing the variable (V) regions of the microbial 16S rDNA gene. The PCR products were studied by restriction fragment length polymorphism (RFLP) analysis and DNA sequence, whose percent of homology with microbial sequences reported in GenBank was verified using bioinformatics tools. The presence of microbes in the colon of rats was quantified by the quantitative PCR (qPCR) technique. We obtained microbial DNA from rat, useful for PCR analysis with the universal primers for the bacteria 16S rDNA. The sequences of PCR products obtained from a colon biopsy of the animal showed homology with the classes bacilli (Lactobacillus spp) and proteobacteria, normally represented in the colon of rats. The proposed methodology allowed the attainment of DNA of bacteria with the quality and integrity for use in qPCR, sequencing, and PCR-RFLP analysis. The selected universal primers provided knowledge of the abundance of microorganisms and the formation of a preliminary test of bacterial diversity in rat colon biopsies. PMID:27382362

  17. The use of 16S and 16S-23S rDNA to easily detect and differentiate common Gram-negative orchard epiphytes.

    PubMed

    Jeng, R S; Svircev, A M; Myers, A L; Beliaeva, L; Hunter, D M; Hubbes, M

    2001-02-01

    The identification of Gram-negative pathogenic and non-pathogenic bacteria commonly isolated from an orchard phylloplane may result in a time consuming and tedious process for the plant pathologist. The paper provides a simple "one-step" protocol that uses the polymerase chain reaction (PCR) to amplify intergenic spacer regions between 16S and 23S genes and a portion of 16S gene in the prokaryotic rRNA genetic loci. Amplified 16S rDNA, and restriction fragment length polymorphisms (RFLP) following EcoRI digestion produced band patterns that readily distinguished between the plant pathogen Erwinia amylovora (causal agent of fire blight in pear and apple) and the orchard epiphyte Pantoea agglomerans (formerly E. herbicola). The amplified DNA patterns of 16S-23S spacer regions may be used to differentiate E. amylovora at the intraspecies level. Isolates of E. amylovora obtained from raspberries exhibited two major fragments while those obtained from apples showed three distinct amplified DNA bands. In addition, the size of the 16S-23S spacer region differs between Pseudomonas syringae and Pseudomonas fluorescens. The RFLP pattern generated by HaeIII digestion may be used to provide a rapid and accurate identification of these two common orchard epiphytes. PMID:11166101

  18. Molecular Analysis of Methanogen Richness in Landfill and Marshland Targeting 16S rDNA Sequences

    PubMed Central

    Yadav, Shailendra; Kundu, Sharbadeb; Ghosh, Sankar K.; Maitra, S. S.

    2015-01-01

    Methanogens, a key contributor in global carbon cycling, methane emission, and alternative energy production, generate methane gas via anaerobic digestion of organic matter. The methane emission potential depends upon methanogenic diversity and activity. Since they are anaerobes and difficult to isolate and culture, their diversity present in the landfill sites of Delhi and marshlands of Southern Assam, India, was analyzed using molecular techniques like 16S rDNA sequencing, DGGE, and qPCR. The sequencing results indicated the presence of methanogens belonging to the seventh order and also the order Methanomicrobiales in the Ghazipur and Bhalsawa landfill sites of Delhi. Sequences, related to the phyla Crenarchaeota (thermophilic) and Thaumarchaeota (mesophilic), were detected from marshland sites of Southern Assam, India. Jaccard analysis of DGGE gel using Gel2K showed three main clusters depending on the number and similarity of band patterns. The copy number analysis of hydrogenotrophic methanogens using qPCR indicates higher abundance in landfill sites of Delhi as compared to the marshlands of Southern Assam. The knowledge about “methanogenic archaea composition” and “abundance” in the contrasting ecosystems like “landfill” and “marshland” may reorient our understanding of the Archaea inhabitants. This study could shed light on the relationship between methane-dynamics and the global warming process. PMID:26568700

  19. Assessment of four DNA fragments (COI, 16S rDNA, ITS2, 12S rDNA) for species identification of the Ixodida (Acari: Ixodida)

    PubMed Central

    2014-01-01

    Background The 5’ region of cytochrome oxidase I (COI) is the standard marker for DNA barcoding. However, COI has proved to be of limited use in identifying some species, and for some taxa, the coding sequence is not efficiently amplified by PCR. These deficiencies lead to uncertainty as to whether COI is the most suitable barcoding fragment for species identification of ticks. Methods In this study, we directly compared the relative effectiveness of COI, 16S ribosomal DNA (rDNA), nuclear ribosomal internal transcribed spacer 2 (ITS2) and 12S rDNA for tick species identification. A total of 307 sequences from 84 specimens representing eight tick species were acquired by PCR. Besides the 1,834 published sequences of 189 tick species from GenBank and the Barcode of Life Database, 430 unpublished sequences representing 59 tick species were also successfully screened by Bayesian analyses. Thereafter, the performance of the four DNA markers to identify tick species was evaluated by identification success rates given by these markers using nearest neighbour (NN), BLASTn, liberal tree-based or liberal tree-based (+threshold) methods. Results Genetic divergence analyses showed that the intra-specific divergence of each marker was much lower than the inter-specific divergence. Our results indicated that the rates of correct sequence identification for all four markers (COI, 16S rDNA, ITS2, 12S rDNA) were very high (> 96%) when using the NN methodology. We also found that COI was not significantly better than the other markers in terms of its rate of correct sequence identification. Overall, BLASTn and NN methods produced higher rates of correct species identification than that produced by the liberal tree-based methods (+threshold or otherwise). Conclusions As the standard DNA barcode, COI should be the first choice for tick species identification, while 16S rDNA, ITS2, and 12S rDNA could be used when COI does not produce reliable results. Besides, NN and BLASTn are

  20. Simultaneous Discrimination between 15 Fish Pathogens by Using 16S Ribosomal DNA PCR and DNA Microarrays

    PubMed Central

    Warsen, Adelaide E.; Krug, Melissa J.; LaFrentz, Stacey; Stanek, Danielle R.; Loge, Frank J.; Call, Douglas R.

    2004-01-01

    We developed a DNA microarray suitable for simultaneous detection and discrimination between multiple bacterial species based on 16S ribosomal DNA (rDNA) polymorphisms using glass slides. Microarray probes (22- to 31-mer oligonucleotides) were spotted onto Teflon-masked, epoxy-silane-derivatized glass slides using a robotic arrayer. PCR products (ca. 199 bp) were generated using biotinylated, universal primer sequences, and these products were hybridized overnight (55°C) to the microarray. Targets that annealed to microarray probes were detected using a combination of Tyramide Signal Amplification and Alexa Fluor 546. This methodology permitted 100% specificity for detection of 18 microbes, 15 of which were fish pathogens. With universal 16S rDNA PCR (limited to 28 cycles), detection sensitivity for purified control DNA was equivalent to <150 genomes (675 fg), and this sensitivity was not adversely impacted either by the presence of competing bacterial DNA (1.1 × 106 genomes; 5 ng) or by the addition of up to 500 ng of fish DNA. Consequently, coupling 16S rDNA PCR with a microarray detector appears suitable for diagnostic detection and surveillance for commercially important fish pathogens. PMID:15240304

  1. Identification of signature and primers specific to genus Pseudomonas using mismatched patterns of 16S rDNA sequences

    PubMed Central

    Purohit, HJ; Raje, DV; Kapley, A

    2003-01-01

    Background Pseudomonas, a soil bacterium, has been observed as a dominant genus that survives in different habitats with wide hostile conditions. We had a basic assumption that the species level variation in 16S rDNA sequences of a bacterial genus is mainly due to substitutions rather than insertion or deletion of bases. Keeping this in view, the aim was to identify a region of 16S rDNA sequence and within that focus on substitution prone stretches indicating species level variation and to derive patterns from these stretches that are specific to the genus. Results Repeating elements that are highly conserved across different species of Pseudomonas were considered as guiding markers to locate a region within the 16S gene. Four repeating patterns showing more than 80% consistency across fifty different species of Pseudomonas were identified. The sub-sequences between the repeating patterns yielded a continuous region of 495 bases. The sub-sequences after alignment and using Shanon's entropy measure yielded a consensus pattern. A stretch of 24 base positions in this region, showing maximum variations across the sampled sequences was focused for possible genus specific patterns. Nine patterns in this stretch showed nearly 70% specificity to the target genus. These patterns were further used to obtain a signature that is highly specific to Pseudomonas. The signature region was used to design PCR primers, which yielded a PCR product of 150 bp whose specificity was validated through a sample experiment. Conclusions The developed approach was successfully applied to genus Pseudomonas. It could be tried in other bacterial genera to obtain respective signature patterns and thereby PCR primers, for their rapid tracking in the environmental samples. PMID:12769821

  2. Paenibacillus larvae 16S-23S rDNA intergenic transcribed spacer (ITS) regions: DNA fingerprinting and characterization.

    PubMed

    Dingman, Douglas W

    2012-07-01

    Paenibacillus larvae is the causative agent of American foulbrood in honey bee (Apis mellifera) larvae. PCR amplification of the 16S-23S ribosomal DNA (rDNA) intergenic transcribed spacer (ITS) regions, and agarose gel electrophoresis of the amplified DNA, was performed using genomic DNA collected from 134 P. larvae strains isolated in Connecticut, six Northern Regional Research Laboratory stock strains, four strains isolated in Argentina, and one strain isolated in Chile. Following electrophoresis of amplified DNA, all isolates exhibited a common migratory profile (i.e., ITS-PCR fingerprint pattern) of six DNA bands. This profile represented a unique ITS-PCR DNA fingerprint that was useful as a fast, simple, and accurate procedure for identification of P. larvae. Digestion of ITS-PCR amplified DNA, using mung bean nuclease prior to electrophoresis, characterized only three of the six electrophoresis bands as homoduplex DNA and indicating three true ITS regions. These three ITS regions, DNA migratory band sizes of 915, 1010, and 1474 bp, signify a minimum of three types of rrn operons within P. larvae. DNA sequence analysis of ITS region DNA, using P. larvae NRRL B-3553, identified the 3' terminal nucleotides of the 16S rRNA gene, 5' terminal nucleotides of the 23S rRNA gene, and the complete DNA sequences of the 5S rRNA, tRNA(ala), and tRNA(ile) genes. Gene organization within the three rrn operon types was 16S-23S, 16S-tRNA(ala)-23S, and l6S-5S-tRNA(ile)-tRNA(ala)-23S and these operons were named rrnA, rrnF, and rrnG, respectively. The 23S rRNA gene was shown by I-CeuI digestion and pulsed-field gel electrophoresis of genomic DNA to be present as seven copies. This was suggestive of seven rrn operon copies within the P. larvae genome. Investigation of the 16S-23S rDNA regions of this bacterium has aided the development of a diagnostic procedure and has helped genomic mapping investigations via characterization of the ITS regions. PMID:22510214

  3. Characterization of the dominant bacterial communities during storage of Norway lobster and Norway lobster tails (Nephrops norvegicus) based on 16S rDNA analysis by PCR-DGGE.

    PubMed

    Bekaert, Karen; Devriese, Lisa; Maes, Sara; Robbens, Johan

    2015-04-01

    The aim of this study was to investigate the microbial quality of whole Norway lobster (Nephrops norvegicus) and Norway lobster tails to optimize handling conditions. This was done by assessing the total viable count (TVC) and characterizing the dominant microbiota. The cultivable microorganisms were quantified via classical microbiological plating methods. To characterize as many bacterial species present as possible, we performed advanced molecular identification techniques (PCR-DGGE). The initial TVC of fresh Norway lobster meat was high (3.0 log cfu/g) as compared to fish. No significant difference between whole Norway lobster and Norway lobster tails could be found during the storage period. From day 6 of storage, a significant difference between Plate Count Agar (PCA) and Marine Agar (MA) was observed. The microbiota of Norway lobster was dominated by members of the Gram-negative genera such as Psychrobacter spp., Pseudoalteromonas spp., Pseudomonas spp., Luteimonas spp., and Aliivibrio spp. From these bacteria, mainly Psychrobacter spp. and Pseudomonas spp. remained present until the end of the storage period. These are known spoilage organisms in fishery products. Other known spoilage organisms of crustaceans such as Photobacterium spp. could not be identified. PMID:25475276

  4. Identification of Thiobacillus ferrooxidans strains based on restriction fragment length polymorphism analysis of 16S rDNA.

    PubMed

    Kamimura, K; Wakai, S; Sugio, T

    2001-01-01

    The 16S rDNA sequences from ten strains of Thiobacillus ferrooxidans were amplified by PCR. The products were compared by performing restriction fragment length polymorphism (RFLP) analysis with restriction endonucleases Alu I, Hap II, Hha I, and Hae III. The RFLP patterns revealed that T. ferrooxidans could be distinguished from other iron- or sulphur-oxidizing bacteria such as T. thiooxidans NB1-3, T. caldus GO-1, Leptospirillum ferrooxidans and the marine iron-oxidizing bacterium strain KU2-11. The RFLP patterns obtained with Alu I, Hap II, and Hae III were the same for nine strains of T. ferrooxidans except for strain ATCC 13661. The RFLP patterns for strains NASF-1 and ATCC 13661 with Hha I were distinct from those for other T. ferrooxidans strains. The 16S rDNA sequence of T. ferrooxidans NASF-1 possessed an additional restriction site for Hha I. These results show that iron-oxidizing bacteria isolated from natural environments were rapidly identified as T. ferrooxidans by the method combining RFLP analysis with physiological analysis. PMID:11414499

  5. Phylogenetic relationships between Bacillus species and related genera inferred from 16s rDNA sequences

    PubMed Central

    Wei Wang, Mi Sun

    2009-01-01

    Neighbor-joining, maximum-parsimony, minimum-evolution, maximum-likelihood and Bayesian trees constructed based on 16S rDNA sequences of 181 type strains of Bacillus species and related taxa manifested nine phylogenetic groups. The phylogenetic analysis showed that Bacillus was not a monophyletic group. B. subtilis was in Group 1. Group 4, 6 and 8 respectively consisted of thermophiles, halophilic or halotolerant bacilli and alkaliphilic bacilli. Group 2, 4 and 8 consisting of Bacillus species and related genera demonstrated that the current taxonomic system did not agree well with the 16S rDNA evolutionary trees. The position of Caryophanaceae and Planococcaceae in Group 2 suggested that they might be transferred into Bacillaceae, and the heterogeneity of Group 2 implied that some Bacillus species in it might belong to several new genera. Group 9 was mainly comprised of the genera (excluding Bacillus) of Bacillaceae, so some Bacillus species in Group 9: B. salarius, B. qingdaonensis and B. thermcloacae might not belong to Bacillus. Four Bacillus species, B. schlegelii, B. tusciae, B. edaphicus and B. mucilaginosus were clearly placed outside the nine groups. PMID:24031394

  6. Obtaining long 16S rDNA sequences using multiple primers and its application on dioxin-containing samples

    PubMed Central

    2015-01-01

    Background Next-generation sequencing (NGS) technology has transformed metagenomics because the high-throughput data allow an in-depth exploration of a complex microbial community. However, accurate species identification with NGS data is challenging because NGS sequences are relatively short. Assembling 16S rDNA segments into longer sequences has been proposed for improving species identification. Current approaches, however, either suffer from amplification bias due to one single primer or insufficient 16S rDNA reads in whole genome sequencing data. Results Multiple primers were used to amplify different 16S rDNA segments for 454 sequencing, followed by 454 read classification and assembly. This permitted targeted sequencing while reducing primer bias. For test samples containing four known bacteria, accurate and near full-length 16S rDNAs of three known bacteria were obtained. For real soil and sediment samples containing dioxins in various concentrations, 16S rDNA sequences were lengthened by 50% for about half of the non-rare microbes, and 16S rDNAs of several microbes reached more than 1000 bp. In addition, reduced primer bias using multiple primers was illustrated. Conclusions A new experimental and computational pipeline for obtaining long 16S rDNA sequences was proposed. The capability of the pipeline was validated on test samples and illustrated on real samples. For dioxin-containing samples, the pipeline revealed several microbes suitable for future studies of dioxin chemistry. PMID:26681335

  7. Unexpected Diagnosis of Cerebral Toxoplasmosis by 16S and D2 Large-Subunit Ribosomal DNA PCR and Sequencing

    PubMed Central

    Kvich, Lasse; Eickhardt, Steffen; Omland, Lars H.; Bjarnsholt, Thomas; Moser, Claus

    2015-01-01

    The protozoan parasite Toxoplasma gondii causes severe opportunistic infections. Here, we report an unexpected diagnosis of cerebral toxoplasmosis. T. gondii was diagnosed by 16S and D2 large-subunit (LSU) ribosomal DNA (rDNA) sequencing of a cerebral biopsy specimen and confirmed by T. gondii-specific PCR and immunohistochemistry. The patient was later diagnosed with HIV/AIDS. PMID:25854484

  8. Phylogenetic analysis of Demodex caprae based on mitochondrial 16S rDNA sequence.

    PubMed

    Zhao, Ya-E; Hu, Li; Ma, Jun-Xian

    2013-11-01

    Demodex caprae infests the hair follicles and sebaceous glands of goats worldwide, which not only seriously impairs goat farming, but also causes a big economic loss. However, there are few reports on the DNA level of D. caprae. To reveal the taxonomic position of D. caprae within the genus Demodex, the present study conducted phylogenetic analysis of D. caprae based on mt16S rDNA sequence data. D. caprae adults and eggs were obtained from a skin nodule of the goat suffering demodicidosis. The mt16S rDNA sequences of individual mite were amplified using specific primers, and then cloned, sequenced, and aligned. The sequence divergence, genetic distance, and transition/transversion rate were computed, and the phylogenetic trees in Demodex were reconstructed. Results revealed the 339-bp partial sequences of six D. caprae isolates were obtained, and the sequence identity was 100% among isolates. The pairwise divergences between D. caprae and Demodex canis or Demodex folliculorum or Demodex brevis were 22.2-24.0%, 24.0-24.9%, and 22.9-23.2%, respectively. The corresponding average genetic distances were 2.840, 2.926, and 2.665, and the average transition/transversion rates were 0.70, 0.55, and 0.54, respectively. The divergences, genetic distances, and transition/transversion rates of D. caprae versus the other three species all reached interspecies level. The five phylogenetic trees all presented that D. caprae clustered with D. brevis first, and then with D. canis, D. folliculorum, and Demodex injai in sequence. In conclusion, D. caprae is an independent species, and it is closer to D. brevis than to D. canis, D. folliculorum, or D. injai. PMID:23996126

  9. Effects of 16S rDNA sampling on estimates of the number of endosymbiont lineages in sucking lice.

    PubMed

    Allen, Julie M; Burleigh, J Gordon; Light, Jessica E; Reed, David L

    2016-01-01

    Phylogenetic trees can reveal the origins of endosymbiotic lineages of bacteria and detect patterns of co-evolution with their hosts. Although taxon sampling can greatly affect phylogenetic and co-evolutionary inference, most hypotheses of endosymbiont relationships are based on few available bacterial sequences. Here we examined how different sampling strategies of Gammaproteobacteria sequences affect estimates of the number of endosymbiont lineages in parasitic sucking lice (Insecta: Phthirapatera: Anoplura). We estimated the number of louse endosymbiont lineages using both newly obtained and previously sequenced 16S rDNA bacterial sequences and more than 42,000 16S rDNA sequences from other Gammaproteobacteria. We also performed parametric and nonparametric bootstrapping experiments to examine the effects of phylogenetic error and uncertainty on these estimates. Sampling of 16S rDNA sequences affects the estimates of endosymbiont diversity in sucking lice until we reach a threshold of genetic diversity, the size of which depends on the sampling strategy. Sampling by maximizing the diversity of 16S rDNA sequences is more efficient than randomly sampling available 16S rDNA sequences. Although simulation results validate estimates of multiple endosymbiont lineages in sucking lice, the bootstrap results suggest that the precise number of endosymbiont origins is still uncertain. PMID:27547523

  10. Effects of 16S rDNA sampling on estimates of the number of endosymbiont lineages in sucking lice

    PubMed Central

    Burleigh, J. Gordon; Light, Jessica E.; Reed, David L.

    2016-01-01

    Phylogenetic trees can reveal the origins of endosymbiotic lineages of bacteria and detect patterns of co-evolution with their hosts. Although taxon sampling can greatly affect phylogenetic and co-evolutionary inference, most hypotheses of endosymbiont relationships are based on few available bacterial sequences. Here we examined how different sampling strategies of Gammaproteobacteria sequences affect estimates of the number of endosymbiont lineages in parasitic sucking lice (Insecta: Phthirapatera: Anoplura). We estimated the number of louse endosymbiont lineages using both newly obtained and previously sequenced 16S rDNA bacterial sequences and more than 42,000 16S rDNA sequences from other Gammaproteobacteria. We also performed parametric and nonparametric bootstrapping experiments to examine the effects of phylogenetic error and uncertainty on these estimates. Sampling of 16S rDNA sequences affects the estimates of endosymbiont diversity in sucking lice until we reach a threshold of genetic diversity, the size of which depends on the sampling strategy. Sampling by maximizing the diversity of 16S rDNA sequences is more efficient than randomly sampling available 16S rDNA sequences. Although simulation results validate estimates of multiple endosymbiont lineages in sucking lice, the bootstrap results suggest that the precise number of endosymbiont origins is still uncertain. PMID:27547523

  11. Bacterial diversity in the rumen of Indian Surti buffalo (Bubalus bubalis), assessed by 16S rDNA analysis.

    PubMed

    Pandya, P R; Singh, K M; Parnerkar, S; Tripathi, A K; Mehta, H H; Rank, D N; Kothari, R K; Joshi, C G

    2010-01-01

    Bacterial communities in buffalo rumen were characterized using a culture-independent approach for a pooled sample of rumen fluid from 3 adult Surti buffaloes. Buffalo rumen is likely to include species of various bacterial phyla, so 16S rDNA sequences were amplified and cloned from the sample. A total of 191 clones were sequenced and similarities to known 16S rDNA sequences were examined. About 62.82% sequences (120 clones) had >90% similarity to the 16S rDNA database sequences. Furthermore, about 34.03% of the sequences (65 clones) were 85-89% similar to 16S rDNA database sequences. For the remaining 3.14%; the similarity was lower than 85% Phylogenetic analyses were also used to infer the makeup of bacterial communities in the rumen of Surti buffalo. As a result, we distinguished 42 operational taxonomic units (OTUs) based on unique 16S r DNA sequences: 19 OTUs affiliated to an unidentified group (45.23% of total OTUs), 11 OTUs of the phylum Firmicutes, also known as the low G+C group (26.19%), 7 OTUs of the Cytophaga-Flexibacter-Bacteroides phylum (16.66%), 4 OTUs of Spirochaetes (9.52%), and 1 OTU of Actinobacteria (2.38%). These include 10 single-clone OTUs, so Good's coverage (94.76%) of 16S rRNA libraries indicated that sequences identified in the libraries represent the majority of bacterial diversity present in rumen. PMID:20720314

  12. Characterization of viable bacteria from Siberian permafrost by 16S rDNA sequencing

    NASA Technical Reports Server (NTRS)

    Shi, T.; Reeves, R. H.; Gilichinsky, D. A.; Friedmann, E. I.

    1997-01-01

    Viable bacteria were found in permafrost core samples from the Kolyma-Indigirka lowland of northeast Siberia. The samples were obtained at different depths; the deepest was about 3 million years old. The average temperature of the permafrost is -10 degrees C. Twenty-nine bacterial isolates were characterized by 16S rDNA sequencing and phylogenetic analysis, cell morphology, Gram staining, endospore formation, and growth at 30 degrees C. The majority of the bacterial isolates were rod shaped and grew well at 30 degrees C; but two of them did not grow at or above 28 degrees C, and had optimum growth temperatures around 20 degrees C. Thirty percent of the isolates could form endospores. Phylogenetic analysis revealed that the isolates fell into four categories: high-GC Gram-positive bacteria, beta-proteobacteria, gamma-proteobacteria, and low-GC Gram-positive bacteria. Most high-GC Gram-positive bacteria and beta-proteobacteria, and all gamma-proteobacteria, came from samples with an estimated age of 1.8-3.0 million years (Olyor suite). Most low-GC Gram-positive bacteria came from samples with an estimated age of 5,000-8,000 years (Alas suite).

  13. Methanogen diversity in the rumen of Indian Surti buffalo (Bubalus bubalis), assessed by 16S rDNA analysis.

    PubMed

    Singh, K M; Tripathi, A K; Pandya, P R; Parnerkar, S; Rank, D N; Kothari, R K; Joshi, C G

    2012-06-01

    The methanogenic communities in buffalo rumen were characterized using a culture-independent approach of a pooled sample of rumen fluid from three adult Surti buffaloes. Buffalo rumen is likely to include species of various methanogens, so 16S rDNA sequences were amplified and cloned from the sample. A total of 171 clones were sequenced to examine 16S rDNA sequence similarity. About 52.63% sequences (90 clones) had ≥ 90% similarity, whereas, 46.78% of the sequences (81 clones) were 75-89% similar to 16S rDNA database sequences, respectively. Phylogenetic analyses were also used to infer the makeup of methanogenic communities in the rumen of Surti buffalo. As a result, we distinguished 23 operational taxonomic units (OTUs) based on unique 16S rDNA sequences: 12 OTUs (52.17%) affiliated to Methanomicrobiales order, 10 OTUs (43.47%) of the order Methanobacteriales and one OTU (4.34%) of Methanosarcina barkeri like clone, respectively. In addition, the population of Methanomicrobiales and Methabacteriales orders were also observed, accounting 4% and 2.17% of total archea. This study has revealed the largest assortment of hydrogenotrophic methanogens phylotypes ever identified from rumen of Surti buffaloes. PMID:21507441

  14. Performance of 16s rDNA Primer Pairs in the Study of Rhizosphere and Endosphere Bacterial Microbiomes in Metabarcoding Studies

    PubMed Central

    Beckers, Bram; Op De Beeck, Michiel; Thijs, Sofie; Truyens, Sascha; Weyens, Nele; Boerjan, Wout; Vangronsveld, Jaco

    2016-01-01

    Next-generation sequencing technologies have revolutionized the methods for studying microbial ecology by enabling high-resolution community profiling. However, the use of these technologies in unraveling the plant microbiome remains challenging. Many bacterial 16S rDNA primer pairs also exhibit high affinity for non-target DNA such as plastid (mostly chloroplast) DNA and mitochondrial DNA. Therefore, we experimentally tested a series of commonly used primers for the analysis of plant-associated bacterial communities using 454 pyrosequencing. We evaluated the performance of all selected primer pairs in the study of the bacterial microbiomes present in the rhizosphere soil, root, stem and leaf endosphere of field-grown poplar trees (Populus tremula × Populus alba) based on (a) co-amplification of non-target DNA, (b) low amplification efficiency for pure chloroplast DNA (real-time PCR), (c) high retrieval of bacterial 16S rDNA, (d) high operational taxonomic unit (OTU) richness and Inverse Simpson diversity and (e) taxonomic assignment of reads. Results indicate that experimental evaluation of primers provide valuable information that could contribute in the selection of suitable primer pairs for 16S rDNA metabarcoding studies in plant-microbiota research. Furthermore, we show that primer pair 799F-1391R outperforms all other primer pairs in our study in the elimination of non-target DNA and retrieval of bacterial OTUs. PMID:27242686

  15. Performance of 16s rDNA Primer Pairs in the Study of Rhizosphere and Endosphere Bacterial Microbiomes in Metabarcoding Studies.

    PubMed

    Beckers, Bram; Op De Beeck, Michiel; Thijs, Sofie; Truyens, Sascha; Weyens, Nele; Boerjan, Wout; Vangronsveld, Jaco

    2016-01-01

    Next-generation sequencing technologies have revolutionized the methods for studying microbial ecology by enabling high-resolution community profiling. However, the use of these technologies in unraveling the plant microbiome remains challenging. Many bacterial 16S rDNA primer pairs also exhibit high affinity for non-target DNA such as plastid (mostly chloroplast) DNA and mitochondrial DNA. Therefore, we experimentally tested a series of commonly used primers for the analysis of plant-associated bacterial communities using 454 pyrosequencing. We evaluated the performance of all selected primer pairs in the study of the bacterial microbiomes present in the rhizosphere soil, root, stem and leaf endosphere of field-grown poplar trees (Populus tremula × Populus alba) based on (a) co-amplification of non-target DNA, (b) low amplification efficiency for pure chloroplast DNA (real-time PCR), (c) high retrieval of bacterial 16S rDNA, (d) high operational taxonomic unit (OTU) richness and Inverse Simpson diversity and (e) taxonomic assignment of reads. Results indicate that experimental evaluation of primers provide valuable information that could contribute in the selection of suitable primer pairs for 16S rDNA metabarcoding studies in plant-microbiota research. Furthermore, we show that primer pair 799F-1391R outperforms all other primer pairs in our study in the elimination of non-target DNA and retrieval of bacterial OTUs. PMID:27242686

  16. Analysis of the unexplored features of rrs (16S rDNA) of the Genus Clostridium

    PubMed Central

    2011-01-01

    Background Bacterial taxonomy and phylogeny based on rrs (16S rDNA) sequencing is being vigorously pursued. In fact, it has been stated that novel biological findings are driven by comparison and integration of massive data sets. In spite of a large reservoir of rrs sequencing data of 1,237,963 entries, this analysis invariably needs supplementation with other genes. The need is to divide the genetic variability within a taxa or genus at their rrs phylogenetic boundaries and to discover those fundamental features, which will enable the bacteria to naturally fall within them. Within the large bacterial community, Clostridium represents a large genus of around 110 species of significant biotechnological and medical importance. Certain Clostridium strains produce some of the deadliest toxins, which cause heavy economic losses. We have targeted this genus because of its high genetic diversity, which does not allow accurate typing with the available molecular methods. Results Seven hundred sixty five rrs sequences (> 1200 nucleotides, nts) belonging to 110 Clostridium species were analyzed. On the basis of 404 rrs sequences belonging to 15 Clostridium species, we have developed species specific: (i) phylogenetic framework, (ii) signatures (30 nts) and (iii) in silico restriction enzyme (14 Type II REs) digestion patterns. These tools allowed: (i) species level identification of 95 Clostridium sp. which are presently classified up to genus level, (ii) identification of 84 novel Clostridium spp. and (iii) potential reduction in the number of Clostridium species represented by small populations. Conclusions This integrated approach is quite sensitive and can be easily extended as a molecular tool for diagnostic and taxonomic identification of any microbe of importance to food industries and health services. Since rapid and correct identification allows quicker diagnosis and consequently treatment as well, it is likely to lead to reduction in economic losses and mortality

  17. Investigating bacterial populations in styrene-degrading biofilters by 16S rDNA tag pyrosequencing.

    PubMed

    Portune, Kevin J; Pérez, M Carmen; Álvarez-Hornos, F Javier; Gabaldón, Carmen

    2015-01-01

    Microbial biofilms are essential components in the elimination of pollutants within biofilters, yet still little is known regarding the complex relationships between microbial community structure and biodegradation function within these engineered ecosystems. To further explore this relationship, 16S rDNA tag pyrosequencing was applied to samples taken at four time points from a styrene-degrading biofilter undergoing variable operating conditions. Changes in microbial structure were observed between different stages of biofilter operation, and the level of styrene concentration was revealed to be a critical factor affecting these changes. Bacterial genera Azoarcus and Pseudomonas were among the dominant classified genera in the biofilter. Canonical correspondence analysis (CCA) and correlation analysis revealed that the genera Brevundimonas, Hydrogenophaga, and Achromobacter may play important roles in styrene degradation under increasing styrene concentrations. No significant correlations (P > 0.05) could be detected between biofilter operational/functional parameters and biodiversity measurements, although biological heterogeneity within biofilms and/or technical variability within pyrosequencing may have considerably affected these results. Percentages of selected bacterial taxonomic groups detected by fluorescence in situ hybridization (FISH) were compared to results from pyrosequencing in order to assess the effectiveness and limitations of each method for identifying each microbial taxon. Comparison of results revealed discrepancies between the two methods in the detected percentages of numerous taxonomic groups. Biases and technical limitations of both FISH and pyrosequencing, such as the binding of FISH probes to non-target microbial groups and lack of classification of sequences for defined taxonomic groups from pyrosequencing, may partially explain some differences between the two methods. PMID:24950754

  18. Molecular systematics of the genus Troglophilus (Rhaphidophoridae, Orthoptera) in Turkey: mitochondrial 16S rDNA evidences

    PubMed Central

    Taylan, Mehmet Sait; Russo, Claudio Di; Rampini, Mauro; Ketmaier, Valerio

    2013-01-01

    Abstract This study focuses on the evolutionary relationships among Turkish species of the cave cricket genus Troglophilus.Fifteen populations were studied for sequence variation in a fragment (543 base pairs) of the mitochondrial DNA (mtDNA) 16S rDNA gene (16S) to reconstruct their phylogenetic relationships and biogeographic history. Genetic data retrieved three main clades and at least three divergent lineages that could not be attributed to any of the taxa known for the area. Molecular time estimates suggest that the diversification of the group took place between the Messinian and the Plio-Pleistocene. PMID:23653493

  19. Assessing diversity of the female urine microbiota by high throughput sequencing of 16S rDNA amplicons

    PubMed Central

    2011-01-01

    Background Urine within the urinary tract is commonly regarded as "sterile" in cultivation terms. Here, we present a comprehensive in-depth study of bacterial 16S rDNA sequences associated with urine from healthy females by means of culture-independent high-throughput sequencing techniques. Results Sequencing of the V1V2 and V6 regions of the 16S ribosomal RNA gene using the 454 GS FLX system was performed to characterize the possible bacterial composition in 8 culture-negative (<100,000 CFU/ml) healthy female urine specimens. Sequences were compared to 16S rRNA databases and showed significant diversity, with the predominant genera detected being Lactobacillus, Prevotella and Gardnerella. The bacterial profiles in the female urine samples studied were complex; considerable variation between individuals was observed and a common microbial signature was not evident. Notably, a significant amount of sequences belonging to bacteria with a known pathogenic potential was observed. The number of operational taxonomic units (OTUs) for individual samples varied substantially and was in the range of 20 - 500. Conclusions Normal female urine displays a noticeable and variable bacterial 16S rDNA sequence richness, which includes fastidious and anaerobic bacteria previously shown to be associated with female urogenital pathology. PMID:22047020

  20. 16S and 23S plastid rDNA phylogenies of Prototheca species and their auxanographic phenotypes1

    PubMed Central

    Ewing, Aren; Brubaker, Shane; Somanchi, Aravind; Yu, Esther; Rudenko, George; Reyes, Nina; Espina, Karen; Grossman, Arthur; Franklin, Scott

    2014-01-01

    Because algae have become more accepted as sources of human nutrition, phylogenetic analysis can help resolve the taxonomy of taxa that have not been well studied. This can help establish algal evolutionary relationships. Here, we compare Auxenochlorella protothecoides and 23 strains of Prototheca based on their complete 16S and partial 23S plastid rDNA sequences along with nutrient utilization (auxanographic) profiles. These data demonstrate that some of the species groupings are not in agreement with the molecular phylogenetic analyses and that auxanographic profiles are poor predictors of phylogenetic relationships. PMID:25937672

  1. Use of acetate for enrichment of electrochemically active microorganisms and their 16S rDNA analyses.

    PubMed

    Lee, Jiyoung; Phung, Nguyet Thu; Chang, In Seop; Kim, Byung Hong; Sung, Ha Chin

    2003-06-27

    A fuel cell-type electrochemical device has been used to enrich microbes oxidizing acetate with concomitant electricity generation without using an electron mediator from activated sludge. The device generated a stable current of around 5 mA with complete oxidation of 5 mM acetate at the hydraulic retention time of 2.5 h after 4 weeks of enrichment. Over 70% of electrons available from acetate oxidation was recovered as current. Carbon monoxide or hydrogen did not influence acetate oxidation or current generation from the microbial fuel cell (MFC). Denaturing gradient gel electrophoresis showed that DNA extracted from the acetate-enriched MFC had different 16S rDNA patterns from those of sludge or glucose+glutamate-enriched MFCs. Nearly complete 16S rDNA sequence analyses showed that diverse bacteria were enriched in the MFC fed with acetate. Electron microscopic observations showed biofilm developed on the electrode, but not microbial clumps observed in MFCs fed with complex fuel such as glucose and wastewater from a corn-processing factory. PMID:12829284

  2. Studying long 16S rDNA sequences with ultrafast-metagenomic sequence classification using exact alignments (Kraken).

    PubMed

    Valenzuela-González, Fabiola; Martínez-Porchas, Marcel; Villalpando-Canchola, Enrique; Vargas-Albores, Francisco

    2016-03-01

    Ultrafast-metagenomic sequence classification using exact alignments (Kraken) is a novel approach to classify 16S rDNA sequences. The classifier is based on mapping short sequences to the lowest ancestor and performing alignments to form subtrees with specific weights in each taxon node. This study aimed to evaluate the classification performance of Kraken with long 16S rDNA random environmental sequences produced by cloning and then Sanger sequenced. A total of 480 clones were isolated and expanded, and 264 of these clones formed contigs (1352 ± 153 bp). The same sequences were analyzed using the Ribosomal Database Project (RDP) classifier. Deeper classification performance was achieved by Kraken than by the RDP: 73% of the contigs were classified up to the species or variety levels, whereas 67% of these contigs were classified no further than the genus level by the RDP. The results also demonstrated that unassembled sequences analyzed by Kraken provide similar or inclusively deeper information. Moreover, sequences that did not form contigs, which are usually discarded by other programs, provided meaningful information when analyzed by Kraken. Finally, it appears that the assembly step for Sanger sequences can be eliminated when using Kraken. Kraken cumulates the information of both sequence senses, providing additional elements for the classification. In conclusion, the results demonstrate that Kraken is an excellent choice for use in the taxonomic assignment of sequences obtained by Sanger sequencing or based on third generation sequencing, of which the main goal is to generate larger sequences. PMID:26812576

  3. Improved performance of the PacBio SMRT technology for 16S rDNA sequencing.

    PubMed

    Mosher, Jennifer J; Bowman, Brett; Bernberg, Erin L; Shevchenko, Olga; Kan, Jinjun; Korlach, Jonas; Kaplan, Louis A

    2014-09-01

    Improved sequencing accuracy was obtained with 16S amplicons from environmental samples and a known pure culture when upgraded Pacific Biosciences (PacBio) hardware and enzymes were used for the single molecule, real-time (SMRT) sequencing platform. The new PacBio RS II system with P4/C2 chemistry, when used with previously constructed libraries (Mosher et al., 2013) surpassed the accuracy of Roche/454 pyrosequencing platform. With accurate read lengths of >1400 base pairs, the PacBio system opens up the possibility of identifying microorganisms to the species level in environmental samples. PMID:24978594

  4. When molecules support morphology: Phylogenetic reconstruction of the family Onuphidae (Eunicida, Annelida) based on 16S rDNA and 18S rDNA.

    PubMed

    Budaeva, Nataliya; Schepetov, Dmitry; Zanol, Joana; Neretina, Tatiana; Willassen, Endre

    2016-01-01

    Onuphid polychaetes are tubicolous marine worms commonly reported worldwide from intertidal areas to hadal depths. They often dominate in benthic communities and have economic importance in aquaculture and recreational fishing. Here we report the phylogeny of the family Onuphidae based on the combined analyses of nuclear (18S rDNA) and mitochondrial (16S rDNA) genes. Results of Bayesian and Maximum Likelihood analyses supported the monophyly of Onuphidae and its traditional subdivision into two monophyletic subfamilies: Onuphinae and Hyalinoeciinae. Ten of 22 recognized genera were monophyletic with strong node support; four more genera included in this study were either monotypic or represented by a single species. None of the genera appeared para- or polyphyletic and this indicates a strong congruence between the traditional morphology-based systematics of the family and the newly obtained molecular-based phylogenetic reconstructions. Intergeneric relationships within Hyalinoeciinae were not resolved. Two strongly supported monophyletic groups of genera were recovered within Onuphinae: ((Onuphis, Aponuphis), Diopatra, Paradiopatra) and (Hirsutonuphis, (Paxtonia, (Kinbergonuphis, Mooreonuphis))). A previously accepted hypothesis on the subdivision of Onuphinae into the Onuphis group of genera and the Diopatra group of genera was largely rejected. PMID:26497420

  5. MOLECULAR TRACKING FECAL CONTAMINATION IN SURFACE WATERS: 16S RDNA VERSUS METAGENOMICS APPROACHES

    EPA Science Inventory

    Microbial source tracking methods need to be sensitive and exhibit temporal and geographic stability in order to provide meaningful data in field studies. The objective of this study was to use a combination of PCR-based methods to track cow fecal contamination in two watersheds....

  6. Description of the male, redescription of the female and 16S rDNA sequence of Ixodes aulacodi (Ixodidae).

    PubMed

    Chiţimia-Dobler, Lidia; D'Amico, Gianluca; Yao, Patrick Kouassi; Kalmár, Zsuzsa; Gherman, Călin Mircea; Mihalca, Andrei Daniel; Estrada-Peña, Agustin

    2016-04-01

    Ixodes (Afrixodes) aulacodiArthur, 1956 is a poorly known species that has been recorded predominantly in the wet countries of western and central Africa, mainly associated to the greater cane rat Thryonomys swinderianus (Temmink). We herein redescribe the female, describe the male (ascribed to the species from specimens found in copula) and provide the 16S rDNA sequence. We also provide complete illustrations of the adults based on specimens found on greater cane rats in Ivory Coast. Ixodes aulacodi is included in the group of species of the subgenus Afrixodes that have horseshoe shaped anal groove, and which lack auriculae and cornua. The female is easily separated when compared with other species because of a unique combination of characters: All the coxae have internal spurs, coxa II has two external spurs, syncoxae are absent, and trochanters I-III have one spur each. The male has a notched hypostome and lacks syncoxae, auriculae and cornua. PMID:26803353

  7. Algae-bacteria association inferred by 16S rDNA similarity in established microalgae cultures.

    PubMed

    Schwenk, Dagmar; Nohynek, Liisa; Rischer, Heiko

    2014-06-01

    Forty cultivable, visually distinct bacterial cultures were isolated from four Baltic microalgal cultures Chlorella pyrenoidosa, Scenedesmus obliquus, Isochrysis sp., and Nitzschia microcephala, which have been maintained for several years in the laboratory. Bacterial isolates were characterized with respect to morphology, antibiotic susceptibility, and 16S ribosomal DNA sequence. A total of 17 unique bacterial strains, almost all belonging to one of three families, Rhodobacteraceae, Rhizobiaceae, and Erythrobacteraceae, were subsequently isolated. The majority of isolated bacteria belong to Rhodobacteraceae. Literature review revealed that close relatives of the bacteria isolated in this study are not only often found in marine environments associated with algae, but also in lakes, sediments, and soil. Some of them had been shown to interact with organisms in their surroundings. A Basic Local Alignment Search Tool study indicated that especially bacteria isolated from the Isochrysis sp. culture were highly similar to microalgae-associated bacteria. Two of those isolates, I1 and I6, belong to the Cytophaga-Flavobacterium-Bacteroides phylum, members of which are known to occur in close communities with microalgae. An UniFrac analysis revealed that the bacterial community of Isochrysis sp. significantly differs from the other three communities. PMID:24799387

  8. Algae–bacteria association inferred by 16S rDNA similarity in established microalgae cultures

    PubMed Central

    Schwenk, Dagmar; Nohynek, Liisa; Rischer, Heiko

    2014-01-01

    Forty cultivable, visually distinct bacterial cultures were isolated from four Baltic microalgal cultures Chlorella pyrenoidosa, Scenedesmus obliquus, Isochrysis sp., and Nitzschia microcephala, which have been maintained for several years in the laboratory. Bacterial isolates were characterized with respect to morphology, antibiotic susceptibility, and 16S ribosomal DNA sequence. A total of 17 unique bacterial strains, almost all belonging to one of three families, Rhodobacteraceae, Rhizobiaceae, and Erythrobacteraceae, were subsequently isolated. The majority of isolated bacteria belong to Rhodobacteraceae. Literature review revealed that close relatives of the bacteria isolated in this study are not only often found in marine environments associated with algae, but also in lakes, sediments, and soil. Some of them had been shown to interact with organisms in their surroundings. A Basic Local Alignment Search Tool study indicated that especially bacteria isolated from the Isochrysis sp. culture were highly similar to microalgae-associated bacteria. Two of those isolates, I1 and I6, belong to the Cytophaga–Flavobacterium–Bacteroides phylum, members of which are known to occur in close communities with microalgae. An UniFrac analysis revealed that the bacterial community of Isochrysis sp. significantly differs from the other three communities. PMID:24799387

  9. Detection of novel organisms associated with salpingitis, by use of 16S rDNA polymerase chain reaction.

    PubMed

    Hebb, Jennifer K; Cohen, Craig R; Astete, Sabina G; Bukusi, Elizabeth A; Totten, Patricia A

    2004-12-15

    Although Chlamydia trachomatis and Neisseria gonorrhoeae are established causes of salpingitis, the majority of cases have no known etiology. We used broad-range 16S rDNA polymerase chain reaction to identify novel, possibly uncultivable, bacteria associated with salpingitis and identified bacterial 16S sequences in Fallopian-tube specimens from 11 (24%) of 45 consecutive women with laparoscopically confirmed acute salpingitis (the case patients) and from 0 of 44 women seeking tubal ligations (the control subjects) at Kenyatta National Hospital, Nairobi, Kenya. Bacterial phylotypes most closely related to Leptotrichia spp. were detected as the sole phylotypes in 1, and mixed with other bacterial phylotypes in 2, specimens. Novel bacterial phylotypes and those associated with bacterial vaginosis, including Atopobium vaginae, were identified in 3 specimens. N. gonorrhoeae and Streptococcus pyogenes were identified in 2 and 1 specimens, respectively. The finding of novel phylotypes associated with salpingitis has important implications for the etiology, pathogenesis, and treatment of this important reproductive-tract disease syndrome. PMID:15551209

  10. Application of Faecalibacterium 16S rDNA genetic marker for accurate identification of duck faeces.

    PubMed

    Sun, Da; Duan, Chuanren; Shang, Yaning; Ma, Yunxia; Tan, Lili; Zhai, Jun; Gao, Xu; Guo, Jingsong; Wang, Guixue

    2016-04-01

    The aim of this study was to judge the legal duty of pollution liabilities by assessing a duck faeces-specific marker, which can exclude distractions of residual bacteria from earlier contamination accidents. With the gene sequencing technology and bioinformatics method, we completed the comparative analysis of Faecalibacterium sequences, which were associated with ducks and other animal species, and found the sequences unique to duck faeces. Polymerase chain reaction (PCR) and agarose gel electrophoresis techniques were used to verify the reliability of both human and duck faeces-specific primers. The duck faeces-specific primers generated an amplicon of 141 bp from 43.3 % of duck faecal samples, 0 % of control samples and 100 % of sewage wastewater samples that contained duck faeces. We present here the initial evidence of Faecalibacterium-based applicability as human faeces-specificity in China. Meanwhile, this study represents the initial report of a Faecalibacterium marker for duck faeces and suggests an independent or supplementary environmental biotechnology of microbial source tracking (MST). PMID:26743644

  11. Distribution and 16S rDNA sequences of Argas monachus (Acari: Argasidae), a soft tick parasite of Myiopsitta monachus (Aves: Psittacidae).

    PubMed

    Mastropaolo, Mariano; Turienzo, Paola; Di Iorio, Osvaldo; Nava, Santiago; Venzal, José M; Guglielmone, Alberto A; Mangold, Atilio J

    2011-11-01

    Specimens of Argas monachus Keirans et al. were collected from Myiopsitta monachus nests in 42 localities in Argentina and Paraguay from 2006 to 2010. A list of localities where this tick has been found is presented. 16S rDNA sequences of specimens of A. monachus from different localities were compared to confirm whether they belong to the same specific taxon. Argas monachus is present in the phytogeographic provinces of Chaco, Espinal, and Monte, but not in the Pampa (all from de Chaco Domain) where the host is well distributed. No differences were found among 16S rDNA sequences of geographically distant specimens. PMID:21739257

  12. 16S rRNA region based PCR protocol for identification and subtyping of Parvimonas micra

    PubMed Central

    Ota-Tsuzuki, C.; Brunheira, A.T.P.; Mayer, M.P.A.

    2008-01-01

    The present study established a PCR protocol in order to identify Parvimonas micra and to evaluate the intra-species diversity by PCR-RFLP of 16S rRNA partial sequence. The data indicated that the protocol was able to identify this species which could be clustered in five genotypes. PMID:24031274

  13. Characterization of Lactobacillus from Algerian Goat'S Milk Based on Phenotypic, 16S rDNA Sequencing and their Technological Properties.

    PubMed

    Marroki, Ahmed; Zúñiga, Manuel; Kihal, Mabrouk; Pérez-Martínez, Gaspar

    2011-01-01

    Nineteen strains of Lactobacillus isolated from goat's milk from farms in north-west of Algeria were characterized. Isolates were identified by phenotypic, physiological and genotypic methods and some of their important technological properties were studied. Phenotypic characterization was carried out by studying physiological, morphological characteristics and carbohydrate fermentation patterns using API 50 CHL system. Isolates were also characterized by partial 16S rDNA sequencing. Results obtained with phenotypic methods were correlated with the genotypic characterization and 13 isolates were identified as L. plantarum, two isolates as L. rhamnosus and one isolate as L. fermentum. Three isolates identified as L. plantarum by phenotypic characterization were found to be L. pentosus by the genotypic method. A large diversity in technological properties (acid production in skim milk, exopolysaccharide production, aminopeptidase activity, antibacterial activity and antibiotic susceptibility) was observed. Based on these results, two strains of L. plantarum (LbMS16 and LbMS21) and one strain of L. rhamnosus (LbMF25) have been tentatively selected for use as starter cultures in the manufacture of artisanal fermented dairy products in Algeria. PMID:24031617

  14. The phylogeny of native and exotic scallops cultured in China based on 16S rDNA sequences

    NASA Astrophysics Data System (ADS)

    Liu, Baozhong; Dong, Bo; Xiang, Jianhai; Wang, Zaizhao

    2007-01-01

    Scallops of the Family Pectinidae are a valuable resource in marine industry of the world. Understanding the phylogeny of the family is important for the development of the industry. In this study, partial 16S mitochondrial rDNA genes were obtained from 8 scallop species that are commonly cultured indigenous and transplanted species in China. Phylogenetic relationships of Pectinidae were analyzed based on the 8 sequences and other 5 published ones in GenBank, representing 9 genera of the family. The molecular phylogeny trees were constructed using 3 methods with software PHYLIP. The results showe that total 13 species of scallops clustered in 4 clades. Pecten maximus joins P. jacobaeus then Amusium pleuronectes in cluster, indicating close relationship of genus Amusium with Pecten in evolution. P. yessoensis is close to Chlamys farreri and C. islandica. No enough material was available to single out genus Patinopecten as an independent monophyletic subfamily. The position of Adamussium colbecki indicates that it is far from genus Pecten but near to genus Chlamys in evolution.

  15. Sharp switches between regular and swinger mitochondrial replication: 16S rDNA systematically exchanging nucleotides A<->T+C<->G in the mitogenome of Kamimuria wangi.

    PubMed

    Seligmann, Hervé

    2016-07-01

    Swinger DNAs are sequences whose homology with known sequences is detected only by assuming systematic exchanges between nucleotides. Nine symmetric (X<->Y, i.e. A<->C) and fourteen asymmetric (X->Y->Z, i.e. A->C->G) exchanges exist. All swinger DNA previously detected in GenBank follow the A<->T+C<->G exchange, while mitochondrial swinger RNAs distribute among different swinger types. Here different alignment criteria detect 87 additional swinger mitochondrial DNAs (86 from insects), including the first swinger gene embedded within a complete genome, corresponding to the mitochondrial 16S rDNA of the stonefly Kamimuria wangi. Other Kamimuria mt genome regions are "regular", stressing unanswered questions on (a) swinger polymerization regulation; (b) swinger 16S rDNA functions; and (c) specificity to rDNA, in particular 16S rDNA. Sharp switches between regular and swinger replication, together with previous observations on swinger transcription, suggest that swinger replication might be due to a switch in polymerization mode of regular polymerases and the possibility of swinger-encoded information, predicted in primordial genes such as rDNA. PMID:25865623

  16. 16S rDNA analysis of archaea indicates dominance of Methanobacterium and high abundance of Methanomassiliicoccaceae in rumen of Nili-Ravi buffalo.

    PubMed

    Paul, S S; Deb, S M; Dey, A; Somvanshi, S P S; Singh, D; Rathore, R; Stiverson, J

    2015-10-01

    The molecular diversity of rumen methanogens was investigated using 16S rDNA gene library prepared from the rumen contents of Nili-Ravi buffaloes. Microbial genomic DNA was isolated from four adult male fistulated buffaloes and PCR conditions were set up using specific primers. Amplified product was cloned into a suitable vector, and the inserts of positive clones were sequenced. A total of 142 clones were examined, and the analysis revealed 46 species level (0.01 distance) operational taxonomic units (OTUs). Twenty six OTUs comprising 89 clones (63% of the total clones) were taxonomically assigned to Methanobacterium genus and the majority of them had highest percent identity with Methanobacterium flexile among cultured methanogens. Five OTUs comprising 27 clones (19% of total clones) were taxonomically assigned to Methanomicrobium genus and these clones showed highest sequence identity with Methanomicrobium mobile. Only two OTUs comprising 6 clones (4% of total clones) were assigned to Methanobrevibacter genus. A total of 17 clones belonging to 10 species level OTUs showed highest percent identity (ranging from 85 to 95%) with Methanomassilicoccus luminyensis and were taxonomically classified as Methanomassiliicocaceae. Out of the 142 rDNA clones, 112 clones, which constitute 79% of the total clones representing 42 OTUs, had less than 98.5% sequence identity with any of the cultured strains of methanogens and represent novel species of methanogens. This study has revealed the largest assortment of hydrogenotrophic methanogen phylotypes ever identified from the rumen of Nili-Ravi buffaloes. The study indicates that Methanobacterium is the most dominant methanogen in the rumen of Nili-Ravi buffalo. This is also the first report on the presence of methanogens phylogenetically close to M. luminyensis, an H2 dependent methylotrophic methanogen, in the rumen of buffaloes at such a high level of abundance. PMID:26103451

  17. Evaluation of direct 16S rDNA sequencing as a metagenomics-based approach to screening bacteria in bottled water.

    PubMed

    Hansen, Trine; Skånseng, Beate; Hoorfar, Jeffrey; Löfström, Charlotta

    2013-09-01

    Deliberate or accidental contamination of food, feed, and water supplies poses a threat to human health worldwide. A rapid and sensitive detection technique that could replace the current labor-intensive and time-consuming culture-based methods is highly desirable. In addition to species-specific assays, such as PCR, there is a need for generic methods to screen for unknown pathogenic microorganisms in samples. This work presents a metagenomics-based direct-sequencing approach for detecting unknown microorganisms, using Bacillus cereus (as a model organism for B. anthracis) in bottled water as an example. Total DNA extraction and 16S rDNA gene sequencing were used in combination with principle component analysis and multicurve resolution to study detection level and possibility for identification. Results showed a detection level of 10(5) to 10(6) CFU/L. Using this method, it was possible to separate 2 B. cereus strains by the principal component plot, despite the close sequence resemblance. A linear correlation between the artificial contamination level and the relative amount of the Bacillus artificial contaminant in the metagenome was observed, and a relative amount value above 0.5 confirmed the presence of Bacillus. The analysis also revealed that background flora in the bottled water varied between the different water types that were included in the study. This method has the potential to be adapted to other biological matrices and bacterial pathogens for fast screening of unknown bacterial threats in outbreak situations. PMID:23971801

  18. 16S rDNA sequence analysis of bacterial isolates from biodeteriorated mural paintings in the Servilia tomb (Necropolis of carmona, Seville, Spain).

    PubMed

    Heyrman, J; Swings, J

    2001-11-01

    Bacteria were isolated from damaged mural paintings of the Servilia tomb (necropolis of Carmona, Seville, Spain). Selected strains, representative for different clusters of isolates with similar fatty acid profiles, were analysed by 16S rDNA sequence analysis. Bacillus is the dominant genus among the isolates: members of the rRNA species complexes of B. megaterium, B. pumilus and B. firmus were found as well as several other Bacillus species. One group of halotolerant isolates falls in the Bacillus sensu lato group, with closest relatedness to the genera Salibacillus and Virgibacillus. Other genera found are Artbrobacter, Micrococcus, Streptomyces, Sphingomonas, Paenibacillus, and a genus closely related to Paracraurococcus. Many isolates showed low 16S rDNA sequence similarities with the closest related database entries, a strong indication for the presence of several new species among the isolates. PMID:11822679

  19. [Investigation of bacterial diversity in the biological desulfurization reactor for treating high salinity wastewater by the 16S rDNA cloning method].

    PubMed

    Liu, Wei-Guo; Liang, Cun-Zhen; Yang, Jin-Sheng; Wang, Gui-Ping; Liu, Miao-Miao

    2013-02-01

    The bacterial diversity in the biological desulfurization reactor operated continuously for 1 year was studied by the 16S rDNA cloning and sequencing method. Forty clones were randomly selected and their partial 16S rDNA genes (ca. 1,400 bp) were sequenced and blasted. The results indicated that there were dominant bacterias in the biological desulfurization reactor, where 33 clones belonged to 3 different published phyla, while 1 clone belonged to unknown phylum. The dominant bacterial community in the system was Proteobacteria, which accounted for 85.3%. The bacterial community succession was as follows: the gamma-Proteobacteria(55.9%), beta-Proteobacteria(17.6%), Actinobacteridae (8.8%), delta-Proteobacteria (5.9%) , alpha-Proteobacteria(5.9%), and Sphingobacteria (2.9%). Halothiobacillus sp. ST15 and Thiobacillus sp. UAM-I were the major desulfurization strains. PMID:23668153

  20. Characterization of bacterial diversity in pulque, a traditional Mexican alcoholic fermented beverage, as determined by 16S rDNA analysis.

    PubMed

    Escalante, Adelfo; Rodríguez, María Elena; Martínez, Alfredo; López-Munguía, Agustín; Bolívar, Francisco; Gosset, Guillermo

    2004-06-15

    The bacterial diversity in pulque, a traditional Mexican alcoholic fermented beverage, was studied in 16S rDNA clone libraries from three pulque samples. Sequenced clones identified as Lactobacillus acidophilus, Lactobacillus strain ASF360, L. kefir, L. acetotolerans, L. hilgardii, L. plantarum, Leuconostoc pseudomesenteroides, Microbacterium arborescens, Flavobacterium johnsoniae, Acetobacter pomorium, Gluconobacter oxydans, and Hafnia alvei, were detected for the first time in pulque. Identity of 16S rDNA sequenced clones showed that bacterial diversity present among pulque samples is dominated by Lactobacillus species (80.97%). Seventy-eight clones exhibited less than 95% of relatedness to NCBI database sequences, which may indicate the presence of new species in pulque samples. PMID:15183874

  1. [Analysis of DNA homology and 16S rDNA sequence of rhizobia, a new phenotypic subgroup, isolated from Xizang Autonomous Region of China].

    PubMed

    Wang, Su-ying; Yang, Xiao-li; Li, Hai-feng; Liu, Jie

    2006-02-01

    Based on the studies of numerical taxonomy, the seven rhizobial strains isolated from the root nodules of leguminous plants Trigonella spp. and Astragalus spp. growing in the Xizang Autonomous Region of China constituted a new phenotypic subgroup, where wide phenotypic and genotypic diversity among legume crops had been reported due to complex terrain and various climate. The new phenotypic subgroup were further identified to clarify its taxonomic position by DNA homology analysis and 16S rDNA gene sequencing. The mol% G + C ratio of the DNA among members of the new subgroup ranged from 59.5 to 63.3 mol% as determined by T (m) assay. The levels of DNA relatedness, determined by using the DNA liquid hybridization method, among the members of the new subgroup were between 74.3% and 92.3%, while level of DNA relatedness between the central strains XZ2-3 of the new subgroup and the type strains of known species of Rhizobium was less than 47.4%. These results indicated that the new phenotypic subgroup is a DNA homological group different from described species of Rhizobium. Therefore, this new phenotypic subgroup was supposed to be a new species in the genus of Rhizobium since the strains in the same species generally exhibit levels of DNA homology ranging from 70 to 100%. A systematic identification method-16S rDNA gene sequence comparison was carried out to determine the phylogenetic relationships of the new subgroup with the described species of Rhizobium. The GenBank accession number for the 16S rDNA sequence of the central strain XZ2-3 of the new subgroup is DQ099745. The full-length 16S rDNA gene sequence were sequenced by chain terminator techniques and analyzed with PHYLIP. The phylogenetic trees were constructed by using the programs DRAWTREE. The phylogenetic analysis indicated that new subgroup occupy a independent sub-branch in phylogenetic tree. The sequence similarities between the center strain XZ2-3 and the closest relatives, strain R. leguminosarum USDA

  2. Isolation and molecular identification of Vibrio spp. by sequencing of 16S rDNA from seafood, meat and meat products in Libya

    PubMed Central

    Azwai, S.M.; Alfallani, E.A.; Abolghait, S.K.; Garbaj, A.M.; Naas, H.T.; Moawad, A.A.; Gammoudi, F.T.; Rayes, H.M.; Barbieri, I.; Eldaghayes, I.M.

    2016-01-01

    The genus Vibrio includes several food-borne pathogens that cause a spectrum of clinical conditions including septicemia, cholera and milder forms of gastroenteritis. Several Vibrio spp. are commonly associated with food-borne transmission including Vibrio cholerae, Vibrio parahemolyticus, and Vibrio vulnificus. Microbiological analysis for enumeration and isolation of Vibrio spp. were carried out for a total of 93 samples of seafood, meat and meat products from different geographic localities in Libya (Tripoli, Regdalin, Janzour and Tobruk). Vibrio spp. were detected by conventional cultural and molecular method using PCR and sequencing of 16S rDNA. Out of the 93 cultured samples only 48 (51.6%) yielded colonies on Thiosulfate Citrate Bile Salt agar (TCBS) with culture characteristics of Vibrio spp. More than half (n=27) of processed seafood samples (n=46) yielded colonies on TCBS, while only 44.6 % of samples of meat and meat products showed colonies on TCBS. Among cultured seafood samples, the highest bacterial count was recorded in clam with a count of 3.8 ×104 CFU\\g. Chicken burger samples showed the highest bacterial count with 6.5 ×104 CFU\\g. Molecular analysis of the isolates obtained in this study, showed that 11 samples out of 48 (22.9%) were Vibrio spp. Vibrio parahemolyticus was isolated from camel meat for the first time. This study is an initial step to provide a baseline for future molecular research targeting Vibrio spp. foodborne illnesses. This data will be used to provide information on the magnitude of such pathogens in Libyan seafood, meat and meat products. PMID:27004169

  3. Isolation and molecular identification of Vibrio spp. by sequencing of 16S rDNA from seafood, meat and meat products in Libya.

    PubMed

    Azwai, S M; Alfallani, E A; Abolghait, S K; Garbaj, A M; Naas, H T; Moawad, A A; Gammoudi, F T; Rayes, H M; Barbieri, I; Eldaghayes, I M

    2016-01-01

    The genus Vibrio includes several food-borne pathogens that cause a spectrum of clinical conditions including septicemia, cholera and milder forms of gastroenteritis. Several Vibrio spp. are commonly associated with food-borne transmission including Vibrio cholerae, Vibrio parahemolyticus, and Vibrio vulnificus. Microbiological analysis for enumeration and isolation of Vibrio spp. were carried out for a total of 93 samples of seafood, meat and meat products from different geographic localities in Libya (Tripoli, Regdalin, Janzour and Tobruk). Vibrio spp. were detected by conventional cultural and molecular method using PCR and sequencing of 16S rDNA. Out of the 93 cultured samples only 48 (51.6%) yielded colonies on Thiosulfate Citrate Bile Salt agar (TCBS) with culture characteristics of Vibrio spp. More than half (n=27) of processed seafood samples (n=46) yielded colonies on TCBS, while only 44.6 % of samples of meat and meat products showed colonies on TCBS. Among cultured seafood samples, the highest bacterial count was recorded in clam with a count of 3.8 ×10(4) CFU\\g. Chicken burger samples showed the highest bacterial count with 6.5 ×10(4) CFU\\g. Molecular analysis of the isolates obtained in this study, showed that 11 samples out of 48 (22.9%) were Vibrio spp. Vibrio parahemolyticus was isolated from camel meat for the first time. This study is an initial step to provide a baseline for future molecular research targeting Vibrio spp. foodborne illnesses. This data will be used to provide information on the magnitude of such pathogens in Libyan seafood, meat and meat products. PMID:27004169

  4. Analysis of bacterial communities in the rhizosphere of chrysanthemum via denaturing gradient gel electrophoresis of PCR-amplified 16S rRNA as well as DNA fragments coding for 16S rRNA.

    PubMed

    Duineveld, B M; Kowalchuk, G A; Keijzer, A; van Elsas, J D; van Veen, J A

    2001-01-01

    The effect of developing chrysanthemum roots on the presence and activity of bacterial populations in the rhizosphere was examined by using culture-independent methods. Nucleic acids were extracted from rhizosphere soil samples associated with the bases of roots or root tips of plants harvested at different stages of development. PCR and reverse transcriptase (RT) PCR were used to amplify 16S ribosomal DNA (rDNA) and 16S rRNA, respectively, and the products were subjected to denaturing gradient gel electrophoresis (DGGE). Prominent DGGE bands were excised and sequenced to gain insight into the identities of predominantly present (PCR) and predominantly active (RT-PCR) bacterial populations. The majority of DGGE band sequences were related to bacterial genera previously associated with the rhizosphere, such as Pseudomonas, Comamonas, Variovorax, and Acetobacter, or typical of root-free soil environments, such as Bacillus and Arthrobacter. The PCR-DGGE patterns observed for bulk soil were somewhat more complex than those obtained from rhizosphere samples, and the latter contained a subset of the bands present in bulk soil. DGGE analysis of RT-PCR products detected a subset of bands visible in the rDNA-based analysis, indicating that some dominantly detected bacterial populations did not have high levels of metabolic activity. The sequences detected by the RT-PCR approach were, however, derived from a wide taxonomic range, suggesting that activity in the rhizosphere was not determined at broad taxonomic levels but rather was a strain- or species-specific phenomenon. Comparative analysis of DGGE profiles grouped all DNA-derived root tip samples together in a cluster, and within this cluster the root tip samples from young plants formed a separate subcluster. Comparison of rRNA-derived bacterial profiles showed no grouping of root tip samples versus root base samples. Rather, all profiles derived from 2-week-old plant rhizosphere soils grouped together regardless of

  5. PCR primers to amplify 16S rRNA genes from cyanobacteria.

    PubMed Central

    Nübel, U; Garcia-Pichel, F; Muyzer, G

    1997-01-01

    We developed and tested a set of oligonucleotide primers for the specific amplification of 16S rRNA gene segments from cyanobacteria and plastids by PCR. PCR products were recovered from all cultures of cyanobacteria and diatoms that were checked but not from other bacteria and archaea. Gene segments selectively retrieved from cyanobacteria and diatoms in unialgal but nonaxenic cultures and from cyanobionts in lichens could be directly sequenced. In the context of growing sequence databases, this procedure allows rapid and phylogenetically meaningful identification without pure cultures or molecular cloning. We demonstrate the use of this specific PCR in combination with denaturing gradient gel electrophoresis to probe the diversity of oxygenic phototrophic microorganisms in cultures, lichens, and complex microbial communities. PMID:9251225

  6. Improved PCR primers to amplify 16S rRNA genes from NC10 bacteria.

    PubMed

    He, Zhanfei; Wang, Jiaqi; Hu, Jiajie; Zhang, Hao; Cai, Chaoyang; Shen, Jiaxian; Xu, Xinhua; Zheng, Ping; Hu, Baolan

    2016-06-01

    Anaerobic oxidation of methane (AOM) coupled to nitrite reduction (AOM-NIR) is ecologically significant for mitigating the methane-induced greenhouse effect. The microbes responsible for this reaction, NC10 bacteria, have been widely detected in diverse ecosystems. However, some defects were discovered in the commonly used NC10-specific primers, 202F and qP1F. In the present work, the primers were redesigned and improved to overcome the defects found in the previous primers. A new nested PCR method was developed using the improved primers to amplify 16S ribosomal RNA (rRNA) genes from NC10 bacteria. In the new nested PCR method, the qP1mF/1492R and 1051F/qP2R primer sets were used in the first and second rounds, respectively. The PCR products were sequenced, and more operational taxonomic units (OTUs) of the NC10 phylum were obtained using the new primers compared to the previous primers. The sensitivity of the new nested PCR was tested by the serial dilution method, and the limit of detection was approximately 10(3) copies g(-1) dry sed. for the environmental samples compared to approximately 10(5) copies g(-1) dry sed. by the previous method. Finally, the improved primer, qP1mF, was used in quantitative PCR (qPCR) to determine the abundance of NC10 bacteria, and the results agreed well with the activity of AOM-NIR measured by isotope tracer experiments. The improved primers are able to amplify NC10 16S rRNA genes more efficiently than the previous primers and useful to explore the microbial community of the NC10 phylum in different systems. PMID:27020287

  7. Specific Detection of Bradyrhizobium and Rhizobium Strains Colonizing Rice (Oryza sativa) Roots by 16S-23S Ribosomal DNA Intergenic Spacer-Targeted PCR

    PubMed Central

    Tan, Zhiyuan; Hurek, Thomas; Vinuesa, Pablo; Müller, Peter; Ladha, Jagdish K.; Reinhold-Hurek, Barbara

    2001-01-01

    In addition to forming symbiotic nodules on legumes, rhizobial strains are members of soil or rhizosphere communities or occur as endophytes, e.g., in rice. Two rhizobial strains which have been isolated from root nodules of the aquatic legumes Aeschynomene fluminensis (IRBG271) and Sesbania aculeata (IRBG74) were previously found to promote rice growth. In addition to analyzing their phylogenetic positions, we assessed the suitability of the 16S-23S ribosomal DNA (rDNA) intergenic spacer (IGS) sequences for the differentiation of closely related rhizobial taxa and for the development of PCR protocols allowing the specific detection of strains in the environment. 16S rDNA sequence analysis (sequence identity, 99%) and phylogenetic analysis of IGS sequences showed that strain IRBG271 was related to but distinct from Bradyrhizobium elkanii. Rhizobium sp. (Sesbania) strain IRBG74 was located in the Rhizobium-Agrobacterium cluster as a novel lineage according to phylogenetic 16S rDNA analysis (96.8 to 98.9% sequence identity with Agrobacterium tumefaciens; emended name, Rhizobium radiobacter). Strain IRBG74 harbored four copies of rRNA operons whose IGS sequences varied only slightly (2 to 9 nucleotides). The IGS sequence analyses allowed intraspecies differentiation, especially in the genus Bradyrhizobium, as illustrated here for strains of Bradyrhizobium japonicum, B. elkanii, Bradyrhizobium liaoningense, and Bradyrhizobium sp. (Chamaecytisus) strain BTA-1. It also clearly differentiated fast-growing rhizobial species and strains, albeit with lower statistical significance. Moreover, the high sequence variability allowed the development of highly specific IGS-targeted nested-PCR assays. Strains IRBG74 and IRBG271 were specifically detected in complex DNA mixtures of numerous related bacteria and in the DNA of roots of gnotobiotically cultured or even of soil-grown rice plants after inoculation. Thus, IGS sequence analysis is an attractive technique for both microbial

  8. Phylogenetic relationships linking Duttaphrynus (Amphibia: Anura: Bufonidae) species based on 12S and 16S rDNA sequences.

    PubMed

    Pratihar, Suman; Bhattacharya, Manojit; Deuti, Kaushik

    2016-07-01

    Genus Duttaphrynus (Amphibia: Anura: Bufonidae) is endemic to southwestern and southern China and throughout southern Asia. Duttaphrynus phylogeny was also under debate for many years. 12S and 16S rDNAs help us to elucidate Duttaphrynus phylogeny. PMID:26155970

  9. Identification of Bacillus Probiotics Isolated from Soil Rhizosphere Using 16S rRNA, recA, rpoB Gene Sequencing and RAPD-PCR.

    PubMed

    Mohkam, Milad; Nezafat, Navid; Berenjian, Aydin; Mobasher, Mohammad Ali; Ghasemi, Younes

    2016-03-01

    Some Bacillus species, especially Bacillus subtilis and Bacillus pumilus groups, have highly similar 16S rRNA gene sequences, which are hard to identify based on 16S rDNA sequence analysis. To conquer this drawback, rpoB, recA sequence analysis along with randomly amplified polymorphic (RAPD) fingerprinting was examined as an alternative method for differentiating Bacillus species. The 16S rRNA, rpoB and recA genes were amplified via a polymerase chain reaction using their specific primers. The resulted PCR amplicons were sequenced, and phylogenetic analysis was employed by MEGA 6 software. Identification based on 16S rRNA gene sequencing was underpinned by rpoB and recA gene sequencing as well as RAPD-PCR technique. Subsequently, concatenation and phylogenetic analysis showed that extent of diversity and similarity were better obtained by rpoB and recA primers, which are also reinforced by RAPD-PCR methods. However, in one case, these approaches failed to identify one isolate, which in combination with the phenotypical method offsets this issue. Overall, RAPD fingerprinting, rpoB and recA along with concatenated genes sequence analysis discriminated closely related Bacillus species, which highlights the significance of the multigenic method in more precisely distinguishing Bacillus strains. This research emphasizes the benefit of RAPD fingerprinting, rpoB and recA sequence analysis superior to 16S rRNA gene sequence analysis for suitable and effective identification of Bacillus species as recommended for probiotic products. PMID:26898909

  10. The world in a river? A preliminary analysis of the 16S rDNA variability of Tubifex species (Clitellata: Tubificidae) from the Lambro River.

    PubMed

    Crottini, Angelica; Marotta, Roberto; Barbuto, Michela; Casiraghi, Maurizio; Ferraguti, Marco

    2008-09-01

    Tubifex tubifex Müller, 1774 is a cosmopolitan freshwater tubificid widely used as a model in ecotoxicology, population dynamics and developmental biology. It is traditionally recognized as a polytypic species and in Lambro River (Milano, Northern Italy) it occurs in two of the three recognized forms, named "tubifex" and "blanchardi", alternatively considered as ecological forms or distinct species. To investigate the genetic differentiation of the populations occurring in the Lambro River we sequenced a fragment of the 16S rDNA mitochondrial gene. T. blanchardi, characterized by a low genetic diversity, was genetically segregated from the other sympatric T. tubifex. The ancestral state reconstruction was used to define the morphological traits that support its distinctness. On the contrary, the other T. tubifex from the Lambro community, although morphologically indistinguishable, revealed an astonishing degree of genetic variability, both between and within the three identified clades that proved to be genetically isolated. Using samples from the mixed Lambro River community and from other countries around the world we present an overview of the species complex' 16S rDNA variability. Our results show that the genetic variability did not sensibly increase widening the data set, suggesting that the Lambro River populations meet the species' worldwide genetic variability. PMID:18625325

  11. Comparison of VITEK2, MALDI-TOF MS, and 16S rDNA sequencing for identification of Myroides odoratus and Myroides odoratimimus.

    PubMed

    Schröttner, Percy; Rudolph, Wolfram W; Eing, Bodo R; Bertram, Sebastian; Gunzer, Florian

    2014-06-01

    The genus Myroides comprises the 2 medically relevant species Myroides odoratus and Myroides odoratimimus that are rare opportunistic pathogens and cause infections in immunocompromised patients. A fast identification of Myroides is of importance because these bacterial strains show multiple resistance against antibiotics and therefore limit treatment options. They are associated, for instance, with urinary tract infections, sepsis, meningitis, pneumonia, and infectious cellulitis. Since more and more Myroides spp. are being described, additional potentially pathogenic bacteria may be identified in the future demanding the need for fast and reliable identification methods at species level. However, to date, only molecular approaches meet these demands. In this study, we, therefore, attempt to define an appropriate method other than DNA fingerprinting that will permit a comparable efficacy and, possibly, a more economical strain identification. For this purpose, we compared 2 widely used automated diagnostic systems (VITEK 2 [bioMérieux, Nürtingen, Germany] and Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry (MALDI-TOF MS) [Bruker Daltonics, Bremen, Germany]) and correlated the results to 16S rDNA sequencing data. In total, we analyzed 22 strains collected in the course of routine diagnostics. In this study, we demonstrate that VITEK 2 reliably identifies the genus Myroides but cannot differentiate between M. odoratimimus and M. odoratus. In contrast to this, both MALDI-TOF MS and 16S rDNA sequencing efficiently distinguish between the 2 species. PMID:24666701

  12. [Communities of Actynomicetes fungy in three vegetation types of the Colombian Amazon: abundance, morphotypes and the 16s rDNA gene].

    PubMed

    Cardona, Gladys Inés; Peña-Venegas, Clara Patricia; Ruiz-García, Manuel

    2009-12-01

    Among soil microorganisms, Actinomycetes play an important role in the sustainability of natural and agricultural systems: decomposition of organic matter; degradation of recalcitrant compounds like lignin; nitrogen fixation; degradation of agricultural chemicals and biological control in plants and animals. We evaluated their diversity in soils under three different vegetation covers (pasture, tropical primary forest and stubble) at two depths in the Southern Colombian Amazon border. We collected five replicates per vegetation type (in each, three samples at 0-20cm and three at 20-30cm; for a total of 30 samples). Abundance and phenotypic diversity were determined by plate counting. Genomic DNA was extracted from the isolates: the 16s rDNA gene was amplified with specific primers, and its genetic diversity was estimated by means of an amplified restriction analysis (ARDRA). Actynomicetes abundance varied with vegetation and depth, possibly reflecting presence of earthworms, macro-fauna and physico-chemical characteristics associated to fertility, as well as organic matter, total bases, and optimal capacity to cationic interchange. Primary forests had the highest diversity. Sixteen morpho-types (six genera) were identified; Streptomyces was the most abundant everywhere. The heterogeneity ofARDRA patterns prevented species identification because of the intra-species variability in sequences of 16s rDNA operons. This community is a biological indicator of landscape alteration and could include new bio-active compounds of pharmaceutical interest. PMID:20073339

  13. Phylogeny of coral-inhabiting barnacles (Cirripedia; Thoracica; Pyrgomatidae) based on 12S, 16S and 18S rDNA analysis.

    PubMed

    Simon-Blecher, N; Huchon, D; Achituv, Y

    2007-09-01

    The traditional phylogeny of the coral-inhabiting barnacles, the Pyrgomatidae, is based on morphological characteristics, mainly of the hard parts. It has been difficult to establish the phylogenetic relationships among Pyrgomatidae because of the apparent convergence of morphological characteristics, and due to the use of non-cladistic systematics, which emphasize ancestor-descendant relationships rather than sister-clade relationships. We used partial sequences of two mithochondrial genes, 12S rDNA and 16S rDNA, and a nuclear gene, 18S rDNA, to infer the molecular phylogeny of the pyrgomatids. Our phylogenetic results allowed us to reject previous classifications of Pyrgomatidae based on morphological characteristics. Our results also suggested the possibility of paraphyly of the Pyrgomatidae. The hydrocoral barnacle Wanella is not found on the same clade as the other pyrgomatids, but rather, with the free-living balanids. The basal position of Megatrema and Ceratoconcha is supported. The archeaobalanid Armatobalanus is grouped with Cantellius at the base of the Indo-Pacific pyrgomatines. Fusion of the shell plate and modification of the opercular valves are homoplasious features that occurred more than three times on different clades. The monophyly of the "Savignium" group, comprising four nominal genera, is also not supported, and the different taxa are placed on different clades. PMID:17560131

  14. Microbial Diversity of Bovine Mastitic Milk as Described by Pyrosequencing of Metagenomic 16s rDNA

    PubMed Central

    Oikonomou, Georgios; Machado, Vinicius Silva; Santisteban, Carlos; Schukken, Ynte Hein; Bicalho, Rodrigo Carvalho

    2012-01-01

    Dairy cow mastitis is an important disease in the dairy industry. Different microbial species have been identified as causative agents in mastitis, and are traditionally diagnosed by bacterial culture. The objective of this study was to use metagenomic pyrosequencing of bacterial 16S rRNA genes to investigate bacterial DNA diversity in milk samples of mastitic and healthy dairy cows and compare the results with those obtained by classical bacterial culture. One hundred and thirty-six milk samples were collected from cows showing signs of mastitis and used for microbiological culture. Additionally, 20 milk samples were collected from healthy quarters. Bacterial DNA was isolated from the same milk samples and the 16S rRNA genes were individually amplified and pyrosequenced. Discriminant analysis showed that the groups of samples that were most clearly different from the rest and thus easily discriminated were the normal milk samples from healthy cows and those characterised by culture as Trueperella pyogenes and Streptococcus spp. The mastitis pathogens identified by culture were generally among the most frequent organisms detected by pyrosequencing, and in some cases (Escherichia coli, Klebsiella spp. and Streptococcus uberis mastitis) the single most prevalent microorganism. Trueperella pyogenes sequences were the second most prevalent sequences in mastitis cases diagnosed as Trueperella pyogenes by culture, Streptococcus dysgalactiae sequences were the second most prevalent sequences in mastitis cases diagnosed as Streptococcus dysgalactiae by culture, and Staphyloccocus aureus sequences were the third most prevalent in mastitis cases diagnosed as Staphylococcus aureus by culture. In samples that were aerobic culture negative, pyrosequencing identified DNA of bacteria that are known to cause mastitis, DNA of bacteria that are known pathogens but have so far not been associated with mastitis, and DNA of bacteria that are currently not known to be pathogens. A

  15. Micelle PCR reduces chimera formation in 16S rRNA profiling of complex microbial DNA mixtures.

    PubMed

    Boers, Stefan A; Hays, John P; Jansen, Ruud

    2015-01-01

    16S rRNA gene profiling has revolutionized the field of microbial ecology. Many researchers in various fields have embraced this technology to investigate bacterial compositions of samples derived from many different ecosystems. However, it is important to acknowledge the current limitations and drawbacks of 16S rRNA gene profiling. Although sample handling, DNA extraction methods and the choice of universal 16S rRNA gene PCR primers are well known factors that could seriously affect the final results of microbiota profiling studies, inevitable amplification artifacts, such as chimera formation and PCR competition, are seldom appreciated. Here we report on a novel micelle based amplification strategy, which overcomes these limitations via the clonal amplification of targeted DNA molecules. Our results show that micelle PCR drastically reduces chimera formation by a factor of 38 (1.5% vs. 56.9%) compared with traditional PCR, resulting in improved microbial diversity estimates. In addition, compartmentalization during micelle PCR prevents PCR competition due to unequal amplification rates of different 16S template molecules, generating robust and accurate 16S microbiota profiles required for comparative studies (e.g. longitudinal surveys). PMID:26373611

  16. Culturable bacteria present in the fluid of the hooded-pitcher plant Sarracenia minor based on 16S rDNA gene sequence data.

    PubMed

    Siragusa, Alex J; Swenson, Janice E; Casamatta, Dale A

    2007-08-01

    The culturable microbial community within the pitcher fluid of 93 Sarracenia minor carnivorous plants was examined over a 2-year study. Many aspects of the plant/bacterial/insect interaction within the pitcher fluid are minimally understood because the bacterial taxa present in these pitchers have not been identified. Thirteen isolates were characterized by 16S rDNA sequencing and subsequent phylogenetic analysis. The Proteobacteria were the most abundant taxa and included representatives from Serratia, Achromobacter, and Pantoea. The Actinobacteria Micrococcus was also abundant while Bacillus, Lactococcus, Chryseobacterium, and Rhodococcus were infrequently encountered. Several isolates conformed to species identifiers (>98% rDNA gene sequence similarity) including Serratia marcescens (isolates found in 27.5% of pitchers), Achromobacter xylosoxidans (37.6%), Micrococcus luteus (40.9%), Bacillus cereus (isolates found in 10.2%), Bacillus thuringiensis (5.4%), Lactococcus lactis (17.2%), and Rhodococcus equi (2.2%). Species-area curves suggest that sampling efforts were sufficient to recover a representative culturable bacterial community. The bacteria present represent a diverse community probably as a result of introduction by insect vectors, but the ecological significance remains under explored. PMID:17380356

  17. Identification of acetic acid bacteria by restriction fragment length polymorphism analysis of a PCR-amplified fragment of the gene coding for 16S rRNA.

    PubMed

    Poblet, M; Rozès, N; Guillamón, J M; Mas, A

    2000-07-01

    Acetic acid bacteria (AAB) irreversibly spoil wines and represent a serious problem. Limited studies on the ecology of AAB during winemaking have been done due to the lack of rapid and precise techniques for their identification. RFLP analysis of PCR-amplified fragment of 16S rDNA was performed on AAB reference strains. The amplified rDNAs were approximately 870-bp long for all AAB species while no amplicons were detected for lactic acid bacteria and yeasts. Out of the four restriction enzymes tested, TaqI was the most efficient one and divided the studied AAB into six groups. However, complete differentiation among collection strains of Acetobacter pasteurianus and Gluconoacetobacter hansenii was not possible. PMID:10886617

  18. Molecular Characterization of Stool Microbiota in HIV-Infected Subjects by Panbacterial and Order-Level 16S Ribosomal DNA (rDNA) Quantification and Correlations with Immune Activation

    PubMed Central

    Ellis, Collin L.; Ma, Zhong-Min; Mann, Surinder K.; Li, Chin-Shang; Wu, Jian; Knight, Thomas H.; Yotter, Tammy; Hayes, Timothy L.; Maniar, Archana H.; Troia-Cancio, Paolo V.; Overman, Heather A; Torok, Natalie J.; Albanese, Anthony; Rutledge, John C.; Miller, Christopher J.; Pollard, Richard B.; Asmuth, David M.

    2011-01-01

    Background The relationship between gut microbial community composition at the higher-taxonomic order-level and local and systemic immunologic abnormalities in HIV disease may provide insight into how bacterial translocation impacts HIV disease. Methods Antiretroviral (ART)-naive HIV patients underwent upper endoscopy before and nine months after starting ART. Duodenal tissue was paraffin-embedded for immunohistochemical analysis (IHC) and digested for FACS for T-cell subsets and immune activation (CD38+/HLA-DR+) enumeration. Stool samples were provided from patients and controls for comparison. Metagenomic microbial DNA was extracted from feces for optimized 16S ribosomal RNA gene (rDNA) real-time qPCR assays designed to quantify panbacterial loads and the relative abundances of proinflammatory Enterobacteriales order, and the dominant Bacteroidales and Clostridiales orders. Results Samples from 10 HIV-subjects prior to initiating, and from 6 subjects receiving, ART were available for analysis. There was a trend for a greater proportion of Enterobacteriales in HIV-positive subjects compared to controls (p=0.099). There were significant negative correlations between total bacterial load and duodenal CD4+ and CD8+ T-cell activation levels (r= −0.74, p= 0.004 and r= −0.67, p=0.013, respectively). The proportions of Enterobacteriales and Bacteroidales were significantly correlated with duodenal CD4+ T-cell depletion and peripheral CD8+ T-cell activation, respectively. Conclusions These data represent the first report of quantitative molecular and cellular correlations between total/universal and order-level gut bacterial populations and GALT levels of immune activation in HIV-infected subjects. The correlations between lower overall 16S rDNA levels and tissue immune activation suggest that the gut microbiome may contribute to immune activation and influence HIV progression. PMID:21436711

  19. Differentiation of Paenibacillus larvae subsp. larvae, the Cause of American Foulbrood of Honeybees, by Using PCR and Restriction Fragment Analysis of Genes Encoding 16S rRNA

    PubMed Central

    Alippi, Adriana M.; López, Ana Claudia; Aguilar, O. Mario

    2002-01-01

    A rapid procedure for the identification of Paenibacillus larvae subsp. larvae, the causal agent of American foulbrood (AFB) disease of honeybees (Apis mellifera L.), based on PCR and restriction fragment analysis of the 16S rRNA genes (rDNA) is described. Eighty-six bacterial strains belonging to 39 species of the genera Paenibacillus, Bacillus, Brevibacillus, and Virgibacillus were characterized. Amplified rDNA was digested with seven restriction endonucleases. The combined data from restriction analysis enabled us to distinguish 35 profiles. Cluster analysis revealed that P. larvae subsp. larvae and Paenibacillus larvae subsp. pulvifaciens formed a group with about 90% similarity; however, the P. larvae subsp. larvae restriction fragment length polymorphism pattern produced by endonuclease HaeIII was found to be unique and distinguishable among other closely related bacteria. This pattern was associated with DNA extracted directly from honeybee brood samples showing positive AFB clinical signs that yielded the restriction profile characteristic of P. larvae subsp. larvae, while no amplification product was obtained from healthy larvae. The method described here is particularly useful because of the short time required to carry it out and because it allows the differentiation of P. larvae subsp. larvae-infected larvae from all other species found in apiarian sources. PMID:12089057

  20. Ureaplasma urealyticum continuous ambulatory peritoneal dialysis-associated peritonitis diagnosed by 16S rRNA gene PCR.

    PubMed

    Yager, Jessica E; Ford, Emily S; Boas, Zachary P; Haseley, Leah A; Cookson, Brad T; Sengupta, Dhruba J; Fang, Ferric C; Gottlieb, Geoffrey S

    2010-11-01

    In some patients with peritonitis complicating continuous ambulatory peritoneal dialysis (CAPD), a causative organism is never identified. We report a case of Ureaplasma urealyticum CAPD-associated peritonitis diagnosed by 16S rRNA gene PCR. Ureaplasma may be an underrecognized cause of peritonitis because it cannot be recovered using routine culture methods. PMID:20739488

  1. Variation in copy number of the 28S rDNA of Aspergillus fumigatus measured by droplet digital PCR and analog quantitative real-time PCR.

    PubMed

    Alanio, Alexandre; Sturny-Leclère, Aude; Benabou, Marion; Guigue, Nicolas; Bretagne, Stéphane

    2016-08-01

    Droplet digital PCR (ddPCR) after DNA digestion yielded a 28S rDNA copy number of 61 to 86 copies/genome when testing 10 unrelated Aspergillus fumigatus isolates, higher than with quantitative PCR. Unfortunately, ddPCR after DNA digestion did not improve the sensitivity of our PCR assay when testing serum patients with invasive aspergillosis. PMID:27316653

  2. Preliminary evaluation of the use of soil bacterial 16S rDNA DNA markers in sediment fingerprinting in two small endorheic lagoons in southern Spain

    NASA Astrophysics Data System (ADS)

    Gomez, Jose Alfonso; Landa del Castillo, Blanca; Guzman, Gema; Petticrew, Ellen L.; Owens, Phillip N.

    2016-04-01

    127 % in Dulce and from 80 to 117 % in Zóñar. These rangesare within values reported for other soil chemical and physical properties, although the higher values are above the most commonly reported CVs which tend to be in the range from 30 to 80 %. Some groups, that are relatively stable to the normalization process, can provide enough information for solving a mixing model, although the specific groups vary between the two catchments as expected from previous studies. Overall, all the models for Zóñar tended to provide similar results with low contributions from source areas 1 and 2, and a much larger contribution from source area 3. For this solution, the mixing model was able to replicate the values of all the OTUs included in the model. The predicted values for Dulce were not as stable. The model with 10 OTUs were similar with a very low contribution from source area 2, a moderate contribution from source area 3 and a maximum contribution from source area 1. However, these values differed from those with only three OTUs, and they also differed between themselves when the normalized and non-normalized values were used. This solution also seemed to replicate the averaged measured values of most of the OTÚs included in the model. These preliminary results demonstrate the potential of soil bacterial 16S rDNA in sediment fingerprinting studies, although some questions need to be addressed in more detail, including: the temporal evolution of the distribution of the bacterial markers with soil depth; the implications of selective transport by runoff; and the relatively large variability of counts among samples from the same area. We are currently repeating the sampling in one of the subcatchments to provide some insight into these issues. Key words: sediment, fingerprinting, soil, microbial, DNA, lagoon References Joe-Strack, J.A., Petticrew, E.L. 2012. Use of LH-PCR as a DNA fingerprint technique to trace sediment-associated microbial communities from various land

  3. Quick identification of acetic acid bacteria based on nucleotide sequences of the 16S-23S rDNA internal transcribed spacer region and of the PQQ-dependent alcohol dehydrogenase gene.

    PubMed

    Trcek, Janja

    2005-10-01

    Acetic acid bacteria (AAB) are well known for oxidizing different ethanol-containing substrates into various types of vinegar. They are also used for production of some biotechnologically important products, such as sorbose and gluconic acids. However, their presence is not always appreciated since certain species also spoil wine, juice, beer and fruits. To be able to follow AAB in all these processes, the species involved must be identified accurately and quickly. Because of inaccuracy and very time-consuming phenotypic analysis of AAB, the application of molecular methods is necessary. Since the pairwise comparison among the 16S rRNA gene sequences of AAB shows very high similarity (up to 99.9%) other DNA-targets should be used. Our previous studies showed that the restriction analysis of 16S-23S rDNA internal transcribed spacer region is a suitable approach for quick affiliation of an acetic acid bacterium to a distinct group of restriction types and also for quick identification of a potentially novel species of acetic acid bacterium (Trcek & Teuber 2002; Trcek 2002). However, with the exception of two conserved genes, encoding tRNAIle and tRNAAla, the sequences of 16S-23S rDNA are highly divergent among AAB species. For this reason we analyzed in this study a gene encoding PQQ-dependent ADH as a possible DNA-target. First we confirmed the expression of subunit I of PQQ-dependent ADH (AdhA) also in Asaia, the only genus of AAB which exhibits little or no ADH-activity. Further we analyzed the partial sequences of adhA among some representative species of the genera Acetobacter, Gluconobacter and Gluconacetobacter. The conserved and variable regions in these sequences made possible the construction of A. acetispecific oligonucleotide the specificity of which was confirmed in PCR-reaction using 45 well-defined strains of AAB as DNA-templates. The primer was also successfully used in direct identification of A. aceti from home made cider vinegar as well as for

  4. Differentiation of anaerobic polycentric fungi by rDNA PCR-RFLP.

    PubMed

    Fliegerová, K; Mrázek, J; Voigt, K

    2006-01-01

    The suitability of restriction fragment length polymorphism (RFLP) analysis of the ribosomal DNA cluster for discriminating two genera of anaerobic polycentric fungi, Orpinomyces and Anaeromyces, was determined. Three PCR-amplified DNA fragments--nuclear small subunit (SSU; 18S rDNA), the nuclear large subunit (LSU; 28S rDNA) and internal transcribed spacer (ITS)--were restricted with endonucleases AluI, DraI, HinfI and MboI. Although the SSU DNA fragment could be restricted successfully by all four enzymes, no differences were observed between restriction patterns of Orpinomyces and Anaeromyces. The most polymorphic restriction pattern between Orpinomyces and Anaeromyces resulted from cleavage of LSU rDNA fragments cut by AluI and HinfI and ITS fragment cut by DraI and HinfI. Genus-specific RFLP patterns were determined for Orpinomyces and Anaeromyces genera; the results showed that the PCR-RFLP analysis of rDNA offers an easy and rapid tool for differentiation of two polycentric genera of anaerobic fungi, which could be hardly separated on the basis of morphology. PMID:17007423

  5. Chimeric 16S rRNA sequence formation and detection in Sanger and 454-pyrosequenced PCR amplicons

    PubMed Central

    Haas, Brian J.; Gevers, Dirk; Earl, Ashlee M.; Feldgarden, Mike; Ward, Doyle V.; Giannoukos, Georgia; Ciulla, Dawn; Tabbaa, Diana; Highlander, Sarah K.; Sodergren, Erica; Methé, Barbara; DeSantis, Todd Z.; Petrosino, Joseph F.; Knight, Rob; Birren, Bruce W.

    2011-01-01

    Bacterial diversity among environmental samples is commonly assessed with PCR-amplified 16S rRNA gene (16S) sequences. Perceived diversity, however, can be influenced by sample preparation, primer selection, and formation of chimeric 16S amplification products. Chimeras are hybrid products between multiple parent sequences that can be falsely interpreted as novel organisms, thus inflating apparent diversity. We developed a new chimera detection tool called Chimera Slayer (CS). CS detects chimeras with greater sensitivity than previous methods, performs well on short sequences such as those produced by the 454 Life Sciences (Roche) Genome Sequencer, and can scale to large data sets. By benchmarking CS performance against sequences derived from a controlled DNA mixture of known organisms and a simulated chimera set, we provide insights into the factors that affect chimera formation such as sequence abundance, the extent of similarity between 16S genes, and PCR conditions. Chimeras were found to reproducibly form among independent amplifications and contributed to false perceptions of sample diversity and the false identification of novel taxa, with less-abundant species exhibiting chimera rates exceeding 70%. Shotgun metagenomic sequences of our mock community appear to be devoid of 16S chimeras, supporting a role for shotgun metagenomics in validating novel organisms discovered in targeted sequence surveys. PMID:21212162

  6. The evolutionary history of the genus Timarcha (Coleoptera, Chrysomelidae) inferred from mitochondrial COII gene and partial 16S rDNA sequences.

    PubMed

    Gómez-Zurita, J; Juan, C; Petitpierre, E

    2000-02-01

    The apterous genus Timarcha consists of three subgenera and more than 100 species in its Palearctic distribution, with specialized feeding on few plant families. Fifty-four sequences sampled from 31 taxa of the genus plus three outgroup leaf beetles were studied for their complete cytochrome oxidase II (COII) and a fragment of 16S rDNA mitochondrial genes, representing a total of about 1200 bp. Phylogenetic analyses using maximum-parsimony and distance methods for each gene separately and for the combined data set gave compatible topologies. The subgenus Metallotimarcha consistently appears in a basal position and is well differentiated from the remaining Timarcha, but no clear monophyletic grouping of Timarchostoma and Timarcha s. str. subgenera can be deduced from our analysis. Calibration of the molecular clock has been done using the opening of the Gibraltar Strait after the Messinian salinity crisis (about 5.5 MYA) as the biogeographic event causing disjunction of two particular taxa. Accordingly, the COII evolutionary rate has been estimated to be of 0.76 x 10(-8) substitution/site/year in Timarcha. Relation between phylogeny and host-plant use indicates widening of trophic regime as a derived character in Timarcha. PMID:10679162

  7. Gastrointestinal Bacterial and Methanogenic Archaea Diversity Dynamics Associated with Condensed Tannin-Containing Pine Bark Diet in Goats Using 16S rDNA Amplicon Pyrosequencing

    PubMed Central

    Min, Byeng R.; Solaiman, Sandra; Shange, Raymon

    2014-01-01

    Eighteen Kiko-cross meat goats (n = 6) were used to collect gastrointestinal (GI) bacteria and methanogenic archaea for diversity measures when fed condensed tannin-containing pine bark (PB). Three dietary treatments were tested: control diet (0% PB and 30% wheat straw (WS); 0.17% condensed tannins (CT) dry matter (DM)); 15% PB and 15% WS (1.6% CT DM), and 30% PB and 0% WS (3.2% CT DM). A 16S rDNA bacterial tag-encoded FLX amplicon pyrosequencing technique was used to characterize and elucidate changes in GI bacteria and methanogenic archaea diversity among the diets. Proteobacteria was the most dominant phylum in goats with mean relative abundance values ranging from 39.7 (30% PB) to 46.5% (control) and 47.1% (15% PB). Other phyla individually accounted for fewer than 25% of the relative abundance observed. Predominant methanogens were Methanobrevibacter (75, 72, and 49%), Methanosphaera (3.3, 2.3, and 3.4%), and Methanobacteriaceae (1.2, 0.6, and 0.7%) population in control, 15, and 30% PB, respectively. Among methanogens, Methanobrevibacter was linearly decreased (P = 0.05) with increasing PB supplementation. These results indicate that feeding PB selectively altered bacteria and methanogenic archaeal populations in the GI tract of goats. PMID:24669219

  8. Evaluation of the bacterial diversity in the feces of cattle using 16S rDNA bacterial tag-encoded FLX amplicon pyrosequencing (bTEFAP)

    PubMed Central

    Dowd, Scot E; Callaway, Todd R; Wolcott, Randall D; Sun, Yan; McKeehan, Trevor; Hagevoort, Robert G; Edrington, Thomas S

    2008-01-01

    Background The microbiota of an animal's intestinal tract plays important roles in the animal's overall health, productivity and well-being. There is still a scarcity of information on the microbial diversity in the gut of livestock species such as cattle. The primary reason for this lack of data relates to the expense of methods needed to generate such data. Here we have utilized a bacterial tag-encoded FLX 16s rDNA amplicon pyrosequencing (bTEFAP) approach that is able to perform diversity analyses of gastrointestinal populations. bTEFAP is relatively inexpensive in terms of both time and labor due to the implementation of a novel tag priming method and an efficient bioinformatics pipeline. We have evaluated the microbiome from the feces of 20 commercial, lactating dairy cows. Results Ubiquitous bacteria detected from the cattle feces included Clostridium, Bacteroides, Porpyhyromonas, Ruminococcus, Alistipes, Lachnospiraceae, Prevotella, Lachnospira, Enterococcus, Oscillospira, Cytophage, Anaerotruncus, and Acidaminococcus spp. Foodborne pathogenic bacteria were detected in several of the cattle, a total of 4 cows were found to be positive for Salmonella spp (tentative enterica) and 6 cows were positive for Campylobacter spp. (tentative lanienae). Conclusion Using bTEFAP we have examined the microbiota in the feces of cattle. As these methods continue to mature we will better understand the ecology of the major populations of bacteria the lower intestinal tract. This in turn will allow for a better understanding of ways in which the intestinal microbiome contributes to animal health, productivity and wellbeing. PMID:18652685

  9. Molecular Diagnosis of Periprosthetic Joint Infection by Quantitative RT-PCR of Bacterial 16S Ribosomal RNA

    PubMed Central

    Lee, Mel S.; Chang, Wen-Hsin; Chen, Su-Chin; Hsieh, Pang-Hsin; Shih, Hsin-Nung; Ueng, Steve W. N.; Lee, Gwo-Bin

    2013-01-01

    The diagnosis of periprosthetic joint infection is sometimes straightforward with purulent discharge from the fistula tract communicating to the joint prosthesis. However it is often difficult to differentiate septic from aseptic loosening of prosthesis because of the high culture-negative rates in conventional microbiologic culture. This study used quantitative reverse transcription polymerase chain reaction (RT-qPCR) to amplify bacterial 16S ribosomal RNA in vitro and in 11 clinical samples. The in vitro analysis demonstrated that the RT-qPCR method was highly sensitive with the detection limit of bacterial 16S rRNA being 0.148 pg/μl. Clinical specimens were analyzed using the same protocol. The RT-qPCR was positive for bacterial detection in 8 culture-positive cases (including aerobic, anaerobic, and mycobacteria) and 2 culture-negative cases. It was negative in one case that the final diagnosis was confirmed without infection. The molecular diagnosis of bacterial infection using RT-qPCR to detect bacterial 16S rRNA around a prosthesis correlated well with the clinical findings. Based on the promising clinical results, we were attempting to differentiate bacterial species or drug-resistant strains by using species-specific primers and to detect the persistence of bacteria during the interim period before the second stage reimplantation in a larger scale of clinical subjects. PMID:24453929

  10. PHYLOGENETIC TREE OF 16S RIBOSOMAL RNA SEQUENCES FROM SULFATE-REDUCING BACTERIA IN A SANDY MARINE ENVIRONMENT

    EPA Science Inventory

    Phylogenetic divergence among sulfate-reducing bacteria in an estuarine sediment sample was investigated by PCR amplification and comparison of partial 16S rDNA sequences. wenty unique 16S RDNA sequences were found, 12 from delta subclass bacteria based on overall sequence simila...

  11. Integrated next-generation sequencing of 16S rDNA and metaproteomics differentiate the healthy urine microbiome from asymptomatic bacteriuria in neuropathic bladder associated with spinal cord injury

    PubMed Central

    2012-01-01

    Background Clinical dogma is that healthy urine is sterile and the presence of bacteria with an inflammatory response is indicative of urinary tract infection (UTI). Asymptomatic bacteriuria (ABU) represents the state in which bacteria are present but the inflammatory response is negligible. Differentiating ABU from UTI is diagnostically challenging, but critical because overtreatment of ABU can perpetuate antimicrobial resistance while undertreatment of UTI can result in increased morbidity and mortality. In this study, we describe key characteristics of the healthy and ABU urine microbiomes utilizing 16S rRNA gene (16S rDNA) sequencing and metaproteomics, with the future goal of utilizing this information to personalize the treatment of UTI based on key individual characteristics. Methods A cross-sectional study of 26 healthy controls and 27 healthy subjects at risk for ABU due to spinal cord injury-related neuropathic bladder (NB) was conducted. Of the 27 subjects with NB, 8 voided normally, 8 utilized intermittent catheterization, and 11 utilized indwelling Foley urethral catheterization for bladder drainage. Urine was obtained by clean catch in voiders, or directly from the catheter in subjects utilizing catheters. Urinalysis, urine culture and 16S rDNA sequencing were performed on all samples, with metaproteomic analysis performed on a subsample. Results A total of 589454 quality-filtered 16S rDNA sequence reads were processed through a NextGen 16S rDNA analysis pipeline. Urine microbiomes differ by normal bladder function vs. NB, gender, type of bladder catheter utilized, and duration of NB. The top ten bacterial taxa showing the most relative abundance and change among samples were Lactobacillales, Enterobacteriales, Actinomycetales, Bacillales, Clostridiales, Bacteroidales, Burkholderiales, Pseudomonadales, Bifidobacteriales and Coriobacteriales. Metaproteomics confirmed the 16S rDNA results, and functional human protein-pathogen interactions were noted in

  12. Bacterial diversity assessment in soil of an active Brazilian copper mine using high-throughput sequencing of 16S rDNA amplicons.

    PubMed

    Rodrigues, Viviane D; Torres, Tatiana T; Ottoboni, Laura M M

    2014-11-01

    Mining activities pose severe environmental risks worldwide, generating extreme pH conditions and high concentrations of heavy metals, which can have major impacts on the survival of organisms. In this work, pyrosequencing of the V3 region of the 16S rDNA was used to analyze the bacterial communities in soil samples from a Brazilian copper mine. For the analysis, soil samples were collected from the slopes (geotechnical structures) and the surrounding drainage of the Sossego mine (comprising the Sossego and Sequeirinho deposits). The results revealed complex bacterial diversity, and there was no influence of deposit geographic location on the composition of the communities. However, the environment type played an important role in bacterial community divergence; the composition and frequency of OTUs in the slope samples were different from those of the surrounding drainage samples, and Acidobacteria, Chloroflexi, Firmicutes, and Gammaproteobacteria were responsible for the observed difference. Chemical analysis indicated that both types of sample presented a high metal content, while the amounts of organic matter and water were higher in the surrounding drainage samples. Non-metric multidimensional scaling (N-MDS) analysis identified organic matter and water as important distinguishing factors between the bacterial communities from the two types of mine environment. Although habitat-specific OTUs were found in both environments, they were more abundant in the surrounding drainage samples (around 50 %), and contributed to the higher bacterial diversity found in this habitat. The slope samples were dominated by a smaller number of phyla, especially Firmicutes. The bacterial communities from the slope and surrounding drainage samples were different in structure and composition, and the organic matter and water present in these environments contributed to the observed differences. PMID:25129578

  13. Evaluation of the effects of intrapartum antibiotic prophylaxis on newborn intestinal microbiota using a sequencing approach targeted to multi hypervariable 16S rDNA regions.

    PubMed

    Aloisio, Irene; Quagliariello, Andrea; De Fanti, Sara; Luiselli, Donata; De Filippo, Carlotta; Albanese, Davide; Corvaglia, Luigi Tommaso; Faldella, Giacomo; Di Gioia, Diana

    2016-06-01

    Different factors are known to influence the early gut colonization in newborns, among them the perinatal use of antibiotics. On the other hand, the effect on the baby of the administration of antibiotics to the mother during labor, referred to as intrapartum antibiotic prophylaxis (IAP), has received less attention, although routinely used in group B Streptococcus positive women to prevent the infection in newborns. In this work, the fecal microbiota of neonates born to mothers receiving IAP and of control subjects were compared taking advantage for the first time of high-throughput DNA sequencing technology. Seven different 16S rDNA hypervariable regions (V2, V3, V4, V6 + V7, V8, and V9) were amplified and sequenced using the Ion Torrent Personal Genome Machine. The results obtained showed significant differences in the microbial composition of newborns born to mothers who had received IAP, with a lower abundance of Actinobacteria and Bacteroidetes as well as an overrepresentation of Proteobacteria. Considering that the seven hypervariable regions showed different discriminant ability in the taxonomic identification, further analyses were performed on the V4 region evidencing in IAP infants a reduced microbial richness and biodiversity, as well as a lower number of bacterial families with a predominance of Enterobacteriaceae members. In addition, this analysis pointed out a significant reduction in Bifidobacterium spp. strains. The reduced abundance of these beneficial microorganisms, together with the increased amount of potentially pathogenic bacteria, may suggest that IAP infants are more exposed to gastrointestinal or generally health disorders later in age. PMID:26971496

  14. A Comparison between Droplet Digital and Quantitative PCR in the Analysis of Bacterial 16S Load in Lung Tissue Samples from Control and COPD GOLD 2

    PubMed Central

    Sze, Marc A.; Abbasi, Meysam; Hogg, James C.; Sin, Don D.

    2014-01-01

    Background Low biomass in the bacterial lung tissue microbiome utilizes quantitative PCR (qPCR) 16S bacterial assays at their limit of detection. New technology like droplet digital PCR (ddPCR) could allow for higher sensitivity and accuracy of quantification. These attributes are needed if specific bacteria within the bacterial lung tissue microbiome are to be evaluated as potential contributors to diseases such as chronic obstructive pulmonary disease (COPD). We hypothesize that ddPCR is better at quantifying the total bacterial load in lung tissue versus qPCR. Methods Control (n = 16) and COPD GOLD 2 (n = 16) tissue samples were obtained from patients who underwent lung resection surgery, were cut on a cryotome, and sections were assigned for use in quantitative histology or for DNA extraction. qPCR and ddPCR were performed on these samples using primers spanning the V2 region on the 16S rRNA gene along with negative controls. Total 16S counts were compared between the two methods. Both methods were assessed for correlations with quantitative histology measurements of the tissue. Results There was no difference in the average total 16S counts (P>0.05) between the two methods. However, the negative controls contained significantly lower counts in the ddPCR (0.55 ± 0.28 16S/uL) than in the qPCR assay (1.00 ± 0.70 16S copies) (P <0.05). The coefficient of variation was significantly lower for the ddPCR assay (0.18 ± 0.14) versus the qPCR assay (0.62 ± 0.29) (P<0.05). Conclusion Overall the ddPCR 16S assay performed better by reducing the background noise in 16S of the negative controls compared with 16S qPCR assay. PMID:25329701

  15. Comparison of Genotypic and Phylogenetic Relationships of Environmental Enterococcus Isolates by BOX-PCR Typing and 16S rRNA Gene Sequencing ▿

    PubMed Central

    Nayak, Bina S.; Badgley, Brian; Harwood, Valerie J.

    2011-01-01

    Environmental Enterococcus spp. were compared by BOX-PCR genotyping and 16S rRNA gene sequencing to clarify the predictive relationship of BOX-PCR fingerprints to species designation. BOX-PCR and 16S rRNA gene relationships agreed for 77% of strains. BOX-PCR provided superior intraspecies discrimination but incorrectly identified some strains to the species level and divided some species into multiple groups. PMID:21622792

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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 ...

  17. Rapid diagnosis of leptospirosis in patients with different clinical manifestations by 16S rRNA gene based nested PCR

    PubMed Central

    Natarajaseenivasan, K.; Raja, V.; Narayanan, R.

    2011-01-01

    Leptospirosis, a zoonosis of global importance and it is underreported in India and more than 50,000 severe cases are reported each year. Here we present the evaluation of 16S rRNA based nested PCR assay for the rapid identification of human leptospires using serum and urine samples. The study includes 261 suspected cases for leptospirosis with different clinical manifestations. 16S rRNA based nested PCR assay was compared and evaluated against the conventional serological methods such as MAT and ELISA. The technique enabled amplification of a 289 bp product with notable percentage of positivity in all sample groups including 94.8 in pediatric cases, 93 in pregnant women, 94.2 in renal failure, 87.8 in jaundice and 94.6 in common febrile cases. The sensitivity and specificity was 94.4% and 100%, respectively. The technique proved to be prompt and effective for the diagnosis of leptospiral infection at the acute phase of the disease. PCR based approach detects leptospiral DNA from the clinical samples both at the acute and leptospiruria phase on comparison with its counter parts where detection is made possible only after 7 days or 7–30 days post-infection. In this regard PCR based diagnosis of leptospirosis should be made available for clinicians for the early diagnosis and prompt treatment of the disease. PMID:23961174

  18. Diversity of 16S-23S rDNA Internal Transcribed Spacer (ITS) Reveals Phylogenetic Relationships in Burkholderia pseudomallei and Its Near-Neighbors

    PubMed Central

    Liguori, Andrew P.; Warrington, Stephanie D.; Ginther, Jennifer L.; Pearson, Talima; Bowers, Jolene; Glass, Mindy B.; Mayo, Mark; Wuthiekanun, Vanaporn; Engelthaler, David; Peacock, Sharon J.; Currie, Bart J.; Wagner, David M.; Keim, Paul; Tuanyok, Apichai

    2011-01-01

    Length polymorphisms within the 16S-23S ribosomal DNA internal transcribed spacer (ITS) have been described as stable genetic markers for studying bacterial phylogenetics. In this study, we used these genetic markers to investigate phylogenetic relationships in Burkholderia pseudomallei and its near-relative species. B. pseudomallei is known as one of the most genetically recombined bacterial species. In silico analysis of multiple B. pseudomallei genomes revealed approximately four homologous rRNA operons and ITS length polymorphisms therein. We characterized ITS distribution using PCR and analyzed via a high-throughput capillary electrophoresis in 1,191 B. pseudomallei strains. Three major ITS types were identified, two of which were commonly found in most B. pseudomallei strains from the endemic areas, whereas the third one was significantly correlated with worldwide sporadic strains. Interestingly, mixtures of the two common ITS types were observed within the same strains, and at a greater incidence in Thailand than Australia suggesting that genetic recombination causes the ITS variation within species, with greater recombination frequency in Thailand. In addition, the B. mallei ITS type was common to B. pseudomallei, providing further support that B. mallei is a clone of B. pseudomallei. Other B. pseudomallei near-neighbors possessed unique and monomorphic ITS types. Our data shed light on evolutionary patterns of B. pseudomallei and its near relative species. PMID:22195045

  19. Species markers for equine strongyles detected in intergenic rDNA by PCR-RFLP.

    PubMed

    Gasser, R B; Stevenson, L A; Chilton, N B; Nansen, P; Bucknell, D G; Beveridge, I

    1996-10-01

    Five species of equine strongyle belonging to the subfamily Strongylinae (Strongylus edentatus, S. equinus, S. vulgaris, Oesophagodontus robustus and Triodontophorus serratus) and 11 species belonging to the subfamily Cyathostominae (Poteriostomum imparidentatum, P. ratzii, Cylicocyclus insignis, Cc. leptostomus, Cc. nassatus, Cylicostephanus calicatus, Cs. longibursatus, Cs. goldi, Cyathostomum catinatum, Cy. labiatum and Cy. pateratum) were characterized using a polymerase chain reaction-linked restriction fragment length polymorphism technique (PCR-RFLP). Internal transcribed spacer ribosomal DNA was amplified from genomic DNA by polymerase chain reaction (PCR) using conserved primers, digested separately with six restriction endonucleases (AluI, BfaI, CfoI, Hae III, VSpI and XbaI) and the fragments separated by agarose gel electrophoresis. The PCR products of the three Strongylus species were approx. 90-100 bp smaller in size compared with those of the other 13 species. The PCR-RFLP analysis of the rDNA region spanning the first and second internal transcribed spacers plus the 5.85 rDNA gene (ITS+) produced characteristic patterns for each of the 16 species examined, and no variation in RFLP patterns was detected within the species Cy. catinatum, where multiple isolates were analysed. The study demonstrates that the internal transcribed spacer sequences provide genetic markers for the species identification of a range of equine strongyles. These markers will be of use for the identification of egg and larval stages, where morphological characters alone are unreliable. The results also indicate that the spacer sequences will be of use to study the systematics of equine strongyles. PMID:8910892

  20. Reassessment of PCR primers targeting 16S rRNA genes of the organohalide-respiring genus Dehalogenimonas.

    PubMed

    Chen, Jie; Bowman, Kimberly S; Rainey, Fred A; Moe, William M

    2014-09-01

    Representatives from the genus Dehalogenimonas have the metabolic capacity to anaerobically transform a variety of environmentally important polychlorinated aliphatic compounds. In light of the recent isolation of additional strains, description of a new species, and an expanded number of uncultured DNA sequences, PCR primers and protocols intended to uniquely target members of this organohalide-respiring genus were reevaluated. Nine of fourteen primer combinations reported previously as genus-specific failed to amplify 16S rRNA genes of recently isolated Dehalogenimonas strains. Use of alternative combinations or modified genus-specific primers, however, allowed detection of all presently known Dehalogenimonas strains. Use of a modified primer set in qPCR revealed an approximately two-order of magnitude increase in concentration of Dehalogenimonas 16S rRNA gene copies following subsurface injection of electron donors at a Louisiana Superfund site, demonstrating the utility of the newly developed protocol and suggesting that the genus Dehalogenimonas can respond to biostimulation remediation strategies in a manner similar to that previously reported for other dechlorinating genera such as Dehalococcoides. PMID:24989478

  1. 16S rRNA PCR-Denaturing Gradient Gel Electrophoresis of Oral Lactobacillus casei Group and Their Phenotypic Appearances

    PubMed Central

    Piwat, S.; Teanpaisan, R.

    2013-01-01

    This study aimed to develop a 16S rRNA PCR-denaturing gradient gel electrophoresis (DGGE) to identify the species level of Lactobacillus casei group and to investigate their characteristics of acid production and inhibitory effect. PCR-DGGE has been developed based on the 16S rRNA gene, and a set of HDA-1-GC and HDA-2, designed at V2-V3 region, and another set of CARP-1-GC and CARP-2, designed at V1 region, have been used. The bacterial strains included L. casei ATCC 393, L. paracasei CCUG 32212, L. rhamnosus ATCC 7469, L. zeae CCUG 35515, and 46 clinical strains of L. casei/paracasei/rhamnosus. Inhibitory effect against Streptococcus mutans and acid production were examined. Results revealed that each type species strain and identified clinical isolate showed its own unique DGGE pattern using CARP1-GC and CARP2 primers. HDA1-GC and HDA2 primers could distinguish the strains of L. paracasei from L. casei. It was found that inhibitory effect of L. paracasei was stronger than L. casei and L. rhamnosus. The acid production of L. paracasei was lower than L. casei and L. rhamnosus. In conclusion, the technique has been proven to be able to differentiate between closely related species in L. casei group and thus provide reliable information of their phenotypic appearances. PMID:24191230

  2. 16S rRNA gene-based identification of microbiota associated with the parthenogenetic troglobiont sand fly Deanemyia maruaga (Diptera, Psychodidae) from central Amazon, Brazil

    PubMed Central

    de Sousa, Katianne Barbosa Alves; da Silva, Túllio Romão Ribeiro; Alencar, Ronildo Baiatone; Baton, Luke Anthony; Naveca, Felipe Gomes; Shimabukuro, Paloma Helena Fernandes

    2013-01-01

    Bacteria associated with the parthenogenetic troglobiont sand fly Deanemyia maruaga were characterized by sequencing cloned 16S rDNA PCR products. Eleven novel partial 16S rDNA sequences, with varying degrees of similarity to Actinobacteria, were identified. None of the sequences identified had homology to those known from parthenogenesis-inducing bacteria. PMID:24159323

  3. Changes in the Composition of Drinking Water Bacterial Clone Libraries Introduced by Using Two Different 16S rRna Gene PCR Primers

    EPA Science Inventory

    Sequence analysis of 16S rRNA gene clone libraries is a popular tool used to describe the composition of natural microbial communities. Commonly, clone libraries are developed by direct cloning of 16S rRNA gene PCR products. Different primers are often employed in the initial amp...

  4. Changes in the Composition of Drinking Water Bacterial Clone Libraries Introduced by Using Two Different 16S rRNA Gene PCR Primers

    EPA Science Inventory

    Sequence analysis of 16S rRNA gene clone libraries is a popular tool used to describe the composition of natural microbial communities. Commonly, clone libraries are developed by direct cloning of 16S rRNA gene PCR products. Different primers are often employed in the initial amp...

  5. Development of a Multiplex PCR Method for Detection of the Genes Encoding 16S rRNA, Coagulase, Methicillin Resistance and Enterotoxins in Staphylococcus aureus

    Technology Transfer Automated Retrieval System (TEKTRAN)

    A multiplex PCR method was developed for simultaneous detection of the genes encoding methicillin resistance (mecA), staphylococcal enterotoxins A, B and C (sea, seb and sec), coagulase (coa) and 16S rRNA. The primers for amplification of the 16S rRNA gene were specific for Staphylococcus spp., and ...

  6. Detection of 16S rDNA of Candidatus Liberibacter asiaticus by quantitative real-time PCR

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Orange juice processed from Huanglongbing (HLB) infected fruit is often associated with bitter taste and/or off-flavor. The widely spread HLB disease in Florida is associated with Candidatus Liberibacter asiaticus (CLas), a phloem limited bacterium. The current standard to diagnose HLB for citrus tr...

  7. Sensitive Detection and Serovar Differentiation of Typhoidal and Nontyphoidal Salmonella enterica Species Using 16S rRNA Gene PCR Coupled with High-Resolution Melt Analysis

    PubMed Central

    Masek, Billie J.; Hardick, Justin; Won, Helen; Yang, Samuel; Hsieh, Yu-Hsiang; Rothman, Richard E.; Gaydos, Charlotte A.

    2015-01-01

    Salmonella enterica species infections are a significant public health problem causing high morbidity rates worldwide and high mortality rates in the developing world. These infections are not always rapidly diagnosed as a cause of bloodstream infections because of the limitations of blood culture, which greatly affects clinical care as a result of treatment delays. A molecular diagnostic assay that could rapidly detect and identify S. enterica species infections as a cause of sepsis is needed. Nine typhoidal and nontyphoidal S. enterica serovars were used to establish the limit of detection (LOD) of a previously published 16S rRNA gene PCR (16S PCR) in mock whole blood specimens. In addition, 16 typhoidal and nontyphoidal S. enterica serovars were used to evaluate the serovar differentiation capability of 16S PCR coupled with high-resolution melt analysis. The overall LOD of 16S PCR for the nine typhoidal and nontyphoidal S. enterica serovars analyzed was <10 colony-forming units per milliliter (CFU/mL) in mock whole blood specimens, with the lowest and highest LOD at <1 CFU/mL and 9 CFU/mL, respectively. By high-resolution melt analysis, the typhoidal and nontyphoidal S. enterica serovar groups analyzed each generated a unique grouping code, allowing for serovar-level identification. 16S PCR coupled with high-resolution melt analysis could be a useful molecular diagnostic that could enhance the current diagnostic, treatment, and surveillance methods of S. enterica bloodstream infections. PMID:24365382

  8. Preliminary evaluation of the use of soil bacterial 16S rDNA DNA markers in sediment fingerprinting in two small endorheic lagoons in southern Spain

    NASA Astrophysics Data System (ADS)

    Gomez, Jose Alfonso; Landa del Castillo, Blanca; Guzman, Gema; Petticrew, Ellen L.; Owens, Phillip N.

    2016-04-01

    bulk community of DNA was extracted from 250 mg of soil samples (three replicates per sample) using the procedure described in Landa et al. (2014). The bacterial 16S rRNA gene V1-V2 hypervariable regions were amplified in polymerase chain reaction (PCR). The sequencing procedure was performed according to the manufacturer's recommendations using MiSeq Reagent Kit v2 for 300 cycles on MiSeq desktop sequencer. The raw dataset for each sample consisted of the number of counts for each of the 6640 operational taxonomic units (OTU) analyzed. All the screening and analysis was performed independently for each lagoon. Given the large number of OTUs, a first screening was made discarding any OTU that did not presented at least five samples with counts >20 for that OTU. This lowered the number of OTUs to 205 in Dulce and 217 in Zoñar. Because of the limited number of samples, we did not perform independent analysis for each soil depth. All the analyses were performed twice; one with the original number of counts and another with the normalized number of counts. We screened the OTU following a 4-step method to determine those with the best ability to discriminate among the three potential source areas. These steps were: 1) eliminate OTUs with no readings or very few, that could be experimental noise; 2) keep only OTUs that are different among source areas; 3) eliminate OTUs that range outside of feasible solutions to explain average values found in sediment; and 4) eliminate OTUs with the largest variability. Afterwards, several over-determined mixing models were solved considering different combinations of OTUs using limSolve (Soetaert et al., 2014) in R. Preliminary results show that 0.2 to 0.6 % of the searched OTUs (i.e. 14 to 42) had the potential for use in the mixing models after the four-step screening process. The results indicate a large variability in the number of counts among the samples from different areas within the subcatchments ranging, on average, from 49 to

  9. Taxonomic identity of type E botulinum toxin-producing Clostridium butyricum strains by sequencing of a short 16S rDNA region.

    PubMed

    Pourshaban, Manoocheher; Franciosa, Giovanna; Fenicia, Lucia; Aureli, Paolo

    2002-08-27

    Several micro-organisms capable of producing botulinum neurotoxin type E, though phenotypically similar to Clostridium butyricum (a normally non-neurotoxigenic organism), have recently been isolated in Italy and China. Some of these micro-organisms had been implicated in food-borne botulism, a serious neuroparalytic disease. The taxonomic identity of the type E botulinum toxin-producing strains is confirmed here, through sequencing of a genus- and species-specific segment of the 16S rRNA gene. Confirmation leads to the conclusion that neurotoxigenic C. butyricum must be regarded as an emergent food-borne pathogen. PMID:12204382

  10. Simultaneous DNA-RNA Extraction from Coastal Sediments and Quantification of 16S rRNA Genes and Transcripts by Real-time PCR

    PubMed Central

    Tatti, Enrico; McKew, Boyd A.; Whitby, Corrine; Smith, Cindy J.

    2016-01-01

    Real Time Polymerase Chain Reaction also known as quantitative PCR (q-PCR) is a widely used tool in microbial ecology to quantify gene abundances of taxonomic and functional groups in environmental samples. Used in combination with a reverse transcriptase reaction (RT-q-PCR), it can also be employed to quantify gene transcripts. q-PCR makes use of highly sensitive fluorescent detection chemistries that allow quantification of PCR amplicons during the exponential phase of the reaction. Therefore, the biases associated with 'end-point' PCR detected in the plateau phase of the PCR reaction are avoided. A protocol to quantify bacterial 16S rRNA genes and transcripts from coastal sediments via real-time PCR is provided. First, a method for the co-extraction of DNA and RNA from coastal sediments, including the additional steps required for the preparation of DNA-free RNA, is outlined. Second, a step-by-step guide for the quantification of 16S rRNA genes and transcripts from the extracted nucleic acids via q-PCR and RT-q-PCR is outlined. This includes details for the construction of DNA and RNA standard curves. Key considerations for the use of RT-q-PCR assays in microbial ecology are included. PMID:27341629

  11. Application of 16s rDNA and cytochrome b ribosomal markers in studies of lineage and fish populations structure of aquatic species.

    PubMed

    Baharum, Syarul Nataqain; Nurdalila, A'wani Aziz

    2012-05-01

    The most economically important form of aquaculture is fish farming, which is an industry that accounts for an ever increasing share of world fishery production. Molecular markers can be used to enhance the productivity of the aquaculture and fish industries to meet the increasing demand. Molecular markers can be identified via a DNA test regardless of the developmental stage, age or environmental challenges experienced by the organism. The application of 16s and cytochrome b markers has enabled rapid progress in investigations of genetic variability and inbreeding, parentage assignments, species and strain identification and the construction of high resolution genetic linkage maps for aquaculture fisheries. In this review, the advantages of principles and potential power tools of 16s and cytochrome b markers are discussed. Main findings in term of trend, aspects and debates on the reviewed issue made from the model of aquatic species for the benefit of aquaculture genomics and aquaculture genetics research are discussed. The concepts in this review are illustrated with various research examples and results that relate theory to reality and provide a strong review of the current status of these biotechnology topics. PMID:22167328

  12. Comparative sequence analyses on the 16S rRNA (rDNA) of Bacillus acidocaldarius, Bacillus acidoterrestris, and Bacillus cycloheptanicus and proposal for creation of a new genus, Alicyclobacillus gen. nov

    NASA Technical Reports Server (NTRS)

    Wisotzkey, J. D.; Jurtshuk, P. Jr; Fox, G. E.; Deinhard, G.; Poralla, K.

    1992-01-01

    Comparative 16S rRNA (rDNA) sequence analyses performed on the thermophilic Bacillus species Bacillus acidocaldarius, Bacillus acidoterrestris, and Bacillus cycloheptanicus revealed that these organisms are sufficiently different from the traditional Bacillus species to warrant reclassification in a new genus, Alicyclobacillus gen. nov. An analysis of 16S rRNA sequences established that these three thermoacidophiles cluster in a group that differs markedly from both the obligately thermophilic organisms Bacillus stearothermophilus and the facultatively thermophilic organism Bacillus coagulans, as well as many other common mesophilic and thermophilic Bacillus species. The thermoacidophilic Bacillus species B. acidocaldarius, B. acidoterrestris, and B. cycloheptanicus also are unique in that they possess omega-alicylic fatty acid as the major natural membranous lipid component, which is a rare phenotype that has not been found in any other Bacillus species characterized to date. This phenotype, along with the 16S rRNA sequence data, suggests that these thermoacidophiles are biochemically and genetically unique and supports the proposal that they should be reclassified in the new genus Alicyclobacillus.

  13. Simple DNA extraction protocol for a 16S rDNA study of bacterial diversity in tropical landfarm soil used for bioremediation of oil waste.

    PubMed

    Maciel, B M; Santos, A C F; Dias, J C T; Vidal, R O; Dias, R J C; Gross, E; Cascardo, J C M; Rezende, R P

    2009-01-01

    Landfarm soil is used to bioremediate oil wastes from petrochemical industries. We developed a simplified protocol for microbial DNA extraction of tropical landfarm soil using only direct lysis of macerated material. Two samples of tropical landfarm soil from a Brazilian refinery were analyzed by this protocol (one consisted of crude oil-contaminated soil; the other was continuously enriched for nine months with petroleum). The soil samples were lysed by maceration with liquid nitrogen, eliminating the need for detergents, organic solvents and enzymatic cell lysis. Then, the DNA from the lysed soil sample was extracted using phenol-chloroform-isoamyl alcohol or guanidium isothiocyanate, giving high DNA yields (more than 1 micro g DNA/g soil) from both soil types. This protocol compared favorably with an established method of DNA template preparation that included mechanical, chemical and enzymatic treatment for cell lysis. The efficiency of this extraction protocol was confirmed by polymerase chain reaction amplification of the 16S rRNA gene, denaturing gradient gel electrophoresis and cloning assays. Fifty-one different clones were obtained; their sequences were classified into at least seven different phyla of the Eubacteria group (Proteobacteria - alpha, gamma and delta, Chloroflexi, Actinobacteria, Acidobac teria, Planctomycetes, Bacteroidetes, and Firmicutes). Forty percent of the sequences could not be classified into these phyla, demonstrating the genetic diversity of this microbial community. Only eight isolates had sequences similar to known sequences of 16S rRNA of cultivable organisms or of known environmental isolates and therefore could be identified to the genus level. This method of DNA extraction is a useful tool for analysis of the bacteria responsible for petroleum degradation in contaminated environments. PMID:19440973

  14. Genus- and Species-Specific PCR-Based Detection of Dairy Propionibacteria in Environmental Samples by Using Primers Targeted to the Genes Encoding 16S rRNA

    PubMed Central

    Rossi, Franca; Torriani, Sandra; Dellaglio, Franco

    1999-01-01

    PCR assays with primers targeted to the genes encoding 16S rRNA were developed for detection of dairy propionibacteria. Propionibacterium thoenii specific oligonucleotide PT3 was selected after partial resequencing. Tests allowed the detection of less than 10 cells per reaction from milk and cheese and 102 cells per reaction from forage and soil. PMID:10473444

  15. Bacterial community profiles on feathers during composting as determined by terminal restriction fragment length polymorphism analysis of 16S rDNA genes.

    PubMed

    Tiquia, S M; Ichida, J M; Keener, H M; Elwell, D L; Burtt, E H; Michel, F C

    2005-05-01

    Composting is one of the more economical and environmentally safe methods of recycling feather waste generated by the poultry industry, since 90% of the feather weight consists of crude keratin protein, and feathers contain 15% N. However, the keratin in waste feathers is resistant to biodegradation and may require the addition of bacterial inocula to enhance the degradation process during composting. Two keratin-degrading bacteria isolated from plumage of wild songbirds and identified as Bacillus licheneformis (OWU 1411T) and Streptomyces sp. (OWU 1441) were inoculated into poultry feather composts (1.13 x 10(8) cfu g(-1) feathers) and co-composted with poultry litter and straw in 200-l compost vessels. Composting temperatures, as well as CO(2) and NH(3) evolution, were measured in these vessels to determine the effects of inoculation on the rate and extent of poultry feather decomposition during composting. Terminal restriction fragment length polymorphisms of 16S rRNA genes were used to follow changes in microbial community structure during composting. The results indicated that extensive carbon conversion occurred in both treatments (55.5 and 56.1%). The addition of the bacterial inocula did not enhance the rate of waste feather composting. The microbial community structure over time was very similar in inoculated and uninoculated waste feather composts. PMID:15614566

  16. Identification of bacteria in a biodegraded wall painting by denaturing gradient gel electrophoresis of PCR-amplified gene fragments coding for 16S rRNA.

    PubMed Central

    Rölleke, S; Muyzer, G; Wawer, C; Wanner, G; Lubitz, W

    1996-01-01

    Medieval wall paintings are often affected by biodecay. An inventory of the existing microorganisms associated with the damage to the paintings is not yet an integral part of the restoration process. This stems from the lack of effective means for such a stocktaking. Nevertheless, fungi and bacteria cause severe damage through mechanical processes from growth into the painting and its grounding and through their metabolism. Detailed information on the bacterial colonization of ancient wall paintings is essential for the protection of the paintings. We used a molecular approach based on the detection and identification of DNA sequences encoding rRNA (rDNA) to identify bacteria present on an ancient wall painting without prior cultivation of the organisms, since it has been shown that most of these bacteria cannot be cultivated under laboratory conditions. To trace the noncultivated fraction of bacteria, total DNA from a biodegraded wall painting sample from a 13th century fresco was extracted and 194-bp fragments of the 16S rDNA were amplified with eubacterial primers. The 16S rDNA fragments of uniform length obtained from the different bacterial species were separated according to their sequence differences by denaturing gradient gel electrophoresis (DGGE). By sequencing excised and reamplified individual DNA bands, we characterized the phylogenetic affiliation of the corresponding bacteria. Using this approach, we identified members or close relatives of the genera Halomonas, Clostridium, and Frankia. To our knowledge, these groups of bacteria have not yet been isolated and implicated by conventional microbiological techniques as contributing to the biodegradation of wall paintings. PMID:8787403

  17. Use of Universal 16S rRNA Gene PCR as a Diagnostic Tool for Venous Access Port-Related Bloodstream Infections

    PubMed Central

    Marín, M.; Martín-Rabadán, P.; Echenagusia, A.; Camúñez, F.; Rodríguez-Rosales, G.; Simó, G.; Echenagusia, M.; Bouza, E.

    2013-01-01

    Amplification of the universal 16S rRNA gene using PCR has improved the diagnostic yield of microbiological samples. However, no data have been reported on the reliability of this technique with venous access ports (VAPs). We assessed the utility of 16S rRNA PCR for the prediction of VAP-related bloodstream infection (VAP-RBSI). During a 2-year period, we prospectively received all VAPs removed by interventional radiologists. PCR and conventional cultures were performed using samples from the different VAP sites. We compared the results of PCR with those of conventional culture for patients with confirmed VAP-RBSI. We collected 219 VAPs from 219 patients. Conventional VAP culture revealed 15 episodes of VAP-RBSI. PCR revealed a further 4 episodes in patients undergoing antibiotic therapy which would have gone undetected using conventional culture. Moreover, it had a negative predictive value of 97.8% for the prediction of VAP-RBSI when it was performed using biofilm from the internal surface of the port. In conclusion, universal 16S rRNA PCR performed with samples from the inside of VAPs proved to be a useful tool for the diagnosis of VAP-RBSI. It increased detection of VAP-RBSI episodes by 21.1% in patients undergoing antibiotic therapy whose episodes would have gone undetected using conventional culture. Therefore, we propose a new application of 16S rRNA PCR as a useful tool for the diagnosis of VAP-RBSI in patients receiving antibiotic therapy. PMID:23254136

  18. Fecal Microbial Diversity in Pre-Weaned Dairy Calves as Described by Pyrosequencing of Metagenomic 16S rDNA. Associations of Faecalibacterium Species with Health and Growth

    PubMed Central

    Oikonomou, Georgios; Teixeira, Andre Gustavo Vieira; Foditsch, Carla; Bicalho, Marcela Lucas; Machado, Vinicius Silva; Bicalho, Rodrigo Carvalho

    2013-01-01

    In this study, we use barcoded pyrosequencing of the 16S rRNA gene to characterize the fecal microbiota of neonatal calves and identify possible relationships of certain microbiota profiles with health and weight gain. Fecal samples were obtained weekly from 61 calves from birth until weaning (seventh week of the calves' life). Firmicutes was the most prevalent phylum, with a prevalence ranging from 63.84% to 81.90%, followed by Bacteroidetes (8.36% to 23.93%), Proteobacteria (3.72% to 9.75%), Fusobacteria (0.76% to 5.67%), and Actinobacteria (1.02% to 2.35%). Chao1 index gradually increased from the first to the seventh postnatal week. Chao1 index was lower during the third, fourth, and fifth week of life in calves that suffered from pneumonia and were treated with antibiotics. Diarrhea incidence during the first four weeks of the calves' life was also associated with a reduction of microbial diversity during the third week of life. Increased fecal microbial diversity after the second week of life was associated with higher weight gain. Using discriminant analysis we were able to show differences in the microbiota profiles between different weeks of life, between high and low weight gain groups of calves, and between calves affected and not affected with diarrhea during the first four weeks life. The prevalence of Faecalibacterium spp. in the first week of life was associated with weight gain and the incidence of diarrhea, with higher prevalence being associated with higher weight gain and less diarrhea. Representative sequences from Faecalibacterium spp. were closely affiliated to Faecalibacterium prausnitzii. Results presented here provide new information regarding the intestinal microbiota of neonatal calves and its association with health and growth. Fecal microbial diversity was associated with calf age, disease status and growth rates. Results suggesting a possible beneficial effect of Faecalibacterium spp. on health and growth are promising. PMID:23646192

  19. Diagnostic genetic markers and evolutionary relationships among invasive dreissenoid and corbiculoid bivalves in North America: phylogenetic signal from mitochondrial 16S rDNA.

    PubMed

    Stepien, C A; Hubers, A N; Skidmore, J L

    1999-10-01

    Diagnostic genetic markers from 486 aligned nucleotide sequences of mitochondrial 16S ribosomal DNA were developed for the four closely related species of dreissenoid and corbiculoid bivalves that have invaded North America; the zebra mussel Dreissena polymorpha, the quagga mussel D. bugensis, and the dark false mussel Mytilopsis leucophaeata of the superfamily Dreissenoidea, and the Asian clam Corbicula fluminea of the sister superfamily Corbiculoidea. Evolutionary relationships were examined among the four genera and comparisons were made with native Eurasian populations of D. polymorpha and D. bugensis. Tests were conducted for gender-specific mitochondrial lineages, which occur in some other bivalves. Genetic variability and divergence rates were tested between stem (paired) and loop (unpaired) regions of secondary structure. There were 251 variable nucleotide sites, of which 99 were phylogenetically informative. Overall transition to transversion ratio was 0.76:1.00 and both accumulated linearly in stem and loop regions, suggesting appropriate phylogenetic signal. Genetic distance calibration with the fossil record estimated the pairwise sequence divergence as 0. 0057 +/- 0.0004 per million years. Mytilopsis and Dreissena appear to have diverged about 20.7 +/- 2.7 million years ago. D. bugensis and D. polymorpha appear separated by about 13.2 +/- 2.2 million years. No intraspecific variation was found, including between Eurasian and North American populations, among shallow and deep morphotypes of D. bugensis and between the sexes. Restriction endonuclease markers were developed to distinguish among the species at all life history stages, allowing rapid identification in areas of sympatric distribution. PMID:10508537

  20. Identification of coagulase-negative staphylococci isolated from ovine milk samples by PCR-RFLP of 16S rRNA and gap genes.

    PubMed

    Onni, T; Sanna, G; Cubeddu, G P; Marogna, G; Lollai, S; Leori, G; Tola, S

    2010-08-26

    The identification of coagulase-negative staphylococci (CNS) causing ovine infections remains problematic, although these bacteria are considered the main etiologic agents of subclinical mastitis in sheep and goats. In this study, 226 CNS isolates were collected from 2201 milking sarda sheep belonging to 15 flocks with high somatic cell count scores. All isolates were subjected to identification with the API Staph ID test, and then to the amplification of staphylococcal 16S rRNA and gap genes by PCR assays. The gap gene was subjected to restriction fragment length polymorphism analysis with the restriction endonuclease AluI, whereas the 16S rRNA gene was subjected to ribosomal fingerprinting with the restriction endonucleases RsaI, PstI and AluI. When PCR-RFLP patterns of CNS isolates were different from those of their reference strains, gap gene amplicons were sequenced for definitive identification. The API Staph ID test, in alternative to the genotypic identification method, produced considerably different results in terms of species identified within each group. Using the PCR-RFLP assay, most of the isolates clustered together with the Staphylococcus epidermidis type strain (131, corresponding to 57.9%), followed by S. caprae (34, corresponding to 15%) and S. chromogenes (30, corresponding to 13.2%). In conclusion, the PCR-RFLP assay of 16S rRNA and gap genes is a more reliable and reproducible method than the API Staph ID test for the identification of CNS causing sheep mastitis. PMID:20167442

  1. Coamplification of eukaryotic DNA with 16S rRNA gene-based PCR primers: possible consequences for population fingerprinting of complex microbial communities.

    PubMed

    Huys, Geert; Vanhoutte, Tom; Joossens, Marie; Mahious, Amal S; De Brandt, Evie; Vermeire, Severine; Swings, Jean

    2008-06-01

    The main aim of this study was to evaluate the specificity of three commonly used 16S rRNA gene-based polymerase chain reaction (PCR) primer sets for bacterial community analysis of samples contaminated with eukaryotic DNA. The specificity of primer sets targeting the V3, V3-V5, and V6-V8 hypervariable regions of the 16S rRNA gene was investigated in silico and by community fingerprinting of human and fish intestinal samples. Both in silico and PCR-based analysis revealed cross-reactivity of the V3 and V3-V5 primers with the 18S rRNA gene of human and sturgeon. The consequences of this primer anomaly were illustrated by denaturing gradient gel electrophoresis (DGGE) profiling of microbial communities in human feces and mixed gut of Siberian sturgeon. DGGE profiling indicated that the cross-reactivity of 16S rRNA gene primers with nontarget eukaryotic DNA might lead to an overestimation of bacterial biodiversity. This study has confirmed previous sporadic indications in literature indicating that several commonly applied 16S rRNA gene primer sets lack specificity toward bacteria in the presence of eukaryotic DNA. The phenomenon of cross-reactivity is a potential source of systematic error in all biodiversity studies where no subsequent analysis of individual community amplicons by cloning and sequencing is performed. PMID:18301945

  2. Telonemia-specific environmental 18S rDNA PCR reveals unknown diversity and multiple marine-freshwater colonizations

    PubMed Central

    2010-01-01

    Background Recent surveys of eukaryote 18S rDNA diversity in marine habitats have uncovered worldwide distribution of the heterotrophic eukaryote phylum Telonemia. Here we investigate the diversity and geographic distribution of Telonemia sequences by in-depth sequencing of several new 18S rDNA clone libraries from both marine and freshwater sites by using a Telonemia-specific PCR strategy. Results In contrast to earlier studies that have employed eukaryote-wide PCR design, we identified a large and unknown diversity of phylotypes and the first rigorous evidence for several freshwater species, altogether comprising 91 unique sequences. Phylogenies of these and publicly available sequences showed 20 statistically supported sub-clades as well as several solitary phylotypes with no clear phylogenetic affiliation. Most of these sub-clades were composed of phylotypes from different geographic regions. Conclusions By using specific PCR primers we reveal a much larger diversity of Telonemia from environmental samples than previously uncovered by eukaryote-wide primers. The new data substantially diminish the geographic structuring of clades identified in earlier studies. Nevertheless, since these clades comprise several distinct phylotypes we cannot exclude endemicity at species level. We identified two freshwater clades and a few solitary phylotypes, implying that Telonemia have colonized freshwater habitats and adapted to the different environmental and ecological conditions at independent occasions. PMID:20534135

  3. Prospective Study of Use of PCR Amplification and Sequencing of 16S Ribosomal DNA from Cerebrospinal Fluid for Diagnosis of Bacterial Meningitis in a Clinical Setting

    PubMed Central

    Schuurman, Tim; de Boer, Richard F.; Kooistra-Smid, Anna M. D.; van Zwet, Anton A.

    2004-01-01

    We have evaluated the use of a broad-range PCR aimed at the 16S rRNA gene in detecting bacterial meningitis in a clinical setting. To achieve a uniform DNA extraction procedure for both gram-positive and gram-negative organisms, a combination of physical disruption (bead beating) and a silica-guanidiniumthiocyanate procedure was used for nucleic acid preparation. To diminish the risk of contamination as much as possible, we chose to amplify almost the entire 16S rRNA gene. The analytical sensitivity of the assay was approximately 1 × 102 to 2 × 102 CFU/ml of cerebrospinal fluid (CSF) for both gram-negative and gram-positive bacteria. In a prospective study of 227 CSF samples, broad-range PCR proved to be superior to conventional methods in detecting bacterial meningitis when antimicrobial therapy had already started. Overall, our assay showed a sensitivity of 86%, a specificity of 97%, a positive predictive value of 80%, and a negative predictive value of 98% compared to culture. We are currently adapting the standard procedures in our laboratory for detecting bacterial meningitis; broad-range 16S ribosomal DNA PCR detection is indicated when antimicrobial therapy has already started at time of lumbar puncture or when cultures remain negative, although the suspicion of bacterial meningitis remains. PMID:14766845

  4. Evaluation of 16S rRNA Gene PCR Sensitivity and Specificity for Diagnosis of Prosthetic Joint Infection: a Prospective Multicenter Cross-Sectional Study

    PubMed Central

    Plouzeau, Chloé; Tande, Didier; Léger, Julie; Giraudeau, Bruno; Valentin, Anne Sophie; Jolivet-Gougeon, Anne; Vincent, Pascal; Corvec, Stéphane; Gibaud, Sophie; Juvin, Marie Emmanuelle; Héry-Arnaud, Genevieve; Lemarié, Carole; Kempf, Marie; Bret, Laurent; Quentin, Roland; Coffre, Carine; de Pinieux, Gonzague; Bernard, Louis; Burucoa, Christophe

    2014-01-01

    There is no standard method for the diagnosis of prosthetic joint infection (PJI). The contribution of 16S rRNA gene PCR sequencing on a routine basis remains to be defined. We performed a prospective multicenter study to assess the contributions of 16S rRNA gene assays in PJI diagnosis. Over a 2-year period, all patients suspected to have PJIs and a few uninfected patients undergoing primary arthroplasty (control group) were included. Five perioperative samples per patient were collected for culture and 16S rRNA gene PCR sequencing and one for histological examination. Three multicenter quality control assays were performed with both DNA extracts and crushed samples. The diagnosis of PJI was based on clinical, bacteriological, and histological criteria, according to Infectious Diseases Society of America guidelines. A molecular diagnosis was modeled on the bacteriological criterion (≥1 positive sample for strict pathogens and ≥2 for commensal skin flora). Molecular data were analyzed according to the diagnosis of PJI. Between December 2010 and March 2012, 264 suspected cases of PJI and 35 control cases were included. PJI was confirmed in 215/264 suspected cases, 192 (89%) with a bacteriological criterion. The PJIs were monomicrobial (163 cases [85%]; staphylococci, n = 108; streptococci, n = 22; Gram-negative bacilli, n = 16; anaerobes, n = 13; others, n = 4) or polymicrobial (29 cases [15%]). The molecular diagnosis was positive in 151/215 confirmed cases of PJI (143 cases with bacteriological PJI documentation and 8 treated cases without bacteriological documentation) and in 2/49 cases without confirmed PJI (sensitivity, 73.3%; specificity, 95.5%). The 16S rRNA gene PCR assay showed a lack of sensitivity in the diagnosis of PJI on a multicenter routine basis. PMID:25056331

  5. Evaluation of 16S rRNA gene PCR sensitivity and specificity for diagnosis of prosthetic joint infection: a prospective multicenter cross-sectional study.

    PubMed

    Bémer, Pascale; Plouzeau, Chloé; Tande, Didier; Léger, Julie; Giraudeau, Bruno; Valentin, Anne Sophie; Jolivet-Gougeon, Anne; Vincent, Pascal; Corvec, Stéphane; Gibaud, Sophie; Juvin, Marie Emmanuelle; Héry-Arnaud, Genevieve; Lemarié, Carole; Kempf, Marie; Bret, Laurent; Quentin, Roland; Coffre, Carine; de Pinieux, Gonzague; Bernard, Louis; Burucoa, Christophe

    2014-10-01

    There is no standard method for the diagnosis of prosthetic joint infection (PJI). The contribution of 16S rRNA gene PCR sequencing on a routine basis remains to be defined. We performed a prospective multicenter study to assess the contributions of 16S rRNA gene assays in PJI diagnosis. Over a 2-year period, all patients suspected to have PJIs and a few uninfected patients undergoing primary arthroplasty (control group) were included. Five perioperative samples per patient were collected for culture and 16S rRNA gene PCR sequencing and one for histological examination. Three multicenter quality control assays were performed with both DNA extracts and crushed samples. The diagnosis of PJI was based on clinical, bacteriological, and histological criteria, according to Infectious Diseases Society of America guidelines. A molecular diagnosis was modeled on the bacteriological criterion (≥ 1 positive sample for strict pathogens and ≥ 2 for commensal skin flora). Molecular data were analyzed according to the diagnosis of PJI. Between December 2010 and March 2012, 264 suspected cases of PJI and 35 control cases were included. PJI was confirmed in 215/264 suspected cases, 192 (89%) with a bacteriological criterion. The PJIs were monomicrobial (163 cases [85%]; staphylococci, n = 108; streptococci, n = 22; Gram-negative bacilli, n = 16; anaerobes, n = 13; others, n = 4) or polymicrobial (29 cases [15%]). The molecular diagnosis was positive in 151/215 confirmed cases of PJI (143 cases with bacteriological PJI documentation and 8 treated cases without bacteriological documentation) and in 2/49 cases without confirmed PJI (sensitivity, 73.3%; specificity, 95.5%). The 16S rRNA gene PCR assay showed a lack of sensitivity in the diagnosis of PJI on a multicenter routine basis. PMID:25056331

  6. Bacterial Diversity and Community Structure in the Pine Wood Nematode Bursaphelenchus xylophilus and B. mucronatus with Different Virulence by High-Throughput Sequencing of the 16S rDNA.

    PubMed

    Xiang, Yang; Wu, Xiao-Qin; Zhou, Ai-Dong

    2015-01-01

    Bursaphelenchus xylophilus is the pathogen of pine wilt disease. Bursaphelenchus mucronatus is similar to B. xylophilus in morphology. Both species share a common niche, but they are quite different in pathogenicity. Presently, the role of bacteria in pine wilt disease development has been widely speculated. The diversity of bacteria associated with B. xylophilus and B. mucronatus with different virulence remains unclear. In this study, virulence of four B. xylophilus and four B. mucronatus strains were evaluated by inoculating Pinus thunbergii. High-throughput sequencing targeted 16S rDNA of different virulence nematode strains was carried out. The associated bacterial community structures of the eight strains were analyzed. The results showed that 634,051 high-quality sequences were obtained from the eight nematode strains. The number of OTUs of bacteria associated with B. mucronatus was generally greater than those of B. xylophilus. The richness of the community of bacteria associated with high virulent B. xylophilus ZL1 and AmA3 was higher than moderately virulent B. xylophilus AA3, HE2, and all B. mucronatus strains. While the diversity of bacteria associated with B. mucronatus was higher than B. xylophilus. Stenotrophomonas, Pseudomonadaceae_Unclassified or Rhizobiaceae_Unclassified were predominant in the nematode strains with different virulence. Oxalobacteraceae and Achromobacter were found more abundant in the low virulent B. xylophilus and non-virulent B. mucronatus strains. PMID:26372013

  7. Bacterial Diversity and Community Structure in the Pine Wood Nematode Bursaphelenchus xylophilus and B. mucronatus with Different Virulence by High-Throughput Sequencing of the 16S rDNA

    PubMed Central

    Xiang, Yang; Wu, Xiao-Qin; Zhou, Ai-Dong

    2015-01-01

    Bursaphelenchus xylophilus is the pathogen of pine wilt disease. Bursaphelenchus mucronatus is similar to B. xylophilus in morphology. Both species share a common niche, but they are quite different in pathogenicity. Presently, the role of bacteria in pine wilt disease development has been widely speculated. The diversity of bacteria associated with B. xylophilus and B. mucronatus with different virulence remains unclear. In this study, virulence of four B. xylophilus and four B. mucronatus strains were evaluated by inoculating Pinus thunbergii. High-throughput sequencing targeted 16S rDNA of different virulence nematode strains was carried out. The associated bacterial community structures of the eight strains were analyzed. The results showed that 634,051 high-quality sequences were obtained from the eight nematode strains. The number of OTUs of bacteria associated with B. mucronatus was generally greater than those of B. xylophilus. The richness of the community of bacteria associated with high virulent B. xylophilus ZL1 and AmA3 was higher than moderately virulent B. xylophilus AA3, HE2, and all B. mucronatus strains. While the diversity of bacteria associated with B. mucronatus was higher than B. xylophilus. Stenotrophomonas, Pseudomonadaceae_Unclassified or Rhizobiaceae_Unclassified were predominant in the nematode strains with different virulence. Oxalobacteraceae and Achromobacter were found more abundant in the low virulent B. xylophilus and non-virulent B. mucronatus strains. PMID:26372013

  8. Who are the active players of the Iberian Margin deep biosphere? Microbial diversity of borehole U1385 through analysis of 16S rDNA and rRNA

    NASA Astrophysics Data System (ADS)

    Russell, J. A.; Orsi, W.; Edgcomb, V. P.; Biddle, J.

    2013-12-01

    Microbial community structure and activity in marine deep subsurface environments across the globe have been assayed using various molecular biology tools including 16S rDNA sequencing, microarrays, FISH/CARD-FISH, and metagenomics. Many studies involving these techniques are DNA-based. This limits study of microbial function in these environments as DNA does not degrade as quickly as RNA and may lead to misinterpreting relic microbial genes as important for present-day activity. In this study, the diversity of bacteria and archaea from sediments of the Iberian Margin IODP borehole U1385 was analyzed from bulk extracted DNA and RNA at seven different depths ranging from 10 to 123 meters below seafloor (mbsf). Presented data suggests that the picture of microbial diversity obtained from DNA is markedly different from that seen through analysis of RNA. IODP borehole U1385 offers a great comparison to ODP Site 1229, a well characterized borehole on the Peru Margin. Similar sediment depositional history and geochemistry will allow exploration of what represents a 'typical' continental margin sediment microbial community or if microbial endemism is established despite similar conditions. This study represents the first molecular exploration of sediment microbial communities from the Iberian Margin IODP Site U1385.

  9. Direct 16S rRNA-seq from bacterial communities: a PCR-independent approach to simultaneously assess microbial diversity and functional activity potential of each taxon.

    PubMed

    Rosselli, Riccardo; Romoli, Ottavia; Vitulo, Nicola; Vezzi, Alessandro; Campanaro, Stefano; de Pascale, Fabio; Schiavon, Riccardo; Tiarca, Maurizio; Poletto, Fabio; Concheri, Giuseppe; Valle, Giorgio; Squartini, Andrea

    2016-01-01

    The analysis of environmental microbial communities has largely relied on a PCR-dependent amplification of genes entailing species identity as 16S rRNA. This approach is susceptible to biases depending on the level of primer matching in different species. Moreover, possible yet-to-discover taxa whose rRNA could differ enough from known ones would not be revealed. DNA-based methods moreover do not provide information on the actual physiological relevance of each taxon within an environment and are affected by the variable number of rRNA operons in different genomes. To overcome these drawbacks we propose an approach of direct sequencing of 16S ribosomal RNA without any primer- or PCR-dependent step. The method was tested on a microbial community developing in an anammox bioreactor sampled at different time-points. A conventional PCR-based amplicon pyrosequencing was run in parallel. The community resulting from direct rRNA sequencing was highly consistent with the known biochemical processes operative in the reactor. As direct rRNA-seq is based not only on taxon abundance but also on physiological activity, no comparison between its results and those from PCR-based approaches can be applied. The novel principle is in this respect proposed not as an alternative but rather as a complementary methodology in microbial community studies. PMID:27577787

  10. Direct 16S rRNA-seq from bacterial communities: a PCR-independent approach to simultaneously assess microbial diversity and functional activity potential of each taxon

    PubMed Central

    Rosselli, Riccardo; Romoli, Ottavia; Vitulo, Nicola; Vezzi, Alessandro; Campanaro, Stefano; de Pascale, Fabio; Schiavon, Riccardo; Tiarca, Maurizio; Poletto, Fabio; Concheri, Giuseppe; Valle, Giorgio; Squartini, Andrea

    2016-01-01

    The analysis of environmental microbial communities has largely relied on a PCR-dependent amplification of genes entailing species identity as 16S rRNA. This approach is susceptible to biases depending on the level of primer matching in different species. Moreover, possible yet-to-discover taxa whose rRNA could differ enough from known ones would not be revealed. DNA-based methods moreover do not provide information on the actual physiological relevance of each taxon within an environment and are affected by the variable number of rRNA operons in different genomes. To overcome these drawbacks we propose an approach of direct sequencing of 16S ribosomal RNA without any primer- or PCR-dependent step. The method was tested on a microbial community developing in an anammox bioreactor sampled at different time-points. A conventional PCR-based amplicon pyrosequencing was run in parallel. The community resulting from direct rRNA sequencing was highly consistent with the known biochemical processes operative in the reactor. As direct rRNA-seq is based not only on taxon abundance but also on physiological activity, no comparison between its results and those from PCR-based approaches can be applied. The novel principle is in this respect proposed not as an alternative but rather as a complementary methodology in microbial community studies. PMID:27577787

  11. PCR assay based on DNA coding for 16S rRNA for detection and identification of mycobacteria in clinical samples.

    PubMed Central

    Kox, L F; van Leeuwen, J; Knijper, S; Jansen, H M; Kolk, A H

    1995-01-01

    A PCR and a reverse cross blot hybridization assay were developed for the detection and identification of mycobacteria in clinical samples. The PCR amplifies a part of the DNA coding for 16S rRNA with a set of primers that is specific for the genus Mycobacterium and that flanks species-specific sequences within the genes coding for 16S rRNA. The PCR product is analyzed in a reverse cross blot hybridization assay with probes specific for M. tuberculosis complex (pTub1), M. avium (pAvi3), M. intracellulare (pInt5 and pInt7), M. kansasii complex-M. scrofulaceum complex (pKan1), M. xenopi (pXen1), M. fortuitum (pFor1), M. smegmatis (pSme1), and Mycobacterium spp. (pMyc5a). The PCR assay can detect 10 fg of DNA, the equivalent of two mycobacteria. The specificities of the probes were tested with 108 mycobacterial strains (33 species) and 31 nonmycobacterial strains (of 17 genera). The probes pAvi3, pInt5, pInt7, pKan1, pXen1, and pMyc5a were specific. With probes pTub1, pFor1, and pSme1, slight cross hybridization occurred. However, the mycobacterial strains from which the cross-hybridizing PCR products were derived belonged to nonpathogenic or nonopportunistic species which do not occur in clinical samples. The test was used on 31 different clinical specimens obtained from patients suspected of having mycobacterial disease, including a patient with a double mycobacterial infection. The samples included sputum, bronchoalveolar lavage, tissue biopsy samples, cerebrospinal fluid, pus, peritoneal fluid, pleural fluid, and blood. The results of the PCR assay agreed with those of conventional identification methods or with clinical data, showing that the test can be used for the direct and rapid detection and identification of mycobacteria in clinical samples. PMID:8586707

  12. PCR-Independent Detection of Bacterial Species-Specific 16S rRNA at 10 fM by a Pore-Blockage Sensor.

    PubMed

    Esfandiari, Leyla; Wang, Siqing; Wang, Siqi; Banda, Anisha; Lorenzini, Michael; Kocharyan, Gayane; Monbouquette, Harold G; Schmidt, Jacob J

    2016-01-01

    A PCR-free, optics-free device is used for the detection of Escherichia coli (E. coli) 16S rRNA at 10 fM, which corresponds to ~100-1000 colony forming units/mL (CFU/mL) depending on cellular rRNA levels. The development of a rapid, sensitive, and cost-effective nucleic acid detection platform is sought for the detection of pathogenic microbes in food, water and body fluids. Since 16S rRNA sequences are species specific and are present at high copy number in viable cells, these nucleic acids offer an attractive target for microbial pathogen detection schemes. Here, target 16S rRNA of E. coli at 10 fM concentration was detected against a total RNA background using a conceptually simple approach based on electromechanical signal transduction, whereby a step change reduction in ionic current through a pore indicates blockage by an electrophoretically mobilized bead-peptide nucleic acid probe conjugate hybridized to target nucleic acid. We investigated the concentration detection limit for bacterial species-specific 16S rRNA at 1 pM to 1 fM and found a limit of detection of 10 fM for our device, which is consistent with our previous finding with single-stranded DNA of similar length. In addition, no false positive responses were obtained with control RNA and no false negatives with target 16S rRNA present down to the limit of detection (LOD) of 10 fM. Thus, this detection scheme shows promise for integration into portable, low-cost systems for rapid detection of pathogenic microbes in food, water and body fluids. PMID:27455337

  13. Direct detection of Brucella spp. in raw milk by PCR and reverse hybridization with 16S-23S rRNA spacer probes.

    PubMed Central

    Rijpens, N P; Jannes, G; Van Asbroeck, M; Rossau, R; Herman, L M

    1996-01-01

    The 16S-23S rRNA spacer regions of Brucella abortus, B. melitensis, and B. suis were cloned and subcloned after PCR amplification. Sequence analysis of the inserts revealed a spacer of about 800 bp with very high ( > 99%) homology among the three species examined. Two genus-specific primer pairs, BRU-P5-BRU-P8 and BRU-P6-BRU-P7, that could be used in a nested PCR format and three genus-specific DNA probes, BRU-ICG2, BRU-ICG3, and BRU-ICG4, were deduced from this spacer. The specificity and sensitivity of both primer sets and probes were examined by testing them against a collection of 18 Brucella strains and 56 strains from other relevant taxa by using PCR and the Line Probe Assay (LiPA), respectively. A method for direct detection of Brucella spp. in 1 ml of raw milk was developed on the basis of enzymatic treatment of the milk components and subsequent PCR and LiPA hybridization. After a single PCR, sensitivities of 2.8 x 10(5) and 2.8 x 10(4) CFU/ml were obtained for detection by agarose gel electrophoresis and LiPA, respectively. Nested PCR yielded a sensitivity of 2.8 x 10(2) CFU/ml for both methods. PMID:8633866

  14. Quantification of Listeria monocytogenes in minimally processed leafy vegetables using a combined method based on enrichment and 16S rRNA real-time PCR.

    PubMed

    Aparecida de Oliveira, Maria; Abeid Ribeiro, Eliana Guimarães; Morato Bergamini, Alzira Maria; Pereira De Martinis, Elaine Cristina

    2010-02-01

    Modern lifestyle markedly changed eating habits worldwide, with an increasing demand for ready-to-eat foods, such as minimally processed fruits and leafy greens. Packaging and storage conditions of those products may favor the growth of psychrotrophic bacteria, including the pathogen Listeria monocytogenes. In this work, minimally processed leafy vegetables samples (n = 162) from retail market from Ribeirão Preto, São Paulo, Brazil, were tested for the presence or absence of Listeria spp. by the immunoassay Listeria Rapid Test, Oxoid. Two L. monocytogenes positive and six artificially contaminated samples of minimally processed leafy vegetables were evaluated by the Most Probable Number (MPN) with detection by classical culture method and also culture method combined with real-time PCR (RTi-PCR) for 16S rRNA genes of L. monocytogenes. Positive MPN enrichment tubes were analyzed by RTi-PCR with primers specific for L. monocytogenes using the commercial preparation ABSOLUTE QPCR SYBR Green Mix (ABgene, UK). Real-time PCR assay presented good exclusivity and inclusivity results and no statistical significant difference was found in comparison with the conventional culture method (p < 0.05). Moreover, RTi-PCR was fast and easy to perform, with MPN results obtained in ca. 48 h for RTi-PCR in comparison to 7 days for conventional method. PMID:19913686

  15. Quantitative identification of fecal water pollution sources by TaqMan real-time PCR assays using Bacteroidales 16S rRNA genetic markers.

    PubMed

    Lee, Dae-Young; Weir, Susan C; Lee, Hung; Trevors, Jack T

    2010-12-01

    PCR-based analysis of Bacteroidales 16S rRNA genes has emerged as a promising tool to identify sources of fecal water pollution. In this study, three TaqMan real-time PCR assays (BacGeneral, BacHuman, and BacBovine) were developed and evaluated for their ability to quantitatively detect general (total), human-specific, and bovine-specific Bacteroidales 16S rRNA genetic markers. The detection sensitivity was determined to be 6.5 copies of 16S rRNA gene for the BacGeneral and BacHuman assays and 10 copies for the BacBovine assay. The assays were capable of detecting approximately one to two cells per PCR. When tested with 70 fecal samples from various sources (human, cattle, pig, deer, dog, cat, goose, gull, horse, and raccoon), the three assays positively identified the target markers in all samples without any false-negative results. The BacHuman and BacBovine assays exhibited false-positive reactions with non-target samples in a few cases. However, the level of the false-positive reactions was about 50 times smaller than that of the true-positive ones, and therefore, these cross-reactions were unlikely to cause misidentifications of the fecal pollution sources. Microbial source-tracking capability was tested at two freshwater streams of which water quality was influenced by human and cattle feces, respectively. The assays accurately detected the presence of the corresponding host-specific markers upon fecal pollution and the persistence of the markers in downstream areas. The assays are expected to reliably determine human and bovine fecal pollution sources in environmental water samples. PMID:20871990

  16. Renibacterium salmoninarum, the causative agent of bacterial kidney disease in salmonid fish, detected by nested reverse transcription-PCR of 16S rRNA sequences.

    PubMed Central

    Magnússon, H B; Fridjónsson, O H; Andrésson, O S; Benediktsdóttir, E; Gudmundsdóttir, S; Andrésdóttir, V

    1994-01-01

    An assay based on reverse transcription and nested PCR amplification of hypervariable regions within the 16S rRNA sequence was used to specifically detect Renibacterium salmoninarum, the slowly growing causative agent of bacterial kidney disease in salmonid fish. This assay detected 1 to 10 bacteria per sample and took 1 to 2 days to perform. The assay was used to detect R. salmoninarum in ovarian fluid obtained from naturally infected fish. The assay was unreliable when it was used to examine kidney tissue. Images PMID:7529017

  17. Renibacterium salmoninarum, the causative agent of bacterial kidney disease in salmonid fish, detected by nested reverse transcription-PCR of 16S rRNA sequences.

    PubMed

    Magnússon, H B; Fridjónsson, O H; Andrésson, O S; Benediktsdóttir, E; Gudmundsdóttir, S; Andrésdóttir, V

    1994-12-01

    An assay based on reverse transcription and nested PCR amplification of hypervariable regions within the 16S rRNA sequence was used to specifically detect Renibacterium salmoninarum, the slowly growing causative agent of bacterial kidney disease in salmonid fish. This assay detected 1 to 10 bacteria per sample and took 1 to 2 days to perform. The assay was used to detect R. salmoninarum in ovarian fluid obtained from naturally infected fish. The assay was unreliable when it was used to examine kidney tissue. PMID:7529017

  18. Detection of Renibacterium salmoninarum in tissue samples by sequence capture and fluorescent PCR based on the 16S rRNA gene.

    PubMed

    Königsson, Malin Heldtander; Ballagi, Andras; Jansson, Eva; Johansson, Karl-Erik

    2005-02-25

    The 16S rRNA genes from eight isolates of Renibacterium salmoninarum with different origins and dates of isolation were sequenced to evaluate the possibility to construct a diagnostic PCR system with target sites within this gene. The sequences were found to be identical but for one single position in one of the isolates, and two regions with an adequate number of nucleotide differences as compared to closely related species were identified. Species-specific fluorescent PCR primers complementary to these regions were constructed as well as oligonucleotides for DNA preparation by sequence capture. A mimic molecule was constructed to be used as an internal control. The PCR was specific and allowed the detection of DNA equivalent to 1-10 R. salmoninarum genomes per reaction. The DNA preparation with sequence capture and analysis by PCR with a mimic was found to be a reliable method for analysis of kidneys from fish with BKD. The amount of PCR inhibiting substances present in the tissue was reduced, and the relevant DNA was concentrated in the capture step. Furthermore, the use of the mimic molecule in the system assured that false negative results could be identified. PMID:15708821

  19. A report of cat scratch disease in Korea confirmed by PCR amplification of the 16S-23S rRNA intergenic region of Bartonella henselae.

    PubMed

    Suh, Borum; Chun, Jin-Kyoung; Yong, Dongeun; Lee, Yang Soon; Jeong, Seok Hoon; Yang, Woo Ick; Kim, Dong Soo

    2010-02-01

    We report a case of cat scratch disease in an 8-yr-old girl who presented with fever and enlargement of both axillary lymph nodes. Both aerobic and anaerobic cultures of the lymph node aspirate were negative for microbial growth. Gram staining and Warthin-Starry silver staining did not reveal any organism. Purified DNA from the PCR-amplicon of the 16S-23S rRNA intergenic region was sequenced and showed 99.7% identity with the corresponding sequence of Bartonella henselae strain Houston-1. Our findings suggest that the internal transcribed spacer is a reliable region for PCR identification of Bartonella species. In patients with lymphadenitis, a history of contact with cats or dogs necessitates the use of diagnostic approaches that employ not only the conventional staining and culture but also molecular methods to detect B. henselae. PMID:20197720

  20. Comparison of two poultry litter qPCR assays targeting the 16S rRNA gene of Brevibacterium sp.

    PubMed

    Ryu, Hodon; Elk, Michael; Khan, Izhar U H; Harwood, Valerie J; Molina, Marirosa; Edge, Thomas A; Domingo, Jorge Santo

    2014-01-01

    Chicken feces commonly contain human pathogens and are also important sources of fecal pollution in environmental waters. Consequently, methods that can detect chicken fecal pollution are needed in public health and environmental monitoring studies. In this study, we compared a previously developed SYBR green qPCR assay (LA35) to a novel TaqMan qPCR assay (CL) for the environmental detection of poultry-associated fecal pollution. We tested both assays against chicken litter (n = 40), chicken fecal samples (n = 186), non-chicken fecal sources (n = 484), and environmental water samples (n = 323). Most chicken litter samples (i.e., ≥ 98%) were positive for both assays with relatively high signal intensities, whereas only 23% and 12% of poultry fecal samples (n = 186) were positive with the LA35 and the CL assays, respectively. Data using fecal samples from non-target animal species showed that the assays are highly host-associated (≥ 95%). Bayesian statistical models showed that the two assays are associated with relatively low probability of false-positive and false-negative signals in water samples. The CL marker had a lower prevalence than the LA35 assay when tested against environmental water samples (i.e., 21% vs. 31% positive signals). However, by combining the results from the two assays the detection levels increased to 41%, suggesting that using multiple assays can improve the detection of chicken-fecal pollution in environmental waters. PMID:24169514

  1. Genetic diversity of Datisca cannabina-compatible Frankia strains as determined by sequence analysis of the PCR-amplified 16S rRNA gene.

    PubMed Central

    Mirza, M S; Hameed, S; Akkermans, A D

    1994-01-01

    The presence of Frankia strains in soil samples collected from northern areas of Pakistan was detected by inoculating Coriaria nepalensis and Datisca cannabina plants. The abundance of compatible Frankia strains in some areas was indicated by profuse nodulation of the host plants, whereas soil samples from other localities failed to result in nodulation. An oligonucleotide probe (COR/DAT) directed against the 16S rRNA gene of the endophytes of Coriaria and Datisca spp. that did not cross-react with the RNA gene of Frankia strains isolated from other hosts was developed. Genetic diversity among Frankia strains nodulating D. cannabina was determined by sequence analysis of the partial 16S rRNA gene amplified from nodules induced by soil samples from different localities by PCR. Four types of Frankia sequences and one non-Frankia sequence were detected by hybridization with a Frankia genus probe and the COR/DAT probe as well as by sequence analysis of the cloned PCR products. Images PMID:7521157

  2. Seasonal and regional diversity of maple sap microbiota revealed using community PCR fingerprinting and 16S rRNA gene clone libraries.

    PubMed

    Filteau, Marie; Lagacé, Luc; LaPointe, Gisèle; Roy, Denis

    2010-04-01

    An arbitrary primed community PCR fingerprinting technique based on capillary electrophoresis was developed to study maple sap microbial community characteristics among 19 production sites in Québec over the tapping season. Presumptive fragment identification was made with corresponding fingerprint profiles of bacterial isolate cultures. Maple sap microbial communities were subsequently compared using a representative subset of 13 16S rRNA gene clone libraries followed by gene sequence analysis. Results from both methods indicated that all maple sap production sites and flow periods shared common microbiota members, but distinctive features also existed. Changes over the season in relative abundance of predominant populations showed evidence of a common pattern. Pseudomonas (64%) and Rahnella (8%) were the most abundantly and frequently represented genera of the 2239 sequences analyzed. Janthinobacterium, Leuconostoc, Lactococcus, Weissella, Epilithonimonas and Sphingomonas were revealed as occasional contaminants in maple sap. Maple sap microbiota showed a low level of deep diversity along with a high variation of similar 16S rRNA gene sequences within the Pseudomonas genus. Predominance of Pseudomonas is suggested as a typical feature of maple sap microbiota across geographical regions, production sites, and sap flow periods. PMID:20202776

  3. Microdiversity of Deep-Sea Bacillales Isolated from Tyrrhenian Sea Sediments as Revealed by ARISA, 16S rRNA Gene Sequencing and BOX-PCR Fingerprinting

    PubMed Central

    Ettoumi, Besma; Guesmi, Amel; Brusetti, Lorenzo; Borin, Sara; Najjari, Afef; Boudabous, Abdellatif; Cherif, Ameur

    2013-01-01

    With respect to their terrestrial relatives, marine Bacillales have not been sufficiently investigated. In this report, the diversity of deep-sea Bacillales, isolated from seamount and non-seamount stations at 3,425 to 3,580 m depth in the Tyrrhenian Sea, was investigated using PCR fingerprinting and 16S rRNA sequence analysis. The isolate collection (n=120) was de-replicated by automated ribosomal intergenic spacer analysis (ARISA), and phylogenetic diversity was analyzed by 16S rRNA gene sequencing of representatives of each ARISA haplotype (n=37). Phylogenetic analysis of isolates showed their affiliation to six different genera of low G+C% content Gram-positive Bacillales: Bacillus, Staphylococcus, Exiguobacterium, Paenibacillus, Lysinibacillus and Terribacillus. Bacillus was the dominant genus represented by the species B. licheniformis, B. pumilus, B. subtilis, B. amyloliquefaciens and B. firmus, typically isolated from marine sediments. The most abundant species in the collection was B. licheniformis (n=85), which showed seven distinct ARISA haplotypes with haplotype H8 being the most dominant since it was identified by 63 isolates. The application of BOX-PCR fingerprinting to the B. licheniformis sub-collection allowed their separation into five distinct BOX genotypes, suggesting a high level of intraspecies diversity among marine B. licheniformis strains. This species also exhibited distinct strain distribution between seamount and non-seamount stations and was shown to be highly prevalent in non-seamount stations. This study revealed the great microdiversity of marine Bacillales and contributes to understanding the biogeographic distribution of marine bacteria in deep-sea sediments. PMID:24005887

  4. Microdiversity of deep-sea Bacillales isolated from Tyrrhenian sea sediments as revealed by ARISA, 16S rRNA gene sequencing and BOX-PCR fingerprinting.

    PubMed

    Ettoumi, Besma; Guesmi, Amel; Brusetti, Lorenzo; Borin, Sara; Najjari, Afef; Boudabous, Abdellatif; Cherif, Ameur

    2013-01-01

    With respect to their terrestrial relatives, marine Bacillales have not been sufficiently investigated. In this report, the diversity of deep-sea Bacillales, isolated from seamount and non-seamount stations at 3,425 to 3,580 m depth in the Tyrrhenian Sea, was investigated using PCR fingerprinting and 16S rRNA sequence analysis. The isolate collection (n=120) was de-replicated by automated ribosomal intergenic spacer analysis (ARISA), and phylogenetic diversity was analyzed by 16S rRNA gene sequencing of representatives of each ARISA haplotype (n=37). Phylogenetic analysis of isolates showed their affiliation to six different genera of low G+C% content Gram-positive Bacillales: Bacillus, Staphylococcus, Exiguobacterium, Paenibacillus, Lysinibacillus and Terribacillus. Bacillus was the dominant genus represented by the species B. licheniformis, B. pumilus, B. subtilis, B. amyloliquefaciens and B. firmus, typically isolated from marine sediments. The most abundant species in the collection was B. licheniformis (n=85), which showed seven distinct ARISA haplotypes with haplotype H8 being the most dominant since it was identified by 63 isolates. The application of BOX-PCR fingerprinting to the B. licheniformis sub-collection allowed their separation into five distinct BOX genotypes, suggesting a high level of intraspecies diversity among marine B. licheniformis strains. This species also exhibited distinct strain distribution between seamount and non-seamount stations and was shown to be highly prevalent in non-seamount stations. This study revealed the great microdiversity of marine Bacillales and contributes to understanding the biogeographic distribution of marine bacteria in deep-sea sediments. PMID:24005887

  5. MULTIPLE ENZYME RESTRICTION FRAGMENT LENGTH POLYMORPHISM ANALYSIS FOR HIGH RESOLUTION DISTINCTION OF PSEUDOMONAS (SENSU STRICTO) 16S RRNA GENES

    EPA Science Inventory

    Pseudomonas specific 16S rDNA PCR amplification and multiple enzyme restriction fragment length polymorphism (MERFLP) analysis using a single digestion mixture of Alu I, Hinf I, Rsa I, and Tru 9I distinguished 150 published sequences and reference strains of authentic Pseudomonas...

  6. Molecular analysis of 16S-23S spacer regions of Acetobacter species.

    PubMed

    Kretová, M; Grones, J

    2005-01-01

    16S-23S rDNA internal transcribed spacer regions (ITS) similarities were determined in 8 Acetobacter and 1 Gluconacetobacter strains. ITS-PCR amplification of the 16S-23S spacers showed 2 products of similar size in 7 strains; only 1 product of similar size was found in the 2 remaining strains. Analysis of the PCR products using restriction endonucleases HaeIII, HpaII and AluI revealed 3 different restriction groups of A. pasteurianus for AluI and HaeIII, and 4 restriction groups for HpaII. ITS nucleotide sequences of all studied strains exhibited a 52-98% similarity. PMID:16408846

  7. Epidemiologic Study of Malassezia Yeasts in Patients with Malassezia Folliculitis by 26S rDNA PCR-RFLP Analysis

    PubMed Central

    Ko, Jong Hyun; Choe, Yong Beom; Ahn, Kyu Joong

    2011-01-01

    Background So far, studies on the inter-relationship between Malassezia and Malassezia folliculitis have been rather scarce. Objective We sought to analyze the differences in body sites, gender and age groups, and to determine whether there is a relationship between certain types of Malassezia species and Malassezia folliculitis. Methods Specimens were taken from the forehead, cheek and chest of 60 patients with Malassezia folliculitis and from the normal skin of 60 age- and gender-matched healthy controls by 26S rDNA PCR-RFLP. Results M. restricta was dominant in the patients with Malassezia folliculitis (20.6%), while M. globosa was the most common species (26.7%) in the controls. The rate of identification was the highest in the teens for the patient group, whereas it was the highest in the thirties for the control group. M. globosa was the most predominant species on the chest with 13 cases (21.7%), and M. restricta was the most commonly identified species, with 17 (28.3%) and 12 (20%) cases on the forehead and cheek, respectively, for the patient group. Conclusion Statistically significant differences were observed between the patient and control groups for the people in their teens and twenties, and in terms of the body site, on the forehead only. PMID:21747616

  8. Epidemiologic Study of Malassezia Yeasts in Acne Patients by Analysis of 26S rDNA PCR-RFLP

    PubMed Central

    Song, Young Chan; Hahn, Hyung Jin; Kim, Ji Young; Ko, Jong Hyun; Choe, Yong Beom; Ahn, Kyu Joong

    2011-01-01

    Background Although acne is a common follicular inflammatory dermatosis, studies of the relationship between Malassezia yeasts and acne have rarely been conducted. Objective We sought to identify Malassezia yeasts from acne patients and establish a relationship between specific types of species of Malassezia and acne. Methods Sixty acne patients were enrolled. Each strain obtained was identified as one of eleven species by 26S rDNA PCR-RFLP. We then compared these data with those of age- and sex-matched healthy subjects. Results Growth of Malassezia was evident in fewer patients with acne (50%) in comparison to controls (70.6%). M. restricta was dominant in patients with acne (23.9%), whereas M. globosa was most common (26.7%) in healthy controls. In the patients group, the rate was the highest (71.7%) in the twenties and, in terms of body site, the rate was the highest (60%) in the chest. In the control group, the rate was the highest (75.0%) in the thirties and in the forehead (85.0%). Conclusion The detection rate of Malassezia yeasts was conspicuously low in the acne patients group. Statistically significant differences were observed between the patient and the control groups in the twenties and thirties, and in terms of body site, in the forehead and chest. PMID:21909202

  9. Analyses of methanogenic archaea populations in swine feces and stored swine manure using 16S rDNA and mcrA PCR and pure culture isolation

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Background: Storage of swine manure is associated with the microbial production of odorous compounds and gaseous emissions which result from anaerobic microbial digestion of materials present in the manure. In the United States, methane emissions from lagoons and manure storage pits are estimated to...

  10. PCR primers and probes for the 16S rRNA gene of most species of pathogenic bacteria, including bacteria found in cerebrospinal fluid.

    PubMed Central

    Greisen, K; Loeffelholz, M; Purohit, A; Leong, D

    1994-01-01

    A set of broad-range PCR primers for the 16S rRNA gene in bacteria were tested, along with three series of oligonucleotide probes to detect the PCR product. The first series of probes is broad in range and consists of a universal bacterial probe, a gram-positive probe, a Bacteroides-Flavobacterium probe, and two probes for other gram-negative species. The second series was designed to detect PCR products from seven major bacterial species or groups frequently causing meningitis: Neisseria meningitidis, Haemophilus influenzae, Streptococcus pneumoniae, S. agalactiae, Escherichia coli and other enteric bacteria, Listeria monocytogenes, and Staphylococcus aureus. The third series was designed for the detection of DNA from species or genera commonly considered potential contaminants of clinical samples, including cerebrospinal fluid (CSF): Bacillus, Corynebacterium, Propionibacterium, and coagulase-negative Staphylococcus spp. The primers amplified DNA from all 124 different species of bacteria tested. Southern hybridization testing of the broad-range probes with washes containing 3 M tetramethylammonium chloride indicated that this set of probes correctly identified all but two of the 102 bacterial species tested, the exceptions being Deinococcus radiopugnans and Gardnerella vaginalis. The gram-negative and gram-positive probes hybridized to isolates of two newly characterized bacteria, Alloiococcus otitis and Rochalimaea henselii, as predicted by Gram stain characteristics. The CSF pathogen and contaminant probe sequences were compared with available sequence information and with sequencing data for 32 different species. Testing of the CSF pathogen and contaminant probes against DNA from over 60 different strains indicated that, with the exception of the coagulase-negative Staphylococcus probes, these probes provided the correct identification of bacterial species known to be found in CSF. Images PMID:7512093

  11. Improved Multiplex PCR Using Conserved and Species-Specific 16S rRNA Gene Primers for Simultaneous Detection of Actinobacillus actinomycetemcomitans, Bacteroides forsythus, and Porphyromonas gingivalis

    PubMed Central

    Tran, Simon Dangtuan; Rudney, Joel D.

    1999-01-01

    Among putative periodontal pathogens, Actinobacillus actinomycetemcomitans, Bacteroides forsythus, and Porphyromonas gingivalis are most convincingly implicated as etiological agents in periodontitis. Therefore, techniques for detection of those three species would be of value. We previously published a description of a multiplex PCR that detects A. actinomycetemcomitans and P. gingivalis. The present paper presents an improvement on that technique, which now allows more sensitive detection of all three periodontal pathogens. Sensitivity was determined by testing serial dilutions of A. actinomycetemcomitans, B. forsythus, and P. gingivalis cells. Primer specificity was tested against (i) all gene sequences from the GenBank-EMBL database, (ii) six A. actinomycetemcomitans, one B. forsythus, and four P. gingivalis strains, (iii) eight different species of oral bacteria, and (iv) supra- and subgingival plaque samples from 20 healthy subjects and subgingival plaque samples from 10 patients with periodontitis. The multiplex PCR had a detection limit of 10 A. actinomycetemcomitans, 10 P. gingivalis, and 100 B. forsythus cells. Specificity was confirmed by the fact that (i) none of our forward primers were homologous to the 16S rRNA genes of other oral species, (ii) amplicons of predicted size were detected for all A. actinomycetemcomitans, B. forsythus, and P. gingivalis strains tested, and (iii) no amplicons were detected for the eight other bacterial species. A. actinomycetemcomitans, B. forsythus, and P. gingivalis were detected in 6 of 20, 1 of 20, and 11 of 20 of supragingival plaque samples, respectively, and 4 of 20, 7 of 20, and 13 of 20 of subgingival plaque samples, respectively, from periodontally healthy subjects. Among patients with periodontitis, the organisms were detected in 7 of 10, 10 of 10, and 7 of 10 samples, respectively. The simultaneous detection of three periodontal pathogens is an advantage of this technique over conventional PCR assays. PMID

  12. Genus-specific primers targeting the 16S rRNA gene for PCR detection of members of the genus Verrucosispora.

    PubMed

    Xie, Qingyi; Hong, Kui; Goodfellow, Michael

    2011-06-01

    Little is known about the genus Verrucosispora though it does contain organisms which produce novel antibiotics. A set of genus-specific oligonucleotide primers was generated to gain an insight into the presence, distribution and taxonomic diversity of members of this genus in diverse samples taken from marine habitats. In silico and pure culture studies showed that the primers matched perfectly with target sequences of the 16S rRNA genes of representatives of the genus Verrucosispora. The primers, designated S-G-Verr-0195-a-S-20 and S-G-Verr-1152-a-A-18, amplified an ≈960 bp stretch of the 16S rRNA genes of Verrucosispora strains but not those of representatives of other genera classified in the family Micromonosporaceae. Genus-specific amplicons were detected from 17 out of 20 community DNA samples prepared from diverse marine sediments and coastal soils. Phylogenetic analysis of over 40% of clones derived from five of the samples indicated they belonged to novel Verrucosispora species. The primers were also used to confirm the identity of Verrucosispora-like strains isolated from two of the environmental samples. The primers can be used to facilitate the isolation of novel Verrucosispora strains by allowing prescreening of environmental samples and the subsequent identification of verrucosisporae on selective isolation plates. For this purpose, a novel medium facilitating the recovery of Verrucosispora strains was formulated and used to recover novel isolates validated using the novel PCR primers. This medium may be useful as the basis for development of a selective medium. PMID:21374042

  13. Molecular analysis of biofilms on the surface of neonatal endotracheal tubes based on 16S rRNA PCR-DGGE and species-specific PCR.

    PubMed

    Li, Hongdong; Song, Chao; Liu, Dong; Ai, Qing; Yu, Jialin

    2015-01-01

    Ventilator-associated pneumonia (VAP) results in considerable morbidity and mortality in neonatal intensive care units. VAP is associated with polymicrobial biofilms that form on endotracheal tubes (ETTs). We aimed to evaluate the diversity and the bacterial community in biofilms on ETTs extubated from mechanically ventilated newborns. ETTs (N = 29) and aerobic sputum cultures were obtained from 20 mechanically ventilated newborns. Denaturing gradient gel electrophoresis (DGGE) was used to characterize the bacterial species in the biofilms on the ETTs. Species-specific PCR was used to detect common oropharyngeal Streptococcus species and known ETT-associated pathogens. DGGE profiling of ETT biofilms showed multiple banding patterns indicating a diverse bacterial community. The dominant bacterial species were Klebsiella spp. (29/29), Streptococcus spp. (27/29), and Pseudomonas spp. (24/29). The most frequently occurring Streptococcus species was Streptococcus mitis (N = 18). Oropharyngeal bacteria were present in 25 of 29 ETT specimens. Streptococcus spp. often co-existed with K. pneumoniae and/or P. aeruginosa. In contrast, only one bacterial species was isolated from each sputum culture, K. pneumoniae or Acinetobacter baumannii. Our results demonstrated that Klebsiella spp., Streptococcus spp., and Pseudomonas spp. were the most frequent microbes on the surface of neonatal ETTs. The co-existence of oral commensals and pathogenic bacteria on the same tubes may play a crucial role for biofilm formation. PMID:26379907

  14. Identification and quantification of Bifidobacterium species isolated from food with genus-specific 16S rRNA-targeted probes by colony hybridization and PCR.

    PubMed Central

    Kaufmann, P; Pfefferkorn, A; Teuber, M; Meile, L

    1997-01-01

    A Bifidobacterium genus-specific target sequence in the V9 variable region of the 16S rRNA has been elaborated and was used to develop a hybridization probe. The specificity of this probe, named lm3 (5'-CGGGTGCTI*CCCACTTTCATG-3'), was used to identify all known type strains and distinguish them from other bacteria. All of the 30 type strains of Bifidobacterium which are available at the German culture collection Deutsche Sammlung von Mikroorganismen und Zellkulturen, 6 commercially available production strains, and 34 closely related relevant strains (as negative controls) were tested. All tested bifidobacteria showed distinct positive signals by colony hybridization, whereas all negative controls showed no distinct dots except Gardnerella vaginalis DSM4944 and Propionibacterium freudenreichii subsp. shermanii DSM4902, which gave slight signals. Furthermore, we established a method for isolation and identification of bifidobacteria from food by using a PCR assay without prior isolation of DNA but breaking the cells with proteinase K. By this method, all Bifidobacterium strains lead to a DNA product of the expected size. We also established a quick assay to quantitatively measure Bifidobacterium counts in food and feces by dilution plating and colony hybridization. We were able to demonstrate that 2.1 x 10(6) to 2.3 x 10(7) colonies/g of sour milk containing bifidobacteria hybridized with the specific nucleotide probe. With these two methods, genus-specific colony hybridization and genus-specific PCR, it is now possible to readily and accurately detect any bifidobacteria in food and fecal samples and to discriminate between them and members of other genera. PMID:9097423

  15. Distribution and diversity of bacteria in a saline meromictic lake as determined by PCR-DGGE of 16S rRNA gene fragments.

    PubMed

    Gugliandolo, Concetta; Lentini, Valeria; Maugeri, Teresa L

    2011-01-01

    The variations in vertical distribution and composition of bacteria in the meromictic Lake Faro (Messina, Italy) were analysed by culture-independent methods in two different mixing conditions. Water samples were collected from a central station from the surface to the bottom (30 m depth) on two different sampling dates--the first characterised by a well-mixed water mass and the second by a marked stratification. A 'red-water' layer, caused by a dense growth of photosynthetic sulphur bacteria, was present at a depth of 25 m in December 2005 and at 15 m in August 2006, defining two different zones in terms of their physicochemical properties. The vertical distribution of bacterioplankton showed that the interface zones were more densely populated than others. In both sampling periods, the highest numbers of live cells were observed within 'red water' layers. The dominant phylotypes of the bacterial community were determined by sequencing the Denaturing Gradient Gel Electrophoresis (DGGE) bands resulting from PCR amplification of 16S rRNA gene fragments. The number of DGGE bands, considered indicative of the total species richness, did not vary predictably across the two different sampling periods. Proteobacteria (α-, γ-, δ- and ε subclass members), Cytophaga-Flavobacterium-Bacteroides, green sulphur bacteria and Cyanobacteria were retrieved from Lake Faro. Most of the bands showed DNA sequences that did not match with other previously described organisms, suggesting the presence of new indigenous bacterial phylotypes. PMID:20544199

  16. Distribution of bacterioplankton in meromictic Lake Saelenvannet, as determined by denaturing gradient gel electrophoresis of PCR-amplified gene fragments coding for 16S rRNA.

    PubMed Central

    Ovreås, L; Forney, L; Daae, F L; Torsvik, V

    1997-01-01

    The community structure of bacterioplankton in meromictic Lake Saelenvannet was examined by PCR amplification of the V3 region of 16S rRNA from microbial communities recovered from various depths in the water column. Two different primer sets were used, one for amplification of DNA from the domain Bacteria and another specific for DNA from the domain Archaea. Amplified DNA fragments were resolved by denaturing gradient gel electrophoresis (DGGE), and the resulting profiles were reproducible and specific for the communities from different depths. Bacterial diversity estimated from the number and intensity of specific fragments in DGGE profiles decreased with depth. The reverse was true for the Archaea, with the diversity increasing with depth. Hybridization of DGGE profiles with oligonucleotide probes specific for phylogenetic groups of microorganisms showed the presence of both sulfate-reducing bacteria and methanogens throughout the water column, but they appeared to be most abundant below the chemocline. Several dominant fragments in the DGGE profiles were excised and sequenced. Among the dominant populations were representatives related to Chlorobium phaeovibrioides, chloroplasts from eukaryotic algae, and unidentified Archaea. PMID:9292986

  17. Molecular characterization of Mycobacterium avium complex isolates giving discordant results in AccuProbe tests by PCR-restriction enzyme analysis, 16S rRNA gene sequencing, and DT1-DT6 PCR.

    PubMed Central

    Devallois, A; Picardeau, M; Paramasivan, C N; Vincent, V; Rastogi, N

    1997-01-01

    Based on cultural and biochemical tests, a total of 84 strains (72 clinical and 12 environmental isolates from the Caribbean Isles, Europe, and the Indian subcontinent) were identified as members of the Mycobacterium avium complex (MAC). They were further characterized with MAC, M. avium, and M. intracellulare probes of the AccuProbe system, and this was followed by selective amplification of DT6 and DT1 sequences. Seventy isolates gave concordant results; 63 were identified as M. avium, 5 were identified as M. intracellulare, and 24 remained untypeable by both methods. Fourteen isolates gave discrepant results, as they were DT1 positive but gave negative results by the M. intracellulare AccuProbe test. Consequently, a detailed molecular analysis of all DT1-positive isolates (14 discrepant strains plus 5 M. intracellulare strains) was performed by PCR-restriction analysis (PRA) of the hsp65 gene and 16S rRNA gene sequencing. The results confirmed the reported heterogeneity of M. intracellulare, as only 6 of 19 isolates (32%) gave PRA results compatible with published M. intracellulare profiles while the rest of the isolates were grouped in four previously unpublished profiles. 16S rRNA gene sequencing showed that only 8 of 19 isolates (42%) were related to M. intracellulare IWGMT 90247 (EMBL accession no. X88917), the rest being related to MCRO19 (EMBL accession no. X93030) and MIWGTMR10 (EMBL accession no. X88915). In conclusion, we have characterized a significant number of MAC isolates which were not identified by the AccuProbe test, PRA, or 16S rRNA sequencing. However, all of them were identifiable by DT1-DT6 PCR (they were DT6 negative and DT1 positive) and could be tentatively identified as M. intracellulare based on previously published observations. It is noteworthy that the majority of such isolates (14 of 19) were from the Indian subcontinent, with 12 of 14 being environmental isolates. Our study confirms the marked heterogeneity of M. intracellulare

  18. Direct PCR amplification of the 16S rRNA gene from single microbial cells isolated from an Antarctic iceberg using laser microdissection microscopy

    NASA Astrophysics Data System (ADS)

    Yanagihara, Katsuhiko; Niki, Hironori; Baba, Tomoya

    2011-09-01

    Here, we describe a technique that allows the genetic linage analysis of 16S rRNA genes in bacteria observed under a microscope. The technique includes the isolation of microbial cells using a laser microdissection microscope, lysis of the cells, and amplification of the 16S rRNA genes in the isolated cells without interference by bacterial DNA contamination from the experimental environment or reagents. Using this technique, we successfully determined 15 16S rRNA gene sequences in cells isolated from an Antarctic iceberg. These sequences showed 94%-100% identity to their closest strains, which included bacteria that occur in aqueous, marine, and soil environments.

  19. Direct Screening of Blood by PCR and Pyrosequencing for a 16S rRNA Gene Target from Emergency Department and Intensive Care Unit Patients Being Evaluated for Bloodstream Infection

    PubMed Central

    Moore, M. S.; McCarroll, M. G.; McCann, C. D.; May, L.; Younes, N.

    2015-01-01

    Here we compared the results of PCR/pyrosequencing to those of culture for detecting bacteria directly from blood. DNA was extracted from 1,130 blood samples from 913 patients suspected of bacteremia (enrollment criteria were physician-ordered blood culture and complete blood count [CBC]), and 102 controls (healthy blood donors). Real-time PCR assays for beta-globin and Universal 16S rRNA gene targets were performed on all 1,232 extracts. Specimens identified by Universal 16S rRNA gene PCR/pyrosequencing as containing staphylococci, streptococci, or enteric Gram-negative rods had target-specific PCR/pyrosequencing performed. Amplifiable beta-globin (melting temperature [Tm], 87.2°C ± 0.2°C) occurred in 99.1% (1,120/1,130) of patient extracts and 100% (102/102) of controls. Concordance between PCR/pyrosequencing and culture was 96.9% (1,085/1,120) for Universal 16S rRNA gene targets, with positivity rates of 9.4% (105/1,120) and 11.3% (126/1,120), respectively. Bacteria cultured included staphylococci (59/126, 46.8%), Gram-negative rods (34/126, 27%), streptococci (32/126, 25.4%), and a Gram-positive rod (1/126, 0.8%). All controls screened negative by PCR/pyrosequencing. Clinical performance characteristics (95% confidence interval [CI]) for Universal 16S rRNA gene PCR/pyrosequencing included sensitivity of 77.8% (69.5 to 84.7), specificity of 99.3% (98.6 to 99.7), positive predictive value (PPV) of 93.3% (86.8 to 97.3), and negative predictive value (NPV) of 97.2% (96.0 to 98.2). Bacteria were accurately identified in 77.8% (98/126) of culture-confirmed sepsis samples with Universal 16S PCR/pyrosequencing and in 76.4% (96/126) with follow-up target-specific PCR/pyrosequencing. The initial PCR/pyrosequencing took ∼5.5 h to complete or ∼7.5 h when including target-specific PCR/pyrosequencing compared to 27.9 ± 13.6 h for Gram stain or 81.6 ± 24.0 h for phenotypic identification. In summary, this molecular approach detected the causative bacteria in over

  20. Bacterial diversity in Philippine fermented mustard (burong mustasa) as revealed by 16S rRNA gene analysis.

    PubMed

    Larcia, L L H; Estacio, R C; Dalmacio, L M M

    2011-12-01

    Previous studies on the bacterial profile of burong mustasa, a traditional Philippine fermented food, had been conducted using culture-dependent techniques. Since these methods may underestimate the total microbiota of a sample, a culture-independent study was done to determine the bacterial diversity in burong mustasa through molecular biology techniques. Bacterial DNA was isolated from fermented mustard samples at different stages of fermentation. The isolated genomic DNA was amplified by PCR using specific primers for the 16S ribosomal RNA gene (16S rDNA). The 1.5 kb amplicons obtained were subjected to nested PCR using primers for the internal variable region of the 16S rDNA. The 585 bp nested PCR amplicons were then subjected to denaturing gradient gel electrophoresis (DGGE) to separate the different bacteria present in each sample. Distinct and unique bands in the DGGE profile were excised, reamplified, purified and sequenced for bacterial identification. Molecular cloning of the 1.5 kb 16S rDNA was also performed using the pGEM-T Easy Vector System. The cloned gene was sequenced for bacterial identification. The identified microbiota in burong mustasa at different stages of fermentation include lactic acid bacteria and several uncultured bacteria (initial up to the final stages); acetic acid bacteria (middle stage); and Streptobacillus and Fusobacterium species (initial stage). The potential probiotic bacteria found in burong mustasa are Weissella and Lactobacillus. PMID:22146686

  1. Comparison of rpoB gene sequencing, 16S rRNA gene sequencing, gyrB multiplex PCR, and the VITEK2 system for identification of Acinetobacter clinical isolates.

    PubMed

    Lee, Min Jung; Jang, Sook Jin; Li, Xue Min; Park, Geon; Kook, Joong-Ki; Kim, Min Jung; Chang, Young-Hyo; Shin, Jong Hee; Kim, Soo Hyun; Kim, Dong-Min; Kang, Seong-Ho; Moon, Dae-Soo

    2014-01-01

    Since accurate identification of species is necessary for proper treatment of Acinetobacter infections, we compared the performances of 4 bacterial identification methods using 167 Acinetobacter clinical isolates to identify the best identification method. To secure more non-baumannii Acinetobacter (NBA) strains as target strains, we first identified Acinetobacter baumannii in a total of 495 Acinetobacter clinical isolates identified using the VITEK 2 system. Because 371 of 495 strains were identified as A. baumannii using gyrB multiplex 1 PCR and blaOXA51-like PCR, we performed rpoB gene sequencing and 16S rRNA gene sequencing on remaining 124 strains belonging to NBA and 52 strains of A. baumannii. For identification of Acinetobacter at the species level, the accuracy rates of rpoB gene sequencing, 16S rRNA gene sequencing, gyrB multiplex PCR, and the VITEK 2 were 98.2%, 93.4%, 77.2%, and 35.9%, respectively. The gyrB multiplex PCR seems to be very useful for the detection of ACB complex because its concordance rates to the final identification of strains of ACB complex were 100%. Both the rpoB gene sequencing and the 16S rRNA gene sequencing may be useful in identifying Acinetobacter. PMID:24157058

  2. Molecular Identification and Differentiation of Fasciola Isolates Using PCR- RFLP Method Based on Internal Transcribed Spacer (ITS1, 5.8S rDNA, ITS2)

    PubMed Central

    Mahami-Oskouei, M; Dalimi, A; Forouzandeh-Moghadam, M; Rokni, MB

    2011-01-01

    Background In this study, we used both ITS1 and ITS2 for molecular identification of Fasciola species. Methods The region between 18S and 28S of ribosomal DNA was used in PCR-RFLP method for molecular identification of Fasciola species. Ninety trematodes of Fasciola were collected during abattoir inspection from livers of naturally infected sheep and cattle from Khorasan, East Azerbaijan, and Fars provinces in Iran. After DNA extraction, PCR was performed to amplify region ITS1, 5.8S rDNA, ITS2. To select a suitable restriction enzyme, we sequenced and analyzed the PCR products of F. hepatica and F. gigantica samples from sheep and cattle. Tsp509I fast digest restriction enzyme was selected for RFLP method that caused the separation specifically of Fasciola species. Results The fragment approximately 1000bp in all of the Fasciola samples was amplified and then digested with the Tsp509I restriction endonuclease. Seventy F. hepatica and 20 F. gigantica were identified of total 90 Fasciola isolates. Conclusion The new PCR-RFLP assay using Tsp509I restriction enzyme provides a simple, practical, fast, low cost, and reliable method for identification and differentiation of Fasciola isolates. PMID:22347295

  3. Detection and identification of Trypanosoma of African livestock through a single PCR based on internal transcribed spacer 1 of rDNA.

    PubMed

    Desquesnes, M; McLaughlin, G; Zoungrana, A; Dávila, A M

    2001-05-01

    Primers hybridising with the rDNA cistron have previously been evaluated for PCR diagnosis specific for kinetoplastids, and shown to detect and differentiate the Trypanosoma brucei complex and Trypanosoma cruzi. Kin1 and Kin2 primers, amplifying internal transcribed spacer 1, were subsequently evaluated for the diagnosis of African livestock trypanosomosis. Based on the size of the PCR products obtained, Kin primers allowed detection and identification of three Trypanosoma congolense types (savannah, forest and Kenya Coast), with distinction among themselves and from the subgenus Trypanozoon (T. brucei spp., Trypanosoma evansi and Trypanosoma equiperdum), Trypanosoma vivax, Trypanosoma simiae and Trypanosoma theileri. These primers were shown to be suitable for the sensitive and type-specific diagnosis of African livestock trypanosome isolates through a single PCR even in the case of multi-taxa samples. With field samples (buffy-coat from cattle blood) sensitivity was close to the sensitivity observed in single reactions with the classical specific primers for the Trypanozoon subgenus and T. congolense-type savannah, but was lower for detection of T. vivax. Additional reaction, improvement of DNA preparation, and/or new primers design are necessary to improve the sensitivity for detection of T. vivax in field samples. However, these primers are suitable for isolate typing through a single PCR. PMID:11334950

  4. Determination of fruit origin by using 26S rDNA fingerprinting of yeast communities by PCR-DGGE: preliminary application to Physalis fruits from Egypt.

    PubMed

    El Sheikha, Aly Farag; Condur, Ana; Métayer, Isabelle; Nguyen, Doan Duy Le; Loiseau, Gérard; Montet, Didier

    2009-10-01

    The determination of geographical origin is a demand of the traceability system of import-export food products. One hypothesis for tracing the source of a product is by global analysis of the microbial communities of the food and statistical linkage of this analysis to the geographical origin of the food. For this purpose, a molecular technique employing 26S rDNA profiles generated by PCR-DGGE was used to detect the variation in yeast community structures of three species of Physalis fruit (Physalis ixocarpa Brat, Physalis pubescens L, Physalis pruinosa L) from four Egyptian regions (Qalyoubia, Minufiya, Beheira and Alexandria Governments). When the 26S rDNA profiles were analysed by multivariate analysis, distinct microbial communities were detected. The band profiles of Physalis yeasts from different Governments were specific for each location and could be used as a bar code to discriminate the origin of the fruits. This method is a new traceability tool which provides fruit products with a unique biological bar code and makes it possible to trace back the fruits to their original location. PMID:19784935

  5. Assessment of fecal pollution sources in a small northern-plains watershed using PCR and phylogenetic analyses of Bacteroidetes 16S rRNA gene

    USGS Publications Warehouse

    Lamendella, R.; Domingo, J.W.S.; Oerther, D.B.; Vogel, J.R.; Stoeckel, D.M.

    2007-01-01

    We evaluated the efficacy, sensitivity, host-specificity, and spatial/temporal dynamics of human- and ruminant-specific 16S rRNA gene Bacteroidetes markers used to assess the sources of fecal pollution in a fecally impacted watershed. Phylogenetic analyses of 1271 fecal and environmental 16S rRNA gene clones were also performed to study the diversity of Bacteroidetes in this watershed. The host-specific assays indicated that ruminant feces were present in 28-54% of the water samples and in all sampling seasons, with increasing frequency in downstream sites. The human-targeted assays indicated that only 3-5% of the water samples were positive for human fecal signals, although a higher percentage of human-associated signals (19-24%) were detected in sediment samples. Phylogenetic analysis indicated that 57% of all water clones clustered with yet-to-be-cultured Bacteroidetes species associated with sequences obtained from ruminant feces, further supporting the prevalence of ruminant contamination in this watershed. However, since several clusters contained sequences from multiple sources, future studies need to consider the potential cosmopolitan nature of these bacterial populations when assessing fecal pollution sources using Bacteroidetes markers. Moreover, additional data is needed in order to understand the distribution of Bacteroidetes host-specific markers and their relationship to water quality regulatory standards. ?? 2006 Federation of European Microbiological Societies.

  6. A universal DNA extraction and PCR amplification method for fungal rDNA sequence-based identification.

    PubMed

    Romanelli, A M; Fu, J; Herrera, M L; Wickes, B L

    2014-10-01

    Accurate identification of fungal pathogens using a sequence-based approach requires an extraction method that yields template DNA pure enough for polymerase chain reaction (PCR) or other types of amplification. Therefore, the objective of this study was to develop and standardise a rapid, inexpensive DNA extraction protocol applicable to the major fungal phyla, which would yield sufficient template DNA pure enough for PCR and sequencing. A total of 519 clinical and culture collection strains, comprised of both yeast and filamentous fungi, were prepared using our extraction method to determine its applicability for PCR, which targeted the ITS and D1/D2 regions in a single PCR amplicon. All templates were successfully amplified and found to yield the correct strain identification when sequenced. This protocol could be completed in approximately 30 min and utilised a combination of physical and chemical extraction methods but did not require organic solvents nor ethanol precipitation. The method reduces the number of tube manipulations and yielded suitable template DNA for PCR amplification from all phyla that were tested. PMID:24865530

  7. Lack of specificity for PCR assays targeting human Bacteroides 16S rRNA gene: cross-amplification with fish feces

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Methods focused on members of the genus Bacteroides have been increasingly utilized in microbial source tracking studies for identifying and quantifying sources of non-point fecal contamination. We present results using real-time PCR to show significant cross-amplification of a human-specific Bacter...

  8. Diversity, dynamics, and activity of bacterial communities during production of an artisanal Sicilian cheese as evaluated by 16S rRNA analysis.

    PubMed

    Randazzo, Cinzia L; Torriani, Sandra; Akkermans, Antoon D L; de Vos, Willem M; Vaughan, Elaine E

    2002-04-01

    The diversity and dynamics of the microbial communities during the manufacturing of Ragusano cheese, an artisanal cheese produced in Sicily (Italy), were investigated by a combination of classical and culture-independent approaches. The latter included PCR, reverse transcriptase-PCR (RT-PCR), and denaturing gradient gel electrophoresis (DGGE) of 16S rRNA genes (rDNA). Bacterial and Lactobacillus group-specific primers were used to amplify the V6 to V8 and V1 to V3 regions of the 16S rRNA gene, respectively. DGGE profiles from samples taken during cheese production indicated dramatic shifts in the microbial community structure. Cloning and sequencing of rDNA amplicons revealed that mesophilic lactic acid bacteria (LAB), including species of Leuconostoc, Lactococcus lactis, and Macrococcus caseolyticus were dominant in the raw milk, while Streptococcus thermophilus prevailed during lactic fermentation. Other thermophilic LAB, especially Lactobacillus delbrueckii and Lactobacillus fermentum, also flourished during ripening. Comparison of the rRNA-derived patterns obtained by RT-PCR to the rDNA DGGE patterns indicated a substantially different degree of metabolic activity for the microbial groups detected. Identification of cultivated LAB isolates by phenotypic characterization and 16S rDNA analysis indicated a variety of species, reflecting to a large extent the results obtained from the 16S rDNA clone libraries, with the significant exception of the Lactobacillus delbrueckii species, which dominated in the ripening cheese but was not detected by cultivation. The present molecular approaches combined with culture can effectively describe the complex ecosystem of natural fermented dairy products, giving useful information for starter culture design and preservation of artisanal fermented food technology. PMID:11916708

  9. 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

  10. Novel 16S rRNA based PCR method targeting Deinococcus spp. and its application to assess the diversity of deinococcal populations in environmental samples.

    PubMed

    Chaturvedi, Ruchi; Archana, G

    2012-09-01

    The members of the genus Deinococcus are extensively studied because of their exemplary radiation resistance. Both ionizing and non-ionizing rays are routinely employed to select upon the radiation resistant deinococcal population and isolate them from the majority of radiation sensitive population. There are no studies on the development of molecular tools for the rapid detection and identification of deinococci from a mixed population without causing the bias of radiation enrichment. Here we present a Deinococcus specific two-step hemi-nested PCR for the rapid detection of deinococci from environmental samples. The method is sensitive and specific to detect deinococci without radiation exposure of the sample. The new protocol was successfully employed to detect deinococci from several soil samples from different geographical regions of India. The PCR method could be adapted to a three-step protocol to study the diversity of the environmental deinococcal population by denaturing gradient gel electrophoresis (DGGE). Sequence analysis of the DGGE bands revealed that the samples harbor diverse populations of deinococci, many of which were not recovered by culturing and may represent novel clades. We demonstrate that the genus specific primers are also suitable for the rapid identification of the bacterial isolates that are obtained from a typical radiation enrichment isolation technique. Therefore the primers and the protocols described in this study can be used to study deinococcal diversity from environmental samples and can be employed for the rapid detection of deinococci in samples or identifying pure culture isolates as Deinococcus species. PMID:22609328

  11. PCR method for the rapid detection and discrimination of Legionella spp. based on the amplification of pcs, pmtA, and 16S rRNA genes.

    PubMed

    Janczarek, Monika; Palusińska-Szysz, Marta

    2016-05-01

    Legionella bacteria are organisms of public health interest due to their ability to cause pneumonia (Legionnaires' disease) in susceptible humans and their ubiquitous presence in water supply systems. Rapid diagnosis of Legionnaires' disease allows the use of therapy specific for the disease. L. pneumophila serogroup 1 is the most common cause of infection acquired in community and hospital environments. The non-L. pneumophila infections are likely under-detected because of a lack of effective diagnosis. In this work, simplex and duplex PCR assays with the use of new molecular markers pcs and pmtA involved in phosphatidylcholine synthesis were specified for rapid and cost-efficient identification and distinguishing Legionella species. The sets of primers developed were found to be sensitive and specific for reliable detection of Legionella belonging to the eight most clinically relevant species. Among these, four primer sets I, II, VI, and VII used for duplex-PCRs proved to have the highest identification power and reliability in the detection of the bacteria. Application of this PCR-based method should improve detection of Legionella spp. in both clinical and environmental settings and facilitate molecular typing of these organisms. PMID:26423783

  12. Real-time PCR assays for the quantitation of rDNA from apricot and other plant species in marzipan.

    PubMed

    Haase, Ilka; Brüning, Philipp; Matissek, Reinhard; Fischer, Markus

    2013-04-10

    Marzipan or marzipan raw paste is a typical German sweet which is consumed directly or is used as an ingredient in the bakery industry/confectionery (e.g., in stollen) and as filling for chocolate candies. Almonds (blanched and pealed) and sugar are the only ingredients for marzipan production according to German food guidelines. Especially for the confectionery industry, the use of persipan, which contains apricot or peach kernels instead of almonds, is preferred due to its stronger aroma. In most of the companies, both raw pastes are produced, in most cases on the same production line, running the risk of an unintended cross contamination. Additionally, due to high almond market values, dilutions of marzipan with cheaper seeds may occur. Especially in the case of apricot and almond, the close relationship of both species is a challenge for the analysis. DNA based methods for the qualitative detection of apricot, peach, pea, bean, lupine, soy, cashew, pistachio, and chickpea in marzipan have recently been published. In this study, different quantitation strategies on the basis of real-time PCR have been evaluated and a relative quantitation method with a reference amplification product was shown to give the best results. As the real-time PCR is based on the high copy rDNA-cluster, even contaminations <1% can be reliably quantitated. PMID:23495652

  13. [Tracing the Fecal Contamination Sources Based on Bacteroides 16S rRNA PCR- DGGE in Karst Groundwater: Taking Laolongdong Underground River System, Nanshan, Chongqing as an Example].

    PubMed

    Zhang, Hong; Jiang, Yong-jun; Zhang, Yuan-zhu; Duan, Yi-fan; Lü, Xian-fu; He, Qiu-fang

    2016-05-15

    Microbial contamination in karst groundwater continually increases and tracing the source researches has become a hot topic for international researchers. In this study, Laolongdong underground river at Nanshan, Chongqing was chosen as an example to adopt filter membrane methods to monitor the fecal microbial contaminations including the total bacterial concentration (TB), the total E. coli concentration (TE), the total fecal coliform (FC) and the total fecal Streptocoocci (FS). Bacteriodes was used as an indicator and PCR-DGGE analysis was used to trace fecal contamination sources in karst groundwater. The results suggested that groundwater in this area was seriously polluted by microbes from feces. The concentrations of microbial parameters exceeded limited levels greatly and the total bacterial amounts ranged 10-2.9 x 10⁷ CFU · mL⁻¹, the concentrations of E. coli were between 4.3-4.0 x 10⁵ CFU · mL⁻¹, the max concentration of FC was 1.1 x 10⁶ CFU · (100 mL)⁻¹ and the max concentration of FS was 1.1 x 10⁵ CFU · (100 mL)⁻¹. The FC/FS ratios were mostly over 2 which suggested that the main fecal source in groundwater was human feces. In addition, PCR-DGGE contrastive analysis of Bacteroides communities showed that the similarities between groundwater samples and human feces were in range of 7. 1% -69. 1% , and the similarity of the groundwater sample from Laolongdong underground river outlet was 69.1% . Bacteroides community similarities between groundwater samples and swine feces were in range of 1.1%-53.4%, and the similarity of Laolongdong underground river outlet was merely 1.5%. The similarity data implied that groundwater contamination resulted mainly from human feces, swine feces contamination composed part of animals' fecal contamination, and other animals' feces participated too. Furthermore, sequencing results of PCR-DGGE products revealed that most Bacteroides in groundwater originated from human intestinal tract and human feces

  14. Genetic Diversity of Bacillus thuringiensis from Different Geo-Ecological Regions of Ukraine by Analyzing the 16S rRNA and gyrB Genes and by AP-PCR and saAFLP

    PubMed Central

    Punina, N. V.; Zotov, V. S.; Parkhomenko, A. L.; Parkhomenko, T. U.; Topunov, A. F.

    2013-01-01

    The Bacillus cereus group consists of closely related species of bacteria and is of interest to researchers due to its importance in industry and medicine. However, it remains difficult to distinguish these bacteria at the intra- and inter-species level. Bacillus thuringiensis (Bt) is a member of the B. cereus group. In this work, we studied the inter-species structure of five entomopathogenic strains and 20 isolates of Bt, which were collected from different geo-ecological regions of Ukraine, using various methods: physiological and biochemical analyses, analysis of the nucleotide sequences of the 16S rRNA and gyrB genes, by AP-PCR (BOX and ERIC), and by saAFLP. The analysis of the 16S rRNA and gyrB genes revealed the existence of six subgroups within theB.cereus group: B anthracis, B. cereus I and II, Bt I and II, and Bt III, and confirmed that these isolates belong to the genus Bacillus. All strains were subdivided into 3 groups. Seventeen strains belong to the group Bt II of commercial, industrial strains. The AP-PCR (BOX and ERIC) and saAFLP results were in good agreement and with the results obtained for the 16S rRNA and gyrB genes. Based on the derived patterns, all strains were reliably combined into 5 groups. Interestingly, a specific pattern was revealed by the saAFLP analysis for the industrial strain Bt 0376 р.о., which is used to produce the entomopathogenic preparation “STAR-t”. PMID:23556134

  15. Development of a Broad-Range 23S rDNA Real-Time PCR Assay for the Detection and Quantification of Pathogenic Bacteria in Human Whole Blood and Plasma Specimens

    PubMed Central

    Gaibani, Paolo; Mariconti, Mara; Bua, Gloria; Bonora, Sonia; Sassera, Davide; Landini, Maria Paola; Mulatto, Patrizia; Novati, Stefano; Bandi, Claudio; Sambri, Vittorio

    2013-01-01

    Molecular methods are important tools in the diagnosis of bloodstream bacterial infections, in particular in patients treated with antimicrobial therapy, due to their quick turn-around time. Here we describe a new broad-range real-time PCR targeting the 23S rDNA gene and capable to detect as low as 10 plasmid copies per reaction of targeted bacterial 23S rDNA gene. Two commercially available DNA extraction kits were evaluated to assess their efficiency for the extraction of plasma and whole blood samples spiked with different amount of either Staphylococcus aureus or Escherichia coli, in order to find the optimal extraction method to be used. Manual QIAmp extraction method with enzyme pre-treatment resulted the most sensitive for detection of bacterial load. Sensitivity of this novel assay ranged between 10 and 103 CFU per PCR reaction for E. coli and S. aureus in human whole blood samples depending on the extraction methods used. Analysis of plasma samples showed a 10- to 100-fold reduction of bacterial 23S rDNA in comparison to the corresponding whole blood specimens, thus indicating that whole blood is the preferential sample type to be used in this real-time PCR protocol. Our results thus show that the 23S rDNA gene represents an optimal target for bacteria quantification in human whole blood. PMID:23586027

  16. Multiplexed Identification of Blood-Borne Bacterial Pathogens by Use of a Novel 16S rRNA Gene PCR-Ligase Detection Reaction-Capillary Electrophoresis Assay▿ †

    PubMed Central

    Pingle, Maneesh R.; Granger, Kathleen; Feinberg, Philip; Shatsky, Rebecca; Sterling, Bram; Rundell, Mark; Spitzer, Eric; Larone, Davise; Golightly, Linnie; Barany, Francis

    2007-01-01

    We have developed a novel high-throughput PCR-ligase detection reaction-capillary electrophoresis (PCR-LDR-CE) assay for the multiplexed identification of 20 blood-borne pathogens (Staphylococcus epidermidis, Staphylococcus aureus, Bacillus cereus, Enterococcus faecalis, Enterococcus faecium, Listeria monocytogenes, Streptococcus pneumoniae, Streptococcus pyogenes, Streptococcus agalactiae, Escherichia coli, Klebsiella pneumoniae, Haemophilus influenzae, Pseudomonas aeruginosa, Acinetobacter baumannii, Neisseria meningitidis, Bacteroides fragilis, Bacillus anthracis, Yersinia pestis, Francisella tularensis, and Brucella abortus), the last four of which are biothreat agents. The method relies on the amplification of two regions within the bacterial 16S rRNA gene, using universal PCR primers and querying the identity of specific single-nucleotide polymorphisms within the amplified regions in a subsequent LDR. The ligation products vary in color and size and are separated by CE. Each organism generates a specific pattern of ligation products, which can be used to distinguish the pathogens using an automated software program we developed for that purpose. The assay has been verified on 315 clinical isolates and demonstrated a detection sensitivity of 98%. Additionally, 484 seeded blood cultures were tested, with a detection sensitivity of 97.7%. The ability to identify geographically variant strains of the organisms was determined by testing 132 isolates obtained from across the United States. In summary, the PCR-LDR-CE assay can successfully identify, in a multiplexed fashion, a panel of 20 blood-borne pathogens with high sensitivity and specificity. PMID:17428930

  17. Epidemiologic Study of Malassezia Yeasts in Seborrheic Dermatitis Patients by the Analysis of 26S rDNA PCR-RFLP

    PubMed Central

    Oh, Byung Ho; Choe, Yong Beom; Ahn, Kyu Joong

    2010-01-01

    Background This case-control study concerns a molecular biological method based on the data gathered from a group of Korean subjects to examine the distribution of Malassezia yeasts in seborrheic dermatitis (SD) patients. Cultures for Malassezia yeasts were taken from the foreheads, cheeks and chests of 60 patients with SD and in 60 healthy controls of equivalent age. Objective The purpose of this study is to identify the relationship between certain species of Malassezia and SD. This was done by analyzing the differences in the distribution of Malassezia species in terms of age and body parts of the host with healthy controls. Methods 26S rDNA PCR-RFLP, a fast and accurate molecular biological method, was used to overcome the limits of morphological and biochemical methods. Results The positive Malassezia culture rate was 51.7% in patients with SD, which was lower than that of healthy adults (63.9%). M. restricta was dominant in patients with SD (19.5%). Likewise, M. restricta was identified as a common species (20.5%) in healthy controls. In the ages 31~40, M. restricta was found to be the most common species (31.6%) among SD patients. Conclusion According to the results of the study, the most frequently isolated species was M. restricta (19.5%) in patients with SD. There was no statistically significant difference in the distribution of Malassezia species between the SD patients and healthy control groups. PMID:20548904

  18. Identification of airborne bacterial and fungal species in the clinical microbiology laboratory of a university teaching hospital employing ribosomal DNA (rDNA) PCR and gene sequencing techniques.

    PubMed

    Nagano, Yuriko; Walker, Jim; Loughrey, Anne; Millar, Cherie; Goldsmith, Colin; Rooney, Paul; Elborn, Stuart; Moore, John

    2009-06-01

    Universal or "broad-range" PCR-based ribosomal DNA (rDNA) was performed on a collection of 58 isolates (n = 30 bacteria + 28 fungi), originating from environmental air from several locations within a busy clinical microbiology laboratory, supporting a university teaching hospital. A total of 10 bacterial genera were identified including both Gram-positive and Gram-negative genera. Gram-positive organisms accounted for 27/30 (90%) of total bacterial species, consisting of seven genera and included (in descending order of frequency) Staphylococcus, Micrococcus, Corynebacterium, Paenibacillus, Arthrobacter, Janibacter and Rothia. Gram-negative organisms were less frequently isolated 3/30 (10%) and comprised three genera, including Moraxella, Psychrobacter and Haloanella. Eight fungal genera were identified among the 28 fungal organisms isolated, including (in descending order of frequency) Cladosporium, Penicillium, Aspergillus, Thanatephorus, Absidia, Eurotium, Paraphaeosphaeria and Tritirachium, with Cladosporium accounting for 10/28 (35.7%) of the total fungal isolates. In conclusion, this study identified the presence of 10 bacterial and eight fungal genera in the air within the laboratory sampled. Although this reflected diversity of the microorganisms present, none of these organisms have been described previously as having an inhalational route of laboratory-acquired infection. Therefore, we believe that the species of organisms identified and the concentration levels of these airborne contaminants determined, do not pose a significant health and safety threat for immunocompotent laboratory personnel and visitors. PMID:20183192

  19. Phylogenetic relationships of the soybean sudden death syndrome pathogen Fusarium solani f. sp. phaseoli inferred from rDNA sequence data and PCR primers for its identification.

    PubMed

    O'Donnell, K; Gray, L E

    1995-01-01

    Phylogenetic relationships of several species within the Fusarium solani-complex were investigated using characters from the nuclear ribosomal DNA. Genetic variation within 24 isolates, including 5 soybean sudden death syndrome (SDS) strains, was assessed using rDNA sequence data and restriction fragment length polymorphic markers. By these techniques, the causal agent of soybean SDS was identified as F. solani f. sp. phaseoli. In separate cladistic analyses, Plectosphaerella cucumerina and Nectria cinnabarina or F. ventricosum were used for rooting purposes. Monophyly of the F. solani-complex was strongly supported by bootstrap and decay analyses. Parsimony analysis indicates that this complex is composed of a number of phylogenetically distinct species, including Neocosmospora vasinfecta, F. solani f. sp. phaseoli, and biological species designated as MPI, MPV, and MPVI of N. haematococca. The results demonstrate complete congruence between biological and phylogenetic species within the N. haematococca-complex. In addition, DNA sequence data were used to design a PCR primer pair which could specifically amplify DNA from isolates of the SDS pathogen from infected plants. PMID:7579615

  20. Genetic Diversity of the Biofilm Covering Montacuta ferruginosa (Mollusca, Bivalvia) as Evaluated by Denaturing Gradient Gel Electrophoresis Analysis and Cloning of PCR-Amplified Gene Fragments Coding for 16S rRNA†

    PubMed Central

    Gillan, David C.; Speksnijder, Arjen G. C. L.; Zwart, Gabriel; De Ridder, Chantal

    1998-01-01

    The shell of the bivalve Montacuta ferruginosa, a symbiont living in the burrow of an echinoid, is covered with a rust-colored biofilm. This biofilm includes different morphotypes of bacteria that are encrusted with a mineral rich in ferric ion and phosphate. The aim of this research was to determine the genetic diversity and phylogenetic affiliation of the biofilm bacteria. Also, the possible roles of the microorganisms in the processes of mineral deposition within the biofilm, as well as their impact on the biology of the bivalve, were assessed by phenotypic inference. The genetic diversity was determined by denaturing gradient gel electrophoresis (DGGE) analysis of short (193-bp) 16S ribosomal DNA PCR products obtained with primers specific for the domain Bacteria. This analysis revealed a diverse consortium; 11 to 25 sequence types were detected depending on the method of DNA extraction used. Individual biofilms analyzed by using the same DNA extraction protocol did not produce identical DGGE profiles. However, different biofilms shared common bands, suggesting that similar bacteria can be found in different biofilms. The phylogenetic affiliations of the sequence types were determined by cloning and sequencing the 16S rRNA genes. Close relatives of the genera Pseudoalteromonas, Colwellia, and Oceanospirillum (members of the γ-Proteobacteria lineage), as well as Flexibacter maritimus (a member of the Cytophaga-Flavobacter-Bacteroides lineage), were found in the biofilms. We inferred from the results that some of the biofilm bacteria could play a role in the mineral formation processes. PMID:9726898

  1. Thinking beside the box: Should we care about the non-coding strand of the 16S rRNA gene?

    PubMed

    Garcia-Mazcorro, Jose F; Barcenas-Walls, Jose R

    2016-08-01

    The 16S rRNA gene (16S rDNA) codes for RNA that plays a fundamental role during translation in the ribosome and is used extensively as a marker gene to establish relationships among bacteria. However, the complementary non-coding 16S rDNA (nc16S rDNA) has been ignored. An idea emerged in the course of analyzing bacterial 16S rDNA sequences in search for nucleotide composition and substitution patterns: Does the nc16S rDNA code? If so, what does it code for? More importantly: Does 16S rDNA evolution reflect its own evolution or the evolution of its counterpart nc16S rDNA? The objective of this minireview is to discuss these thoughts. nc strands often encode small RNAs (sRNAs), ancient components of gene regulation. nc16S rDNA sequences from different bacterial groups were used to search for possible matches in the Bacterial Small Regulatory RNA Database. Intriguingly, the sequence of one published sRNA obtained from Legionella pneumophila (GenBank: AE0173541) showed high non-random similarity with nc16S rDNA corresponding in part to the V5 region especially from Legionella and relatives. While the target(s) of this sRNA is unclear at the moment, its mere existence might open up a new chapter in the use of the 16S rDNA to study relationships among bacteria. PMID:27412167

  2. Bacterial diversity of a Carolina bay as determined by 16S rRNA gene analysis: confirmation of novel taxa.

    PubMed Central

    Wise, M G; McArthur, J V; Shimkets, L J

    1997-01-01

    Carolina bays are naturally occurring shallow elliptical depressions largely fed by rain and shallow ground water. To identify members of the domain Bacteria which inhibit such an environment, we used PCR to construct a library of 16S rRNA genes (16S rDNAs) cloned from DNA extracted from the sediments of Rainbow bay, located on the Savannah River Site, near Aiken, S.C. Oligonucleotides complementary to conserved regions of 16S rDNA were used as primers for PCR, and gel-purified PCR products were cloned into vector pGEM-T. Partial sequencing of the cloned 16S rDNAs revealed an extensive amount of phylogenetic diversity within this system. Of the 35 clones sequenced, 32 were affiliated with five bacterial groups: 11 clustered with the Proteobacteria division (including members of the alpha, beta, and delta subdivisions), 8 clustered with the Acidobacterium subdivision of the Fibrobacter division (as categorized by the Ribosomal Database Project's taxonomic scheme, version 5.0), 7 clustered with the Verrucomicrobium subdivision of the Planctomyces division, 3 clustered with the gram-positive bacteria (Clostridium and relatives subdivision), and 3 clustered with the green nonsulfur bacteria. One sequence branched very deeply from the Bacteria and was found not to be associated with any of the major divisions when phylogenetic trees were constructed. Two clones did not consistently cluster with specific groups and may be chimeric sequences. None of the clones exhibited an exact match to any of the 16S rDNA sequences deposited in the databases, suggesting that most of the bacteria in Rainbow Bay are novel species. In particular, the clones related to the Acidobacterium subdivision and the Verrucomicrobium subdivision confirm the presence of novel taxa discovered previously in other molecular surveys of this type. PMID:9097448

  3. Bacterial diversity of a Carolina Bay as determined by 16S rRNA gene analysis: Confirmation of novel taxa

    SciTech Connect

    Wise, M.G.; Shimkets, L.J.; McArthur, J.V.

    1997-04-01

    Carolina bays are naturally occurring shallow elliptical depressions largely fed by rain and shallow ground water. To identify members of the domain Bacteria which inhabit such an environment, we used PCR to construct a library of 16S rRNA genes (16S rDNAs) cloned from DNA extracted from the sediments of Rainbow Bay, located on the Savannah River Site, near Aiken, S.C. Oligonucleotides complementary to conserved regions of 16S rDNA were used as primers for PCR, and gel-purified PCR products were cloned into vector pGEM-T. Partial sequencing of the cloned 16S rDNAs revealed an extensive amount of phylogenetic diversity within this system. Of the 35 clones sequenced, 32 were affiliated with five bacterial groups: 11 clustered with the Proteobacteria division (including members of the alpha, beta, and delta subdivisions), 8 clustered with the Acidobacterium subdivision of the Fibrobacter division (as categorized by the Ribosomal Database Project`s taxonomic scheme, version 5.0), 7 clustered with the Verrucomicrobium subdivision of the Planctomyces division, 3 clustered with the gram-positive bacteria (Clostridium and relatives subdivision), and 3 clustered with the green nonsulfur bacteria. One sequence branched very deeply from the Bacteria and was found not to be associated with any of the major divisions when phylogenetic trees were constructed. Two clones did not consistently cluster with specific groups and may be chimeric sequences. None of the clones exhibited an exact match to any of the 16S rDNA sequences deposited in the databases, suggesting that most of the bacteria in Rainbow Bay are novel species. In particular, the clones related to the Acidobacterium subdivision and the Verrucomicrobium subdivision confirm the presence of novel taxa discovered previously in other molecular surveys of this type. 50 refs., 7 figs., 1 tab.

  4. Bacterial diversity of a Carolina bay as determined by 16S rRNA gene analysis: confirmation of novel taxa.

    PubMed

    Wise, M G; McArthur, J V; Shimkets, L J

    1997-04-01

    Carolina bays are naturally occurring shallow elliptical depressions largely fed by rain and shallow ground water. To identify members of the domain Bacteria which inhibit such an environment, we used PCR to construct a library of 16S rRNA genes (16S rDNAs) cloned from DNA extracted from the sediments of Rainbow bay, located on the Savannah River Site, near Aiken, S.C. Oligonucleotides complementary to conserved regions of 16S rDNA were used as primers for PCR, and gel-purified PCR products were cloned into vector pGEM-T. Partial sequencing of the cloned 16S rDNAs revealed an extensive amount of phylogenetic diversity within this system. Of the 35 clones sequenced, 32 were affiliated with five bacterial groups: 11 clustered with the Proteobacteria division (including members of the alpha, beta, and delta subdivisions), 8 clustered with the Acidobacterium subdivision of the Fibrobacter division (as categorized by the Ribosomal Database Project's taxonomic scheme, version 5.0), 7 clustered with the Verrucomicrobium subdivision of the Planctomyces division, 3 clustered with the gram-positive bacteria (Clostridium and relatives subdivision), and 3 clustered with the green nonsulfur bacteria. One sequence branched very deeply from the Bacteria and was found not to be associated with any of the major divisions when phylogenetic trees were constructed. Two clones did not consistently cluster with specific groups and may be chimeric sequences. None of the clones exhibited an exact match to any of the 16S rDNA sequences deposited in the databases, suggesting that most of the bacteria in Rainbow Bay are novel species. In particular, the clones related to the Acidobacterium subdivision and the Verrucomicrobium subdivision confirm the presence of novel taxa discovered previously in other molecular surveys of this type. PMID:9097448

  5. Phylogeny and classification of bacteria in the genera Clavibacter and Rathayibacter on the basis of 16s rRNA gene sequence analyses.

    PubMed Central

    Lee, I M; Bartoszyk, I M; Gundersen-Rindal, D E; Davis, R E

    1997-01-01

    A phylogenetic analysis by parsimony of 16S rRNA gene sequences (16S rDNA) revealed that species and subspecies of Clavibacter and Rathayibacter form a discrete monophyletic clade, paraphyletic to Corynebacterium species. Within the Clavibacter-Rathayibacter clade, four major phylogenetic groups (subclades) with a total of 10 distinct taxa were recognized: (I) species C. michiganensis; (II) species C. xyli; (III) species R. iranicus and R. tritici; and (IV) species R. rathayi. The first three groups form a monophyletic cluster, paraphyletic to R. rathayi. On the basis of the phylogeny inferred, reclassification of members of Clavibacter-Rathayibacter group is proposed. A system for classification of taxa in Clavibacter and Rathayibacter was developed based on restriction fragment length polymorphism (RFLP) analysis of the PCR-amplified 16S rDNA sequences. The groups delineated on the basis of RFLP patterns of 16S rDNA coincided well with the subclades delineated on the basis of phylogeny. In contrast to previous classification systems, which are based primarily on phenotypic properties and are laborious, the RFLP analyses allow for rapid differentiation among species and subspecies in the two genera. PMID:9212413

  6. The Characterization of Novel Tissue Microbiota Using an Optimized 16S Metagenomic Sequencing Pipeline

    PubMed Central

    Païssé, Sandrine; Valle, Carine; Valière, Sophie; Kuchly, Claire; Vilchez, Gaëlle; Donnadieu, Cécile; Courtney, Michael; Burcelin, Rémy; Amar, Jacques; Bouchez, Olivier; Lelouvier, Benjamin

    2015-01-01

    Background Substantial progress in high-throughput metagenomic sequencing methodologies has enabled the characterisation of bacteria from various origins (for example gut and skin). However, the recently-discovered bacterial microbiota present within animal internal tissues has remained unexplored due to technical difficulties associated with these challenging samples. Results We have optimized a specific 16S rDNA-targeted metagenomics sequencing (16S metabarcoding) pipeline based on the Illumina MiSeq technology for the analysis of bacterial DNA in human and animal tissues. This was successfully achieved in various mouse tissues despite the high abundance of eukaryotic DNA and PCR inhibitors in these samples. We extensively tested this pipeline on mock communities, negative controls, positive controls and tissues and demonstrated the presence of novel tissue specific bacterial DNA profiles in a variety of organs (including brain, muscle, adipose tissue, liver and heart). Conclusion The high throughput and excellent reproducibility of the method ensured exhaustive and precise coverage of the 16S rDNA bacterial variants present in mouse tissues. This optimized 16S metagenomic sequencing pipeline will allow the scientific community to catalogue the bacterial DNA profiles of different tissues and will provide a database to analyse host/bacterial interactions in relation to homeostasis and disease. PMID:26544955

  7. USE OF BACTEROIDES PCR-BASED METHODS TO EXAMINE FECAL CONTAMINATION SOURCES IN TROPICAL COASTAL WATERS

    EPA Science Inventory

    Several library independent Microbial Source Tracking methods have been developed to rapidly determine the source of fecal contamination. Thus far, none of these methods have been tested in tropical marine waters. In this study, we used a Bacteroides 16S rDNA PCR-based...

  8. REAL-TIME PCR METHOD TO DETECT ENTEROCOCCUS FAECALIS IN WATER

    EPA Science Inventory

    A 16S rDNA real-time PCR method was developed to detect Enterococcus faecalis in water samples. The dynamic range for cell detection spanned five logs and the detection limit was determined to be 6 cfu/reaction. The assay was capable of detecting E. faecalis cells added to biof...

  9. High diversity of bacterial pathogens and antibiotic resistance in salmonid fish farm pond water as determined by molecular identification employing 16S rDNA PCR, gene sequencing and total antibiotic susceptibility techniques.

    PubMed

    Moore, John E; Huang, Junhua; Yu, Pengbo; Ma, Chaofeng; Moore, Peter Ja; Millar, Beverley C; Goldsmith, Colin E; Xu, Jiru

    2014-10-01

    The aim of this study was to examine the microbiological and related parameters (antibiotic resistance and pathogen identification) of water at two salmonid fish farms in Northern Ireland. Total Bacterial Counts at the Movanagher Fish Farm was 1730 colony forming units (cfu)/ml water (log10 3.24cfu/ml) and 3260cfu/ml (log10 3.51cfu/ml) at the Bushmills Salmon Station. Examination of resulting organisms revealed 10 morphological phenotypes, which were subsequently sequenced to determine their identification. All these organisms were Gram-negative and no Gram-positive organisms were isolated from any water sample. From these phenotypes, eight different genera were identified including Acinetobacter, Aeromonas, Chryseobacterium, Erwinia, Flavobacterium, Pseudomonas and Rheinheimera. One unnamed novel taxon was identified from water at the Movanagher Fish Farm, belonging to the genus Acinetobacter and has been tentatively named Acinetobacter movanagherensis. No other novel taxa were observed. All but one of these environmental organisms (Erwinia) are potential pathogens of fish disease. Total antibiotic resistance was observed to varying degrees in water specimens. The most resistant populations were observed in water taken from the Bushmills Salmon Station inlet, followed by water from the Movanagher Fish Farm. No resistance was observed against tetracycline and there was only one occurrence of resistance against ciprofloxacin. Overall, this study indicates that potential fish pathogens made up the majority of environmental organisms identified, even in the absence of recorded fish disease. There was also relatively high levels of total antibiotic resistance in the bacterial water populations examined, where tetracycline was the only antibiotic with zero resistance. These data indicate that the threat of bacterial disease is relatively close due to the indigenous colonization of farm water and that husbandry standards should be maintained at a high standard to avert bacterial disease outbreaks, rather than relying on the absence of specific pathogens in the immediate farm environment. PMID:25105488

  10. Evaluation of 16S rDNA-Based Community Profiling for Human Microbiome Research

    PubMed Central

    2012-01-01

    The Human Microbiome Project will establish a reference data set for analysis of the microbiome of healthy adults by surveying multiple body sites from 300 people and generating data from over 12,000 samples. To characterize these samples, the participating sequencing centers evaluated and adopted 16S rDNA community profiling protocols for ABI 3730 and 454 FLX Titanium sequencing. In the course of establishing protocols, we examined the performance and error characteristics of each technology, and the relationship of sequence error to the utility of 16S rDNA regions for classification- and OTU-based analysis of community structure. The data production protocols used for this work are those used by the participating centers to produce 16S rDNA sequence for the Human Microbiome Project. Thus, these results can be informative for interpreting the large body of clinical 16S rDNA data produced for this project. PMID:22720093

  11. Assessment of bacterial diversity in selected Philippine fermented food products through PCR-DGGE.

    PubMed

    Dalmacio, L M M; Angeles, A K J; Larcia, L L H; Balolong, M P; Estacio, R C

    2011-12-01

    The bacterial population in several Philippine fermented food preparations was assessed by PCR-denaturing gradient gel electrophoresis (PCR-DGGE) of the 16S rRNA gene (16S rDNA). Genomic DNA was isolated directly from alamang (fermented shrimp paste), burong isda (fermented fish and rice), burong hipon (fermented shrimp and rice), burong mustasa (fermented mustard leaves), tuba (sugar cane wine), suka (vinegar) and sinamak (spiced vinegar) using one of two protocols, namely - MoBio DNA Extraction Kit procedure and a cetyltrimethylammonium bromide-based method. Samples recalcitrant to both methods underwent enrichment in three culture broths prior to DNA isolation. Isolated DNA was amplified using nested primer pairs targeting the bacterial 16S rDNA. PCR products were subjected to DGGE to elucidate the bacterial diversity in each fermented food. 16S rDNA sequence analyses revealed that lactic acid bacteria (LAB) and acetic acid bacteria (AAB) were dominant in the food samples. The LAB identified were Lactobacillus fermentum, Lactobacillus plantarum, Lactobacillus panis, Lactobacillus pontis and Weissella cibaria. Identified AAB were Acetobacter pomorum, Acetobacter ghanensis, Acetobacter orientalis, and Acetobacter pasteurianus. Among these, L. fermentum, L. plantarum and W. cibaria are established probiotic bacteria, while L. panis and L. pontis are potential probiotic bacteria. This finding would increase the appeal and significance of local fermented foods to consumers. Furthermore, the majority of the identified bacteria in the study have not been reported before in culture-dependent studies of similar food preparations. As such, some of the bacterial 16S rDNA obtained were cloned to have an initial partial bacterial 16S rDNA library for Philippine fermented foods. PMID:22146687

  12. Universal bacterial identification by mass spectrometry of 16S ribosomal RNA cleavage products

    NASA Astrophysics Data System (ADS)

    Jackson, George W.; McNichols, Roger J.; Fox, George E.; Willson, Richard C.

    2007-03-01

    The public availability of over 180,000 bacterial 16S ribosomal RNA (rRNA) sequences has facilitated microbial identification and classification using nucleic acid hybridization and other molecular approaches. Species-specific PCR, microarrays, and in situ hybridization are based on the presence of unique subsequences in the target sequence and therefore require prior knowledge of what organisms are likely to be present in a sample. Mass spectrometry is not limited by a pre-synthesized inventory of probe/primer sequences. It has already been demonstrated that organism identification can be recovered from mass spectra using various methods including base-specific cleavage of nucleic acids. The feasibility of broad bacterial identification by comparing such mass spectral patterns to predictive databases derived from virtually all previously sequenced strains has yet to be demonstrated, however. Herein, we present universal bacterial identification by base-specific cleavage, mass spectrometry, and an efficient coincidence function for rapid spectral scoring against a large database of predicted "mass catalogs". Using this approach in conjunction with universal PCR of the 16S rDNA gene, four bacterial isolates and an uncultured clone were successfully identified against a database of predicted cleavage products derived 6rom over 47,000 16S rRNA sequences representing all major bacterial taxaE At present, the conventional DNA isolation and PCR steps require approximately 2 h, while subsequent transcription, enzymatic cleavage, mass spectrometric analysis, and database comparison require less than 45 min. All steps are amenable to high-throughput implementation.

  13. Phylogeny of the Sphaerotilus-Leptothrix group inferred from morphological comparisons, genomic fingerprinting, and 16S ribosomal DNA sequence analyses.

    PubMed

    Siering, P L; Ghiorse, W C

    1996-01-01

    Phase-contrast light microscopy revealed that only one of eight cultivated strains belonging to the Sphaerotilus-Leptothrix group of sheathed bacteria actually produced a sheath in standard growth media. Two Sphaerotilus natans strains produced branched cells, but other morphological characteristics that were used to identify these bacteria were consistent with previously published descriptions. Genomic fingerprints, which were obtained by performing PCR amplification with primers corresponding to enterobacterial repetitive intergenic consensus sequences, were useful for distinguishing between the genera Sphaerotilus and Leptothrix, as well as among individual strains. The complete 16S ribosomal DNA (rDNA) sequences of two strains of "Leptothrix discophora" (strains SP-6 and SS-1) were determined. In addition, partial sequences (approximately 300 nucleotides) of one strain of Leptothrix cholodnii (strain LMG 7171), an unidentified Leptothrix strain (strain NC-1), and four strains of Sphaerotilus natans (strains ATCC 13338T [T = type strain], ATCC 15291, ATCC 29329, and ATCC 29330) were determined. We found that two of the S. natans strains (ATCC 15291 and ATCC 13338T), which differed in morphology and in their genomic fingerprints, had identical sequences in the 300-nucleotide region sequenced. Both parsimony and distance matrix methods were used to infer the evolutionary relationships of the eight strains in a comparison of the 16S rDNA sequences of these organisms with 16S rDNA sequences obtained from ribosomal sequence databases. All of the strains clustered in the Rubrivivax subdivision of the beta subclass of the Proteobacteria, which confirmed previously published conclusions concerning selected individual strains. Additional analyses revealed that all of the S. natans strains clustered in one closely related group, while the Leptothrix strains clustered in two separate lineages that were approximately equidistant from the S. natans cluster. This finding

  14. The feline oral microbiome: a provisional 16S rRNA gene based taxonomy with full-length reference sequences.

    PubMed

    Dewhirst, Floyd E; Klein, Erin A; Bennett, Marie-Louise; Croft, Julie M; Harris, Stephen J; Marshall-Jones, Zoe V

    2015-02-25

    The human oral microbiome is known to play a significant role in human health and disease. While less well studied, the feline oral microbiome is thought to play a similarly important role. To determine roles oral bacteria play in health and disease, one first has to be able to accurately identify bacterial species present. 16S rRNA gene sequence information is widely used for molecular identification of bacteria and is also useful for establishing the taxonomy of novel species. The objective of this research was to obtain full 16S rRNA gene reference sequences for feline oral bacteria, place the sequences in species-level phylotypes, and create a curated 16S rRNA based taxonomy for common feline oral bacteria. Clone libraries were produced using "universal" and phylum-selective PCR primers and DNA from pooled subgingival plaque from healthy and periodontally diseased cats. Bacteria in subgingival samples were also cultivated to obtain isolates. Full-length 16S rDNA sequences were determined for clones and isolates that represent 171 feline oral taxa. A provisional curated taxonomy was developed based on the position of each taxon in 16S rRNA phylogenetic trees. The feline oral microbiome curated taxonomy and 16S rRNA gene reference set will allow investigators to refer to precisely defined bacterial taxa. A provisional name such as "Propionibacterium sp. feline oral taxon FOT-327" is an anchor to which clone, strain or GenBank names or accession numbers can point. Future next-generation-sequencing studies of feline oral bacteria will be able to map reads to taxonomically curated full-length 16S rRNA gene sequences. PMID:25523504

  15. Phylogenetic Diversity of Lactic Acid Bacteria Associated with Paddy Rice Silage as Determined by 16S Ribosomal DNA Analysis

    PubMed Central

    Ennahar, Saïd; Cai, Yimin; Fujita, Yasuhito

    2003-01-01

    A total of 161 low-G+C-content gram-positive bacteria isolated from whole-crop paddy rice silage were classified and subjected to phenotypic and genetic analyses. Based on morphological and biochemical characters, these presumptive lactic acid bacterium (LAB) isolates were divided into 10 groups that included members of the genera Enterococcus, Lactobacillus, Lactococcus, Leuconostoc, Pediococcus, and Weissella. Analysis of the 16S ribosomal DNA (rDNA) was used to confirm the presence of the predominant groups indicated by phenotypic analysis and to determine the phylogenetic affiliation of representative strains. The virtually complete 16S rRNA gene was PCR amplified and sequenced. The sequences from the various LAB isolates showed high degrees of similarity to those of the GenBank reference strains (between 98.7 and 99.8%). Phylogenetic trees based on the 16S rDNA sequence displayed high consistency, with nodes supported by high bootstrap values. With the exception of one species, the genetic data was in agreement with the phenotypic identification. The prevalent LAB, predominantly homofermentative (66%), consisted of Lactobacillus plantarum (24%), Lactococcus lactis (22%), Leuconostoc pseudomesenteroides (20%), Pediococcus acidilactici (11%), Lactobacillus brevis (11%), Enterococcus faecalis (7%), Weissella kimchii (3%), and Pediococcus pentosaceus (2%). The present study, the first to fully document rice-associated LAB, showed a very diverse community of LAB with a relatively high number of species involved in the fermentation process of paddy rice silage. The comprehensive 16S rDNA-based approach to describing LAB community structure was valuable in revealing the large diversity of bacteria inhabiting paddy rice silage and enabling the future design of appropriate inoculants aimed at improving its fermentation quality. PMID:12514026

  16. Identification, Detection, and Enumeration of Human Bifidobacterium Species by PCR Targeting the Transaldolase Gene

    PubMed Central

    Requena, Teresa; Burton, Jeremy; Matsuki, Takahiro; Munro, Karen; Simon, Mary Alice; Tanaka, Ryuichiro; Watanabe, Koichi; Tannock, Gerald W.

    2002-01-01

    Methods that enabled the identification, detection, and enumeration of Bifidobacterium species by PCR targeting the transaldolase gene were tested. Bifidobacterial species isolated from the feces of human adults and babies were identified by PCR amplification of a 301-bp transaldolase gene sequence and comparison of the relative migrations of the DNA fragments in denaturing gradient gel electrophoresis (DGGE). Two subtypes of Bifidobacterium longum, five subtypes of Bifidobacterium adolescentis, and two subtypes of Bifidobacterium pseudocatenulatum could be differentiated using PCR-DGGE. Bifidobacterium angulatum and B. catenulatum type cultures could not be differentiated from each other. Bifidobacterial species were also detected directly in fecal samples by this combination of PCR and DGGE. The number of species detected was less than that detected by PCR using species-specific primers targeting 16S ribosomal DNA (rDNA). Real-time quantitative PCR targeting a 110-bp transaldolase gene sequence was used to enumerate bifidobacteria in fecal samples. Real-time quantitative PCR measurements of bifidobacteria in fecal samples from adults correlated well with results obtained by culture when either a 16S rDNA sequence or the transaldolase gene sequence was targeted. In the case of samples from infants, 16S rDNA-targeted PCR was superior to PCR targeting the transaldolase gene for the quantification of bifidobacterial populations. PMID:11976117

  17. 16S rDNA-based probes for two polycyclic aromatic hydrocarbon (PAH)-degrading soil Mycobacteria

    SciTech Connect

    Govindaswami, M.; Feldhake, D.J.; Loper, J.C.

    1994-12-31

    PAHs are a class of widespread pollutants, some of which have been shown to be genotoxic, hence the fate of these compounds in the environment is of considerable interest. Research on the biodegradation of 4 and 5 ring PAHs has been limited by the general lack of microbial isolates or consortia which can completely degrade these toxicants. Heitkamp and Cerniglia have described an oxidative soil Mycobacterium-strain PYR-1 that metabolizes pyrene and fluoranthene more rapidly than the 2 and 3 ring naphthalene and phenanthrene; although some metabolites of benzo-(a)-pyrene (BaP) were detected, no mineralization of BaP was observed. In 1991 Grosser et al. reported the isolation of a Mycobacterium sp. which mineralizes pyrene and also causing some mineralization of BaP. Their study describes a comparative analysis of these two strains, which show very similar colony morphology, growth rate and yellow-orange pigmentation. Genetic differences were shown by DNA amplification fingerprinting (DAF) using two arbitrary GC-rich octanucleotide primers, and by sequence comparison of PCR amplified 16S rDNA, although both strains show similarity closest to that of the genus Mycobacteria. These 16S rDNA sequences are in use for the construction of strain-specific DNA probes to monitor the presence, survival and growth of these isolates in PAH-contaminated soils in studies of biodegradation.

  18. A Real-Time PCR Assay Based on 5.8S rRNA Gene (5.8S rDNA) for Rapid Detection of Candida from Whole Blood Samples.

    PubMed

    Guo, Yi; Yang, Jing-Xian; Liang, Guo-Wei

    2016-06-01

    The prevalence of Candida in bloodstream infections (BSIs) has increased. To date, the identification of Candida in BSIs still mainly relies on blood culture and serological tests, but they have various limitations. Therefore, a real-time PCR assay for the detection of Candida from whole blood is presented. The unique primers/probe system was designed on 5.8S rRNA gene (5.8S rDNA) of Candida genus. The analytical sensitivity was determined by numbers of positive PCRs in 12 repetitions. At the concentration of 10(1) CFU/ml blood, positive PCR rates of 100 % were obtained for C. albicans, C. parapsilosis, C. tropicalis, and C. krusei. The detection rate for C. glabrata was 75 % at 10(1) CFU/ml blood. The reaction specificity was 100 % when evaluating the assay using DNA samples from clinical isolates and human blood. The maximum CVs of intra-assay and inter-assay for the detection limit were 1.22 and 2.22 %, respectively. To assess the clinical applicability, 328 blood samples from 82 patients were prospectively tested and real-time PCR results were compared with results from blood culture. Diagnostic sensitivity of the PCR was 100 % using as gold standard blood culture, and specificity was 98.4 %. Our data suggest that the developed assay can be used in clinical laboratories as an accurate and rapid screening test for the Candida from whole blood. Although further evaluation is warranted, our assay holds promise for earlier diagnosis of candidemia. PMID:26687075

  19. Characterization of polybacterial clinical samples using a set of group-specific broad-range primers targeting the 16S rRNA gene followed by DNA sequencing and RipSeq analysis

    PubMed Central

    Lekang, Katrine; Langeland, Nina; Wiker, Harald G.

    2011-01-01

    The standard use of a single universal broad-range PCR in direct 16S rDNA sequencing from polybacterial samples leaves the minor constituents at risk of remaining undetected because all bacterial DNA will be competing for the same reagents. In this article we introduce a set of three broad-range group-specific 16S rDNA PCRs that together cover the clinically relevant bacteria and apply them in the investigation of 25 polybacterial clinical samples. Mixed DNA chromatograms from samples containing more than one species per primer group were analysed using RipSeq Mixed (iSentio, Norway), a web-based application for the interpretation of chromatograms containing up to three different species. The group-specific PCRs reduced complexity in the resulting DNA chromatograms and made the assay more sensitive in situations with unequal species concentrations. Together this allowed for identification of a significantly higher number of bacterial species than did standard direct sequencing with a single universal primer pair and RipSeq analysis (95 vs 51). The method could improve microbiological diagnostics for important groups of patients and can be established in any laboratory with experience in direct 16S rDNA sequencing. PMID:21436365

  20. Worldwide distribution of Nitrosococcus oceani, a marine ammonia-oxidizing gamma-proteobacterium, detected by PCR and sequencing of 16S rRNA and amoA genes.

    PubMed

    Ward, Bess B; O'Mullan, Gregory D

    2002-08-01

    Diversity of cultured ammonia-oxidizing bacteria in the gamma-subdivision of the Proteobacteria was investigated by using strains isolated from various parts of the world ocean. All the strains were very similar to each other on the basis of the sequences of both the 16S rRNA and ammonia monooxygenase genes and could be characterized as a single species. Sequences were also cloned directly from environmental DNA from coastal Pacific and Atlantic sites, and these sequences represented the first Nitrosococcus oceani-like sequences obtained directly from the ocean. Most of the environmental sequences clustered tightly with those of the cultivated strains, but some sequences could represent new species of NITROSOCOCCUS: These findings imply that organisms similar to the cultivated N. oceani strains have a worldwide distribution. PMID:12147525

  1. The specific isolation of complete 5S rDNA units from chromosome 1A of hexaploid, tetraploid, and diploid wheat species using PCR with head-to-head oriented primers.

    PubMed

    Van Campenhout, S; Stappen, J V; Volckaert, G

    2001-08-01

    The presence of 5S rDNA units on chromosome 1A of Triticum aestivum was shown by the development of a specific PCR test, using head-to-head oriented primers. This primer set allowed the amplification of complete 5S DNA units and was used to isolate SS-Rrna-A1 sequences from polyploid and diploid wheat species. Multiple-alignment and parsimony analyses of the 132 sequences divided the sequences into four types. The isolates from T. aestivum and the tetraploid species (T. dicoccoides, T. dicoccum, T durum, T. araraticum, and T timopheevi) were all of one type, which was shown to be closely related to the type mainly characteristic for T. urartu. The other two types were isolated exclusively from the diploid species T. monococcum, T aegilopoides, T. thaoudar, and T. sinskajae and the hexaploid species T. zhukovski. Triticum monococcum was the only species for which representatives of each of the four sequence types were found to be present. Further, we discuss the possible multicluster arrangement of the 5S-Rrna-A1 array. PMID:11550886

  2. Efficient Detection of Pathogenic Leptospires Using 16S Ribosomal RNA

    PubMed Central

    Lindow, Janet; Wunder, Elsio A.; Reis, Mitermayer G.; Usmani-Brown, Sahar; Ledizet, Michel; Ko, Albert; Pal, Utpal

    2015-01-01

    Pathogenic Leptospira species cause a prevalent yet neglected zoonotic disease with mild to life-threatening complications in a variety of susceptible animals and humans. Diagnosis of leptospirosis, which primarily relies on antiquated serotyping methods, is particularly challenging due to presentation of non-specific symptoms shared by other febrile illnesses, often leading to misdiagnosis. Initiation of antimicrobial therapy during early infection to prevent more serious complications of disseminated infection is often not performed because of a lack of efficient diagnostic tests. Here we report that specific regions of leptospiral 16S ribosomal RNA molecules constitute a novel and efficient diagnostic target for PCR-based detection of pathogenic Leptospira serovars. Our diagnostic test using spiked human blood was at least 100-fold more sensitive than corresponding leptospiral DNA-based quantitative PCR assays, targeting the same 16S nucleotide sequence in the RNA and DNA molecules. The sensitivity and specificity of our RNA assay against laboratory-confirmed human leptospirosis clinical samples were 64% and 100%, respectively, which was superior then an established parallel DNA detection assay. Remarkably, we discovered that 16S transcripts remain appreciably stable ex vivo, including untreated and stored human blood samples, further highlighting their use for clinical detection of L. interrogans. Together, these studies underscore a novel utility of RNA targets, specifically 16S rRNA, for development of PCR-based modalities for diagnosis of human leptospirosis, and also may serve as paradigm for detection of additional bacterial pathogens for which early diagnosis is warranted. PMID:26091292

  3. [Determination of 16S rRNA gene sequence for a new ANAMMOX bacterial species].

    PubMed

    Zu, Bo; Zhang, Dai-jun; Yan, Qing

    2008-02-01

    The anaerobic ammonium oxidation (ANAMMOX) activity of the sludge was about 9.84 x 10(-4) mg x (mg x h)(-1) by measuring the simultaneous consumption of ammonium and nitrite under anoxic conditions in the batch tests. The consumption of NO2(-) -N and NH4+ -N was 1.311 for ANAMMOX bacteria. The partial 16S rDNA sequence was obtained by using molecule biology methods. Crude DNA of the total bacteria in granular sludge from EGSB reactor was extracted and purified. Then, PCR amplification by using specific primer, clone and sequence determination was performed. ANAMMOX bacterial species(anaerobic ammonium-oxidizing Planctomycete cquenviron-1) which was enrichment cultivated from EGSB reactor were the same genera with Candidatus "Anammoxoglobus propionicus" and Candidatus "Jettenia asiatica" by analyzing phylogenetic tree. The maximum identities of anaerobic ammonium-oxidizing Planctomycete cquenviron-1 with other ANAMMOX bacterial species was about 93%. The results showed that a new ANAMMOX bacterial species which was enrichment cultivated from EGSB reactor was found and anaerobic ammonium-oxidizing Planctomycete cquenviron-1 was denominated. PMID:18613522

  4. 16S Ribosomal DNA Sequence Analysis of a Large Collection of Environmental and Clinical Unidentifiable Bacterial Isolates

    PubMed Central

    Drancourt, Michel; Bollet, Claude; Carlioz, Antoine; Martelin, Rolland; Gayral, Jean-Pierre; Raoult, Didier

    2000-01-01

    Some bacteria are difficult to identify with phenotypic identification schemes commonly used outside reference laboratories. 16S ribosomal DNA (rDNA)-based identification of bacteria potentially offers a useful alternative when phenotypic characterization methods fail. However, as yet, the usefulness of 16S rDNA sequence analysis in the identification of conventionally unidentifiable isolates has not been evaluated with a large collection of isolates. In this study, we evaluated the utility of 16S rDNA sequencing as a means to identify a collection of 177 such isolates obtained from environmental, veterinary, and clinical sources. For 159 isolates (89.8%) there was at least one sequence in GenBank that yielded a similarity score of ≥97%, and for 139 isolates (78.5%) there was at least one sequence in GenBank that yielded a similarity score of ≥99%. These similarity score values were used to defined identification at the genus and species levels, respectively. For isolates identified to the species level, conventional identification failed to produce accurate results because of inappropriate biochemical profile determination in 76 isolates (58.7%), Gram staining in 16 isolates (11.6%), oxidase and catalase activity determination in 5 isolates (3.6%) and growth requirement determination in 2 isolates (1.5%). Eighteen isolates (10.2%) remained unidentifiable by 16S rDNA sequence analysis but were probably prototype isolates of new species. These isolates originated mainly from environmental sources (P = 0.07). The 16S rDNA approach failed to identify Enterobacter and Pantoea isolates to the species level (P = 0.04; odds ratio = 0.32 [95% confidence interval, 0.10 to 1.14]). Elsewhere, the usefulness of 16S rDNA sequencing was compromised by the presence of 16S rDNA sequences with >1% undetermined positions in the databases. Unlike phenotypic identification, which can be modified by the variability of expression of characters, 16S rDNA sequencing provides

  5. Molecular and functional diversity of PGPR fluorescent Pseudomonads based on 16S rDNA-RFLP and RAPD markers.

    PubMed

    Singh, Bhim Pratap

    2015-09-01

    The genetic and functional diversity of plant growth promoting rhizobacterial (PGPR) fluorescent pseudomonads associated with chickpea (Cicer arietinum L.) rhizosphere was analyzed. In total, 34 isolates along with two reference isolates were screened for various plant growth promoting traits (phosphorous solubilization, ACC deaminase, HCN, IAA and siderophore productions) and antagonist activity against four fungal phytopathogens and three bacterial pathogens. Most of the isolates, that showed PGPR activity, also showed antagonistic activity against all the three fungal pathogens. The genetic relationship was assessed by using random amplification of polymorphic DNA (RAPD) and PCR-restriction fragment length polymorphism (16S rDNA-RFLP). Relationship between both the markers was analyzed based on mantel test and a negative correlation was observed. The study concluded that PGPR traits appeared to be strain specific rather than specific to any phylogenetic group. The study also reported that 16S rDNA based profiling differentiated PGPR fluorescent Pseudomonas on the basis of location rather than biological trait. RAPD profiling could be useful to differentiate among the closely related isolates. The genetic and functional diversity of fluorescent pseudomonads, associated with the chickpea rhizosphere, has useful ecological role and potential utilization in sustainable agriculture. PMID:26521562

  6. PCR-DGGE analysis of intestinal bacteria and effect of Bacillus spp. on intestinal microbial diversity in kuruma shrimp ( Marsupenaeus japonicus)

    NASA Astrophysics Data System (ADS)

    Liu, Huaide; Liu, Mei; Wang, Baojie; Jiang, Keyong; Jiang, Shan); Sun, Shujuan; Wang, Lei

    2010-07-01

    In this study, the intestinal microbiota of kuruma shrimp ( Marsupenaeus japonicus) was examined by molecular analysis of the 16S rDNA to identify the dominant intestinal bacteria and to investigate the effects of Bacillus spp. on intestinal microbial diversity. Samples of the intestines of kuruma shrimp fed normal feed and Bacillus spp. amended feed. PCR and denaturing gradient gel electrophoresis (DGGE) analyses were then performed on DNA extracted directly from the guts. Population fingerprints of the predominant organisms were generated by DGGE analysis of the universal V3 16S rDNA amplicons, and distinct bands in the gels were sequenced. The results suggested that the gut of kuruma shrimp was dominated by Vibrio sp. and uncultured gamma proteobacterium. Overall, the results of this study suggest that PCR-DGGE is a possible method of studying the intestinal microbial diversity of shrimp.

  7. Nested-quantitative PCR approach with improved sensitivity for the detection of low titer levels of Candidatus Liberibacter asiaticus in the Asian citrus psyllid, Diaphorina citri Kuwayama.

    PubMed

    Coy, M R; Hoffmann, M; Kingdom Gibbard, H N; Kuhns, E H; Pelz-Stelinski, K S; Stelinski, L L

    2014-07-01

    Candidatus Liberibacter asiaticus (CLas) is a phloem-limited bacterium transmitted by the Asian citrus psyllid, Diaphorina citri, and the presumptive causal agent of citrus greening disease. The current method of detection for CLas within plant and insect samples is by a presence/absence qPCR assay using the CLas 16S rDNA gene target. Although qPCR is highly sensitive, low bacterial titers or suboptimal qPCR conditions can result in false-negatives. Using a nested qPCR assay, we determined the false-negative rate of the 16S presence/absence qPCR assay was greater than 50%. Studies to determine the performance parameters of the qPCR assays for CLas 16S and Wingless (Wg), the D. citri endogenous gene, using plasmid and psyllid DNA, revealed suboptimal and variable performance of the 16S assay in psyllid samples. Average efficiencies and sensitivity limits of the plasmid assays were 99.0% and 2.7 copies of template for Wg, respectively, and 98.5% and 2.2-22.1 copies for 16S, respectively. Variability in efficiency was significantly greater in psyllid samples for both gene targets compared to the corresponding plasmid assays, and efficiencies as low as 76% were obtained for 16S. A secondary structure analysis revealed the formation of two stem-loop structures that block the forward and probe binding sites in the 16S template, which could hinder amplification. In summary, our results suggest that suboptimal qPCR efficiency is not uncommon for the 16S presence/absence qPCR assay, which combined with lowCLas titers in some samples, could contribute significantly to the under-reporting of CLas infection in psyllid and plant samples. PMID:24769405

  8. Characterization of cucumber fermentation spoilage bacteria by enrichment culture and 16S rDNA cloning

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Commercial cucumber fermentations are typically carried out in 40000 L fermentation tanks. A secondary fermentation can occur after sugars are consumed that results in the formation of acetic, propionic, and butyric acids, concomitantly with the loss of lactic acid and an increase in pH. Spoilage fe...

  9. PHYLOGENETIC AFFILIATION OF WATER DISTRIBUTION SYSTEM BACTERIAL ISOLATES USING 16S RDNA SEQUENCE ANALYSIS

    EPA Science Inventory

    In a previously described study, only 15% of the bacterial strains isolated from a water distribution system (WDS) grown on R2A agar were identifiable using fatty acid methyl esthers (FAME) profiling. The lack of success was attributed to the use of fatty acid databases of bacter...

  10. Characterization of Bacterial Communities in Selected Smokeless Tobacco Products Using 16S rDNA Analysis

    PubMed Central

    Tyx, Robert E.; Stanfill, Stephen B.; Keong, Lisa M.; Rivera, Angel J.; Satten, Glen A.; Watson, Clifford H.

    2016-01-01

    The bacterial communities present in smokeless tobacco (ST) products have not previously reported. In this study, we used Next Generation Sequencing to study the bacteria present in U.S.-made dry snuff, moist snuff and Sudanese toombak. Sample diversity and taxonomic abundances were investigated in these products. A total of 33 bacterial families from four phyla, Actinobacteria, Firmicutes, Proteobacteria and Bacteroidetes, were identified. U.S.-produced dry snuff products contained a diverse distribution of all four phyla. Moist snuff products were dominated by Firmicutes. Toombak samples contained mainly Actinobacteria and Firmicutes (Aerococcaceae, Enterococcaceae, and Staphylococcaceae). The program PICRUSt (Phylogenetic Investigation of Communities by Reconstruction of Unobserved States) was used to impute the prevalence of genes encoding selected bacterial toxins, antibiotic resistance genes and other pro-inflammatory molecules. PICRUSt also predicted the presence of specific nitrate reductase genes, whose products can contribute to the formation of carcinogenic nitrosamines. Characterization of microbial community abundances and their associated genomes gives us an indication of the presence or absence of pathways of interest and can be used as a foundation for further investigation into the unique microbiological and chemical environments of smokeless tobacco products. PMID:26784944

  11. TURKEY FECAL MICROBIAL COMMUNITY STRUCTURE AND ECOLOGICAL FUNCTIONS REVEALED BY 16S RDNA AND METAGENOME SEQUENCES

    EPA Science Inventory

    Turkey feces are an important source of fecal waste in the United States. With the exception of isolated studies on bacterial pathogens, little is known about the type of bacteria inhabiting the turkey gut. In order to understand the microbial diversity and functional genes assoc...

  12. Development of a real-time PCR assay (SYBR Green I) for rapid identification and quantification of scyphomedusae Aurelia sp.1 planulae

    NASA Astrophysics Data System (ADS)

    Wang, Jianyan; Zhen, Yu; Mi, Tiezhu; Yu, Zhigang; Wang, Guoshan

    2015-07-01

    The complicated life cycle of Aurelia spp., comprising benthic asexually-reproducing polyps and sexually-reproducing medusae, makes it hard for researchers to identify and track them, especially for early stage individuals, such as planulae. To solve this problem, we developed a real-time PCR assay (SYBR Green I) to identify planulae in both cultured and natural seawater samples. Species-specific primers targeting Aurelia sp.1 mitochondrial 16S rDNA (mt 16S rDNA) regions were designed. Using a calibration curve constructed with plasmids containing the Aurelia sp.1 mt 16S rDNA fragment and a standard curve for planulae, the absolute number of mt 16S rDNA copies per planula was determined and from that the total number of planulae per sample was estimated. For the field samples, a 100-fold dilution of the sample DNA combined with a final concentration of 0.2 μg/μL BSA in the PCR reaction mixture was used to remove real-time PCR inhibitors. Samples collected in Jiaozhou Bay from July to September 2012 were subsequently analyzed using this assay. Peak Aurelia sp.1 planula abundance occurred in July 2012 at stations near Hongdao Island and Qingdao offshore; abundances were very low in August and September. The real-time PCR assay (SYBR Green I) developed here negates the need for traditional microscopic identification, which is laborious and time-consuming, and can detect and quantify jellyfish planulae in field plankton samples rapidly and specifically.

  13. Identification of Nonpoint Sources of Fecal Pollution in Coastal Waters by Using Host-Specific 16S Ribosomal DNA Genetic Markers from Fecal Anaerobes

    PubMed Central

    Bernhard, Anne E.; Field, Katharine G.

    2000-01-01

    We describe a new PCR-based method for distinguishing human and cow fecal contamination in coastal waters without culturing indicator organisms, and we show that the method can be used to track bacterial marker sequences in complex environments. We identified two human-specific genetic markers and five cow-specific genetic markers in fecal samples by amplifying 16S ribosomal DNA (rDNA) fragments from members of the genus Bifidobacterium and the Bacteroides-Prevotella group and performing length heterogeneity PCR and terminal restriction fragment length polymorphism analyses. Host-specific patterns suggested that there are species composition differences in the Bifidobacterium and Bacteroides-Prevotella populations of human and cow feces. The patterns were highly reproducible among different hosts belonging to the same species. Additionally, all host-specific genetic markers were detected in water samples collected from areas frequently contaminated with fecal pollution. Ease of detection and longer survival in water made Bacteroides-Prevotella indicators better than Bifidobacterium indicators. Fecal 16S rDNA sequences corresponding to our Bacteroides-Prevotella markers comprised closely related gene clusters, none of which exactly matched previously published Bacteroides or Prevotella sequences. Our method detected host-specific markers in water at pollutant concentrations of 2.8 × 10−5 to 2.8 × 10−7 g (dry weight) of feces/liter and 6.8 × 10−7 g (dry weight) of sewage/liter. Although our aim was to identify nonpoint sources of fecal contamination, the method described here should be widely applicable for monitoring spatial and temporal fluctuations in specific bacterial groups in natural environments. PMID:10742246

  14. Diagnosis of Bacterial Bloodstream Infections: A 16S Metagenomics Approach

    PubMed Central

    Van Puyvelde, Sandra; De Block, Tessa; Maltha, Jessica; Palpouguini, Lompo; Tahita, Marc; Tinto, Halidou; Jacobs, Jan; Deborggraeve, Stijn

    2016-01-01

    Background Bacterial bloodstream infection (bBSI) is one of the leading causes of death in critically ill patients and accurate diagnosis is therefore crucial. We here report a 16S metagenomics approach for diagnosing and understanding bBSI. Methodology/Principal Findings The proof-of-concept was delivered in 75 children (median age 15 months) with severe febrile illness in Burkina Faso. Standard blood culture and malaria testing were conducted at the time of hospital admission. 16S metagenomics testing was done retrospectively and in duplicate on the blood of all patients. Total DNA was extracted from the blood and the V3–V4 regions of the bacterial 16S rRNA genes were amplified by PCR and deep sequenced on an Illumina MiSeq sequencer. Paired reads were curated, taxonomically labeled, and filtered. Blood culture diagnosed bBSI in 12 patients, but this number increased to 22 patients when combining blood culture and 16S metagenomics results. In addition to superior sensitivity compared to standard blood culture, 16S metagenomics revealed important novel insights into the nature of bBSI. Patients with acute malaria or recovering from malaria had a 7-fold higher risk of presenting polymicrobial bloodstream infections compared to patients with no recent malaria diagnosis (p-value = 0.046). Malaria is known to affect epithelial gut function and may thus facilitate bacterial translocation from the intestinal lumen to the blood. Importantly, patients with such polymicrobial blood infections showed a 9-fold higher risk factor for not surviving their febrile illness (p-value = 0.030). Conclusions/Significance Our data demonstrate that 16S metagenomics is a powerful approach for the diagnosis and understanding of bBSI. This proof-of-concept study also showed that appropriate control samples are crucial to detect background signals due to environmental contamination. PMID:26927306

  15. IDENTIFICATION OF ACTIVE BACTERIAL COMMUNITIES IN A MODEL DRINKING WATER BIOFILM SYSTEM USING 16S RRNA-BASED CLONE LIBRARIES

    EPA Science Inventory

    Recent phylogenetic studies have used DNA as the target molecule for the development of environmental 16S rDNA clone libraries. As DNA may persist in the environment, DNA-based libraries cannot be used to identify metabolically active bacteria in water systems. In this study, a...

  16. Design and Evaluation of PCR Primers for Analysis of Bacterial Populations in Wine by Denaturing Gradient Gel Electrophoresis

    PubMed Central

    Lopez, Isabel; Ruiz-Larrea, Fernanda; Cocolin, Luca; Orr, Erica; Phister, Trevor; Marshall, Megan; VanderGheynst, Jean; Mills, David A.

    2003-01-01

    Denaturing gradient gel electrophoresis (DGGE) of PCR-amplified ribosomal DNA (rDNA) is routinely used to compare levels of diversity of microbial communities and to monitor population dynamics. While using PCR-DGGE to examine the bacteria in wine fermentations, we noted that several commonly used PCR primers for amplifying bacterial 16S rDNA also coamplified yeast, fungal, or plant DNA present in samples. Unfortunately, amplification of nonbacterial DNA can result in a masking of bacterial populations in DGGE profiles. To surmount this problem, we developed two new primer sets for specific amplification of bacterial 16S rDNA in wine fermentation samples without amplification of eukaryotic DNA. One primer set, termed WLAB1 and WLAB2, amplified lactic acid bacteria, while another, termed WBAC1 and WBAC2, amplified both lactic acid bacterial and acetic acid bacterial populations found in wine. Primer specificity and efficacy were examined with DNA isolated from numerous bacterial, yeast, and fungal species commonly found in wine and must samples. Importantly, both primer sets effectively distinguished bacterial species in wine containing mixtures of yeast and bacteria. PMID:14602643

  17. Direct Analysis of Genes Encoding 16S rRNA from Complex Communities Reveals Many Novel Molecular Species within the Human Gut

    PubMed Central

    Suau, Antonia; Bonnet, Régis; Sutren, Malène; Godon, Jean-Jacques; Gibson, Glenn R.; Collins, Matthew D.; Doré, Joel

    1999-01-01

    The human intestinal tract harbors a complex microbial ecosystem which plays a key role in nutrition and health. Although this microbiota has been studied in great detail by culture techniques, microscopic counts on human feces suggest that 60 to 80% of the observable bacteria cannot be cultivated. Using comparative analysis of cloned 16S rRNA gene (rDNA) sequences, we have investigated the bacterial diversity (both cultivated and noncultivated bacteria) within an adult-male fecal sample. The 284 clones obtained from 10-cycle PCR were classified into 82 molecular species (at least 98% similarity). Three phylogenetic groups contained 95% of the clones: the Bacteroides group, the Clostridium coccoides group, and the Clostridium leptum subgroup. The remaining clones were distributed among a variety of phylogenetic clusters. Only 24% of the molecular species recovered corresponded to described organisms (those whose sequences were available in public databases), and all of these were established members of the dominant human fecal flora (e.g., Bacteroides thetaiotaomicron, Fusobacterium prausnitzii, and Eubacterium rectale). However, the majority of generated rDNA sequences (76%) did not correspond to known organisms and clearly derived from hitherto unknown species within this human gut microflora. PMID:10543789

  18. Three Group-I introns in 18S rDNA of Endosymbiotic Algae of Paramecium bursaria from Japan

    NASA Astrophysics Data System (ADS)

    Hoshina, Ryo; Kamako, Shin-ichiro; Imamura, Nobutaka

    2004-08-01

    In the nuclear encoded small subunit ribosomal DNA (18S rDNA) of symbiotic alga of Paramecium bursaria (F36 collected in Japan) possesses three intron-like insertions (Hoshina et al., unpubl. data, 2003). The present study confirmed these exact lengths and insertion sites by reverse transcription-PCR. Two of them were inserted at Escherichia coli 16S rRNA genic position 943 and 1512 that are frequent intron insertion positions, but another insertion position (nearly 1370) was the first finding. Their secondary structures suggested they belong to Group-I intron; one belongs to subgroup IE, others belong to subgroup IC1. Similarity search indicated these introns are ancestral ones.

  19. Nested PCR for ultrasensitive detection of the potato ring rot bacterium, Clavibacter michiganensis subsp. sepedonicus.

    PubMed Central

    Lee, I M; Bartoszyk, I M; Gundersen, D E; Mogen, B; Davis, R E

    1997-01-01

    Oligonucleotide primers derived from sequences of the 16S rRNA gene (CMR16F1, CMR16R1, CMR16F2, and CMR16R2) and insertion element IS1121 of Clavibacter michiganensis subsp. sepedonicus (CMSIF1, CMSIR1, CMSIF2, and CMISR2) were used in nested PCR to detect the potato ring rot bacterium C. michiganensis subsp. sepedonicus. Nested PCR with primer pair CMSIF1-CMSIR1 followed by primer pair CMSIF2-CMSIR2 specifically detected C. michiganensis subsp. sepedonicus, while nested PCR with CMR16F1-CMR16R1 followed by CMR16F2-CMR16R2 detected C. michiganensis subsp. sepedonicus and the other C. michiganensis subspecies. In the latter case, C. michiganensis subsp. sepedonicus can be differentiated from the other subspecies by restriction fragment length polymorphism (RFLP) analyses of the nested PCR products (16S rDNA sequences). The nested PCR assays developed in this work allow ultrasensitive detection of very low titers of C. michiganensis subsp. sepedonicus which may be present in symptomiess potato plants or tubers and which cannot be readily detected by direct PCR (single PCR amplification). RFLP analysis of PCR products provides for an unambiguous confirmation of the identify of C. michiganensis subsp. sepedonicus. PMID:9212412

  20. Differentiation of Listeria monocytogenes and Listeria innocua by 16S rRNA genes and intraspecies discrimination of Listeria monocytogenes strains by random amplified polymorphic DNA polymorphisms.

    PubMed Central

    Czajka, J; Bsat, N; Piani, M; Russ, W; Sultana, K; Wiedmann, M; Whitaker, R; Batt, C A

    1993-01-01

    Differences in the 16S rRNA genes (16S rDNA) which can be used to discriminate Listeria monocytogenes from Listeria innocua have been detected. The 16S rDNA were amplified by polymerase chain reaction with a set of oligonucleotide primers which flank a 1.5-kb fragment. Sequence differences were observed in the V2 region of the 16S rDNA both between L. monocytogenes Scott A and L. innocua and between different L. monocytogenes serotypes. Although L. monocytogenes SLCC2371 had the same V2 region sequence as L. innocua, the two species were different within the V9 region at nucleotides 1259 and 1292, in agreement with previous studies (R.-F. Wang, W.-W. Cao, and M.G. Johnson, Appl. Environ. Microbiol. 57:3666-3670, 1991). Intraspecies discrimination of L. monocytogenes strains was achieved by using the patterns generated by random amplified polymorphic DNA primers. Although some distinction can be made within the L. monocytogenes species by their 16S rDNA sequence, a far greater discrimination within species could be made by generating random amplified polymorphic DNA patterns from chromosomal DNA. By using a number of 10-bp primers, unique patterns for each isolate which in all cases examined differentiate between various L. monocytogenes serotypes, even though they may have the same 16S rRNA sequences, could be generated. Images PMID:8439157

  1. Analysis of ammonia-oxidizing bacteria from hypersaline Mono Lake, California, on the basis of 16S rRNA sequences.

    PubMed

    Ward, B B; Martino, D P; Diaz, M C; Joye, S B

    2000-07-01

    Ammonia-oxidizing bacteria were detected by PCR amplification of DNA extracted from filtered water samples throughout the water column of Mono Lake, California. Ammonia-oxidizing members of the beta subdivision of the division Proteobacteria (beta-subdivision Proteobacteria) were detected using previously characterized PCR primers; target sequences were detected by direct amplification in both surface water and below the chemocline. Denaturing gradient gel electrophoresis analysis indicated the presence of at least four different beta-subdivision ammonia oxidizers in some samples. Subsequent sequencing of amplified 16S rDNA fragments verified the presence of sequences very similar to those of cultured Nitrosomonas strains. Two separate analyses, carried out under different conditions (different reagents, locations, PCR machines, sequencers, etc.), 2 years apart, detected similar ranges of sequence diversity in these samples. It seems likely that the physiological diversity of nitrifiers exceeds the diversity of their ribosomal sequences and that these sequences represent members of the Nitrosomonas europaea group that are acclimated to alkaline, high-salinity environments. Primers specific for Nitrosococcus oceanus, a marine ammonia-oxidizing bacterium in the gamma subdivision of the Proteobacteria, did not amplify target from any samples. PMID:10877781

  2. Comparative Phylogenetic Assignment of Environmental Sequences of Genes Encoding 16S rRNA and Numerically Abundant Culturable Bacteria from an Anoxic Rice Paddy Soil

    PubMed Central

    Hengstmann, Ulf; Chin, Kuk-Jeong; Janssen, Peter H.; Liesack, Werner

    1999-01-01

    We used both cultivation and direct recovery of bacterial 16S rRNA gene (rDNA) sequences to investigate the structure of the bacterial community in anoxic rice paddy soil. Isolation and phenotypic characterization of 19 saccharolytic and cellulolytic strains are described in the accompanying paper (K.-J. Chin, D. Hahn, U. Hengstmann, W. Liesack, and P. H. Janssen, Appl. Environ. Microbiol. 65:5042–5049, 1999). Here we describe the phylogenetic positions of these strains in relation to 57 environmental 16S rDNA clone sequences. Close matches between the two data sets were obtained for isolates from the culturable populations determined by the most-probable-number counting method to be large (3 × 107 to 2.5 × 108 cells per g [dry weight] of soil). This included matches with 16S rDNA similarity values greater than 98% within distinct lineages of the division Verrucomicrobia (strain PB90-1) and the Cytophaga-Flavobacterium-Bacteroides group (strains XB45 and PB90-2), as well as matches with similarity values greater than 95% within distinct lines of descent of clostridial cluster XIVa (strain XB90) and the family Bacillaceae (strain SB45). In addition, close matches with similarity values greater than 95% were obtained for cloned 16S rDNA sequences and bacteria (strains DR1/8 and RPec1) isolated from the same type of rice paddy soil during previous investigations. The correspondence between culture methods and direct recovery of environmental 16S rDNA suggests that the isolates obtained are representative geno- and phenotypes of predominant bacterial groups which account for 5 to 52% of the total cells in the anoxic rice paddy soil. Furthermore, our findings clearly indicate that a dual approach results in a more objective view of the structural and functional composition of a soil bacterial community than either cultivation or direct recovery of 16S rDNA sequences alone. PMID:10543822

  3. Design of Vibrio 16S rRNA gene specific primers and their application in the analysis of seawater Vibrio community

    NASA Astrophysics Data System (ADS)

    Yong, Liu; Guanpin, Yang; Hualei, Wang; Jixiang, Chen; Xianming, Shi; Guiwei, Zou; Qiwei, Wei; Xiuqin, Sun

    2006-04-01

    The pathogenic species of genus Vibrio cause vibriosis, one of the most prevalent diseases of maricultured animals and seafood consumers. Monitoring their kinetics in the chain of seafood production, processing and consumption is of great importance for food and mariculture safety. In order to enrich Vibrio-representing 16S ribosomal RNA gene (rDNA) fragments and identify these bacteria further real-timely and synchronously among bacterial flora in the chain, a pair of primers that selectively amplify Vibrio 16S rDNA fragments were designed with their specificities and coverage testified in the analysis of seawater Vibrio community. The specificities and coverage of two primers, VF169 and VR744, were determined theoretically among bacterial 16S rDNAs available in GenBank by using BLAST program and practically by amplifying, Vibrio 16S rDNA fragments from seawater DNA. More than 88.3% of sequences in GenBank, which showed identical matches with VR744, belong to Vibrio genus. A total of 33 clones were randomly selected and sequenced. All of the sequences showed their highest similarities to and clustered around those of diverse known Vibrio species. The primers designed are capable of retrieving a wide range of Vibrio 16S rDNA fragments specifically among bacterial flora in seawater, the most important natural environment of seafood cultivation.

  4. 'Candidatus Phytoplasmas pruni', a novel taxon associated with X-disease of stone fruits, Prunus spp.: multilocus characterization based on 16S rRNA, secY, and ribosomal protein genes

    Technology Transfer Automated Retrieval System (TEKTRAN)

    X-disease is one of the most serious diseases known in peach (Prunus persica). Based on RFLP analysis of 16S rRNA gene sequences, peach X-disease phytoplasma strains from eastern and western United States and eastern Canada were classified in 16S rDNA RFLP group 16SrIII, subgroup A. Phylogenetic a...

  5. Application of 16S rDNA-DGGE and plate culture to characterization of bacterial communities associated with the sawfly, Acantholyda erythrocephala (Hymenoptera, Pamphiliidae).

    PubMed

    Zahner, Viviane; Lucarotti, Christopher J; McIntosh, Douglas

    2008-12-01

    Culture-based analysis was employed in parallel with PCR amplification of 16S rDNA, coupled with denaturing gradient gel electrophoresis (DGGE), to profile bacterial species associated with different developmental stages of the pine false webworm (PFW), Acantholyda erythrocephala, a sawfly pest responsible for incidents of severe defoliation in commercially important tree plantations in North America. Culture-based analysis revealed that Pseudomonas spp. along with Bacillus sphaericus and Arthrobacter sp. were the predominant components of the microflora of the internal organs and identified life-stage-specific associations including Photorhabdus temperata with egg and larval samples and a Janthinobacterium sp. with eonymphs. PCR-DGGE confirmed the predominance of Pseudomonas spp. and B. sphaericus in the majority of samples but did not detect Arthrobacter sp., P. temperate, or Janthinobacterium sp. In contrast, DGGE revealed the presence of a Chryseobacterium sp. as the predominant component of the PFW micoflora at all life stages, with the exception of adults. This species had been infrequently cultured, at low levels, from a limited number of samples and the existence of a possible relationship between this bacterium and the PFW had gone unnoticed using the culture-based approach. Our findings highlight the advantages of applying a dual approach to the study of microbe-insect associations and demonstrate that the benefits of one system can be used to overcome some of the limitations of the other. PMID:18769850

  6. TaqMan qPCR for detection and quantification of mitochondrial DNA from toxic pufferfish species.

    PubMed

    Luekasemsuk, Tassanee; Panvisavas, Nathinee; Chaturongakul, Soraya

    2015-08-01

    Outbreaks of pufferfish food poisoning have been reported worldwide, most were from unsuitable food preparation and adulteration. In order to rapidly detect pufferfish adulterant in processed foods, we developed and proposed the use of a TaqMan probe-based quantitative PCR. The designed detection oligos targeted a unique region in 16S rDNA of toxic marine pufferfish in Tetraodontidae Family and gave a positive signal at ≥1.75 pg of genomic DNA. Non-target DNA samples from other fish, chicken, and beef were negative. PMID:26026622

  7. Collection of small subunit (16S- and 16S-like) ribosomal RNA structures: 1994.

    PubMed Central

    Gutell, R R

    1994-01-01

    A collection of diverse 16S and 16S-like rRNA secondary structure diagrams are available. This set of rRNAs contains representative structures from all of the major phylogenetic groupings--Archaea, (eu)Bacteria, and the nucleus, mitochondrion, and chloroplast of Eucarya. Within this broad phylogenetic sampling are examples of the major forms of structural diversity currently known for this class of rRNAs. These structure diagrams are available online through our computer-network WWW server and anonymous ftp, as well as from the author in hardcopy format. PMID:7524024

  8. IM-TORNADO: A Tool for Comparison of 16S Reads from Paired-End Libraries

    PubMed Central

    Jeraldo, Patricio; Kalari, Krishna; Chen, Xianfeng; Bhavsar, Jaysheel; Mangalam, Ashutosh; White, Bryan; Nelson, Heidi; Kocher, Jean-Pierre; Chia, Nicholas

    2014-01-01

    Motivation 16S rDNA hypervariable tag sequencing has become the de facto method for accessing microbial diversity. Illumina paired-end sequencing, which produces two separate reads for each DNA fragment, has become the platform of choice for this application. However, when the two reads do not overlap, existing computational pipelines analyze data from read separately and underutilize the information contained in the paired-end reads. Results We created a workflow known as Illinois Mayo Taxon Organization from RNA Dataset Operations (IM-TORNADO) for processing non-overlapping reads while retaining maximal information content. Using synthetic mock datasets, we show that the use of both reads produced answers with greater correlation to those from full length 16S rDNA when looking at taxonomy, phylogeny, and beta-diversity. Availability and Implementation IM-TORNADO is freely available at http://sourceforge.net/projects/imtornado and produces BIOM format output for cross compatibility with other pipelines such as QIIME, mothur, and phyloseq. PMID:25506826

  9. A system to simultaneously detect tick-borne pathogens based on the variability of the 16S ribosomal genes

    PubMed Central

    2013-01-01

    Background DNA microarrays can be used to quickly and sensitively identify several different pathogens in one step. Our previously developed DNA microarray, based on the detection of variable regions in the 16S rDNA gene (rrs), which are specific for each selected bacterial genus, allowed the concurrent detection of Borrelia spp., Anaplasma spp., Francisella spp., Rickettsia spp. and Coxiella spp. Methods In this study, we developed a comprehensive detection system consisting of a second generation DNA microarray and quantitative PCRs. New oligonucleotide capture probes specific for Borrelia burgdorferi s.l. genospecies and Candidatus Neoehrlichia mikurensis were included. This new DNA microarray system required substantial changes in solution composition, hybridization conditions and post-hybridization washes. Results This second generation chip displayed high specificity and sensitivity. The specificity of the capture probes was tested by hybridizing the DNA microarrays with Cy5-labeled, PCR-generated amplicons encoding the rrs genes of both target and non-target bacteria. The detection limit was determined to be 103 genome copies, which corresponds to 1–2 pg of DNA. A given sample was evaluated as positive if its mean fluorescence was at least 10% of the mean fluorescence of a positive control. Those samples with fluorescence close to the threshold were further analyzed using quantitative PCRs, developed to identify Francisella spp., Rickettsia spp. and Coxiella spp. Like the DNA microarray, the qPCRs were based on the genus specific variable regions of the rrs gene. No unspecific cross-reactions were detected. The detection limit for Francisella spp. was determined to be only 1 genome copy, for Coxiella spp. 10 copies, and for Rickettsia spp., 100 copies. Conclusions Our detection system offers a rapid method for the comprehensive identification of tick-borne bacteria, which is applicable to clinical samples. It can also be used to identify both pathogenic

  10. Use of PCR-DGGE Based Molecular Methods to Analyse Microbial Community Diversity and Stability during the Thermophilic Stages of an ATAD Wastewater Sludge Treatment Process as an Aid to Performance Monitoring

    PubMed Central

    Piterina, Anna V.; Pembroke, J. Tony

    2013-01-01

    PCR and PCR-DGGE techniques have been evaluated to monitor biodiversity indexes within an ATAD (autothermal thermophilic aerobic digestion) system treating domestic sludge for land spread, by examining microbial dynamics in response to elevated temperatures during treatment. The ATAD process utilises a thermophilic population to generate heat and operates at elevated pH due to degradation of sludge solids, thus allowing pasteurisation and stabilisation of the sludge. Genera-specific PCR revealed that Archaea, Eukarya and Fungi decline when the temperature reaches 59°C, while the bacterial lineage constitutes the dominant group at this stage. The bacterial community at the thermophilic stage, its similarity index to the feed material, and the species richness present were evaluated by PCR-DGGE. Parameters such as choice of molecular target (16S rDNA or rpoB genes), and electrophoresis condition, were optimised to maximise the resolution of the method for ATAD. Dynamic analysis of microbial communities was best observed utilising PCR-DGGE analysis of the V6-V8 region of 16S rDNA, while rpoB gene profiles were less informative. Unique thermophilic communities were shown to quickly adapt to process changes, and shown to be quite stable during the process. Such techniques may be used as a monitoring technique for process health and efficiency. PMID:25937969

  11. Sequence heterogeneity in the two 16S rRNA genes of Phormium yellow leaf phytoplasma.

    PubMed Central

    Liefting, L W; Andersen, M T; Beever, R E; Gardner, R C; Forster, R L

    1996-01-01

    Phormium yellow leaf (PYL) phytoplasma causes a lethal disease of the monocotyledon, New Zealand flax (Phormium tenax). The 16S rRNA genes of PYL phytoplasma were amplified from infected flax by PCR and cloned, and the nucleotide sequences were determined. DNA sequencing and Southern hybridization analysis of genomic DNA indicated the presence of two copies of the 16S rRNA gene. The two 16S rRNA genes exhibited sequence heterogeneity in 4 nucleotide positions and could be distinguished by the restriction enzymes BpmI and BsrI. This is the first record in which sequence heterogeneity in the 16S rRNA genes of a phytoplasma has been determined by sequence analysis. A phylogenetic tree based on 16S rRNA gene sequences showed that PYL phytoplasma is most closely related to the stolbur and German grapevine yellows phytoplasmas, which form the stolbur subgroup of the aster yellows group. This phylogenetic position of PYL phytoplasma was supported by 16S/23S spacer region sequence data. PMID:8795200

  12. One-step PCR amplification of complete arthropod mitochondrial genomes.

    PubMed

    Hwang, U W; Park, C J; Yong, T S; Kim, W

    2001-06-01

    A new PCR primer set which enables one-step amplification of complete arthropod mitochondrial genomes was designed from two conserved 16S rDNA regions for the long PCR technique. For this purpose, partial 16S rDNAs amplified with universal primers 16SA and 16SB were newly sequenced from six representative arthropods: Armadillidium vulgare and Macrobrachium nipponense (Crustacea), Anopheles sinensis (Insecta), Lithobius forficatus and Megaphyllum sp. (Myriapoda), and Limulus polyphemus (Chelicerata). The genomic locations of two new primers, HPK16Saa and HPK16Sbb, correspond to positions 13314-13345 and 12951-12984, respectively, in the Drosophila yakuba mitochondrial genome. The usefulness of the primer set was experimentally examined and confirmed with five of the representative arthropods, except for A. vulgare, which has a linearized mitochondrial genome. With this set, therefore, we could easily and rapidly amplify complete mitochondrial genomes with small amounts of arthropod DNA. Although the primers suggested here were examined only with arthropod groups, a possibility of successful application to other invertebrates is very high, since the high degree of sequence conservation is shown on the primer sites in other invertebrates. Thus, this primer set can serve various research fields, such as molecular evolution, population genetics, and molecular phylogenetics based on DNA sequences, RFLP, and gene rearrangement of mitochondrial genomes in arthropods and other invertebrates. PMID:11399145

  13. Deodorization of pig slurry and characterization of bacterial diversity using 16S rDNA sequence analysis.

    PubMed

    Hwang, Ok-Hwa; Raveendar, Sebastian; Kim, Young-Ju; Kim, Ji-Hun; Choi, Jung-Woo; Kim, Tae-Hun; Choi, Dong-Yoon; Jeon, Che Ok; Cho, Sung-Back; Lee, Kyung-Tai

    2014-11-01

    The concentration of major odor-causing compounds including phenols, indoles, short-chain fatty acids (SCFAs) and branched chain fatty acids (BCFAs) in response to the addition of powdered horse radish (PHR) and spent mushroom compost (SMC) was compared with control non-treated slurry (CNS) samples. A total of 97,465 rDNAs sequence reads were generated from three different samples (CNS, n = 2; PHR, n = 3; SMC, n = 3) using bar-coded pyrosequencing. The number of operational taxonomic units (OTUs) was lower in the PHR slurry compared with the other samples. A total of 11 phyla were observed in the slurry samples, while the phylogenetic analysis revealed that the slurry microbiome predominantly comprised members of the Bacteroidetes, Firmicutes, and Proteobacteria phyla. The rarefaction analysis showed the bacterial species richness varied among the treated samples. Overall, at the OTU level, 2,558 individual genera were classified, 276 genera were found among the three samples, and 1,832 additional genera were identified in the individual samples. A principal component analysis revealed the differences in microbial communities among the CNS, PHR, and SMC pig slurries. Correlation of the bacterial community structure with the Kyoto Encyclopedia of Genes and Genomes (KEGG) predicted pathways showed that the treatments altered the metabolic capabilities of the slurry microbiota. Overall, these results demonstrated that the PHR and S MC treatments significantly reduced the malodor compounds in pig slurry (P < 0.05). PMID:25359269

  14. Virtual RFLP analysis of 16S rDNA sequences identifies new subgroups in the clover proliferation phytoplasma group

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Phytoplasmas are insect-transmitted, phloem-inhabiting, cell wall-less bacteria that cause numerous diseases in several hundred plant species. In adaptation to diverse plant hosts and insect vectors, phytoplasmas have evolved to give rise to widely divergent lineages. As phytoplasmas are uncultura...

  15. Bacterial community dynamics in a swine wastewater anaerobic reactor revealed by 16S rDNA sequence analysis.

    PubMed

    Liu, An-Chi; Chou, Chu-Yang; Chen, Ling-Ling; Kuo, Chih-Horng

    2015-01-20

    Anaerobic digestion is a microbiological process of converting organic wastes into digestate and biogas in the absence of oxygen. In practice, disturbance to the system (e.g., organic shock loading) may cause imbalance of the microbial community and lead to digester failure. To examine the bacterial community dynamics after a disturbance, this study simulated an organic shock loading that doubled the chemical oxygen demand (COD) loading using a 4.5L swine wastewater anaerobic completely stirred tank reactor (CSTR). Before the shock (loading rate=0.65gCOD/L/day), biogas production rate was about 1-2L/L/day. After the shock, three periods representing increased biogas production rates were observed during days 1-7 (∼4.0L/L/day), 13 (3.3L/L/day), and 21-23 (∼6.1L/L/day). For culture-independent assessments of the bacterial community composition, the 454 pyrosequencing results indicated that the community contained >2500 operational taxonomic units (OTUs) and was dominated by three phyla: Bacteroidetes, Firmicutes, and Proteobacteria. The shock induced dynamic changes in the community composition, which was re-stabilized after approximately threefold hydraulic retention time (HRT). Intriguingly, upon restabilization, the community composition became similar to that observed before the shock, rather than reaching a new equilibrium. PMID:25500375

  16. Phylogenetic analysis of the kenaf fiber microbial retting community by semiconductor sequencing of 16S rDNA amplicons

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Kenaf, hemp, and jute have been used for cordage and fiber production since prehistory. To obtain the fibers, harvested plants are soaked in ponds where indigenous microflora digests pectins and other heteropolysaccharides, releasing fibers in a process called retting. Renewed interest in “green” ...

  17. Gyr B versus 16s rDNA sequencing for the identification of Campylobacter jejuni, Campylobacter coli, and Campylobacter lari

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Species of the genus Campylobacter are the causative agents of a sizable number of the cases of food-borne illness in the developed world. The majority of this disease is caused by three of the thermotolerant Campylobacter species: Campylobacter jejuni, Campylobacter coli, and Campylobacter lari. ...

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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...

  19. Differentiation of Phylogenetically Related Slowly Growing Mycobacteria Based on 16S-23S rRNA Gene Internal Transcribed Spacer Sequences

    PubMed Central

    Roth, Andreas; Fischer, Marga; Hamid, Mohamed E.; Michalke, Sabine; Ludwig, Wolfgang; Mauch, Harald

    1998-01-01

    Interspecific polymorphisms of the 16S rRNA gene (rDNA) are widely used for species identification of mycobacteria. 16S rDNA sequences, however, do not vary greatly within a species, and they are either indistinguishable in some species, for example, in Mycobacterium kansasii and M. gastri, or highly similar, for example, in M. malmoense and M. szulgai. We determined 16S-23S rDNA internal transcribed spacer (ITS) sequences of 60 strains in the genus Mycobacterium representing 13 species (M. avium, M. conspicuum, M. gastri, M. genavense, M. kansasii, M. malmoense, M. marinum, M. shimoidei, M. simiae, M. szulgai, M. triplex, M. ulcerans, and M. xenopi). An alignment of these sequences together with additional sequences available in the EMBL database (for M. intracellulare, M. phlei, M. smegmatis, and M. tuberculosis) was established according to primary- and secondary-structure similarities. Comparative sequence analysis applying different treeing methods grouped the strains into species-specific clusters with low sequence divergence between strains belonging to the same species (0 to 2%). The ITS-based tree topology only partially correlated to that based on 16S rDNA, but the main branching orders were preserved, notably, the division of fast-growing from slowly growing mycobacteria, separate branching for M. simiae, M. genavense, and M. triplex, and distinct branches for M. xenopi and M. shimoidei. Comparisons of M. gastri with M. kansasii and M. malmoense with M. szulgai revealed ITS sequence similarities of 93 and 88%, respectively. M. marinum and M. ulcerans possessed identical ITS sequences. Our results show that ITS sequencing represents a supplement to 16S rRNA gene sequences for the differentiation of closely related species. Slowly growing mycobacteria show a high sequence variation in the ITS; this variation has the potential to be used for the development of probes as a rapid approach to mycobacterial identification. PMID:9431937

  20. Identification and characterization of alkaline protease producing Bacillus firmus species EMBS023 by 16S rRNA gene sequencing.

    PubMed

    Wishard, Rohan; wishard, Rohan; Jaiswal, Mahak; Parveda, Maheshwari; Amareshwari, P; Bhadoriya, Sneha Singh; Rathore, Pragya; Yadav, Mukesh; Nayarisseri, Anuraj; Nair, Achuthsankar S

    2014-12-01

    Probiotic microorganisms are those which exert a positive exect on the growth of the host, when administered as a dietary mixture in an adequate amount. They form the best alternative to the use of antibiotics for controlling enteric diseases in poultry farm animals, especially in the light of the gruesome problems of development of antibiotic resistance in enteric pathogens and the contamination of poultry products with antibiotics. 16S rDNA sequencing which has gained wide popularity amongst microbiologists for the molecular characterization and identification of newly discovered isolates provides accurate identification of isolates down to the level of sub-species (strain). It's most important advantage over the traditional biochemical characterization methods are that it can provide an accurate identification of strains with atypical phenotypic characters as well. The following work is an application of 16S rRNA gene sequencing approach to identify a novel, alkaline protease producing bacteria, from poultry farm waste. The sample was collected from a local poultry farm in the Guntur district, Andhra Pradesh, India. Subsequently the sample was serially diluted and the aliquots were incubated for a suitable time period following which the suspected colony was subjected to 16S rDNA sequencing. The results showed the isolate to be a novel, high alkaline protease producing bacteria, which was named Bacillus firmus isolate EMBS023, after characterization the sequence of isolate was deposited in GenBank with accession number JN990980. PMID:25118655

  1. Direct detection of 16S rRNA in soil extracts by using oligonucleotide microarrays.

    PubMed

    Small, J; Call, D R; Brockman, F J; Straub, T M; Chandler, D P

    2001-10-01

    We report on the development and validation of a simple microarray method for the direct detection of intact 16S rRNA from unpurified soil extracts. Total RNAs from Geobacter chapellei and Desulfovibrio desulfuricans were hybridized to an oligonucleotide array consisting of universal and species-specific 16S rRNA probes. PCR-amplified products from Geobacter and Desulfovibrio were easily and specifically detected under a range of hybridization times, temperatures, and buffers. However, reproducible, specific hybridization and detection of intact rRNA could be accomplished only by using a chaperone-detector probe strategy. With this knowledge, assay conditions were developed for rRNA detection using a 2-h hybridization time at room temperature. Hybridization specificity and signal intensity were enhanced using fragmented RNA. Formamide was required in the hybridization buffer in order to achieve species-specific detection of intact rRNA. With the chaperone detection strategy, we were able to specifically hybridize and detect G. chapellei 16S rRNA directly from a total-RNA soil extract, without further purification or removal of soluble soil constituents. The detection sensitivity for G. chapellei 16S rRNA in soil extracts was at least 0.5 microg of total RNA, representing approximately 7.5 x 10(6) Geobacter cell equivalents of RNA. These results suggest that it is now possible to apply microarray technology to the direct detection of microorganisms in environmental samples, without using PCR. PMID:11571176

  2. Mitochondrial 16S ribosomal RNA gene for forensic identification of crocodile species.

    PubMed

    Naga Jogayya, K; Meganathan, P R; Dubey, Bhawna; Haque, I

    2013-05-01

    All crocodilians are under various threats due to over exploitation and these species have been listed in Appendix I or II of CITES. Lack of molecular techniques for the forensic identification of confiscated samples makes it difficult to enforce the law. Therefore, we herein present a molecular method developed on the basis on 16S rRNA gene of mitochondrial DNA for identification of crocodile species. We have developed a set of 16S rRNA primers for PCR based identification of crocodilian species. These novel primers amplify partial 16S rRNA sequences of six crocodile species which can be later combined to obtain a larger region (1290 bp) of 16S rRNA gene. This 16S rRNA gene could be used as an effective tool for forensic authentication of crocodiles. The described primers hold great promise in forensic identification of crocodile species, which can aid in the effective enforcement of law and conservation of these species. PMID:23622485

  3. Flow Cytometric and 16S Sequencing Methodologies for Monitoring the Physiological Status of the Microbiome in Powdered Infant Formula Production

    PubMed Central

    Anvarian, Amir H. P.; Cao, Yu; Srikumar, Shabarinath; Fanning, Séamus; Jordan, Kieran

    2016-01-01

    The aim of this study was to develop appropriate protocols for flow cytometric (FCM) and 16S rDNA sequencing investigation of the microbiome in a powdered infant formula (PIF) production facility. Twenty swabs were collected from each of the three care zones of a PIF production facility and used for preparing composite samples. For FCM studies, the swabs were washed in 200 mL phosphate buffer saline (PBS). The cells were harvested by three-step centrifugation followed by a single stage filtration. Cells were dispersed in fresh PBS and analyzed with a flow cytometer for membrane integrity, metabolic activity, respiratory activity and Gram characteristics of the microbiome using various fluorophores. The samples were also plated on agar plates to determine the number of culturable cells. For 16S rDNA sequencing studies, the cells were harvested by centrifugation only. Genomic DNA was extracted using a chloroform-based method and used for 16S rDNA sequencing studies. Compared to the dry low and high care zones, the wet medium care zone contained a greater number of viable, culturable, and metabolically active cells. Viable but non-culturable cells were also detected in dry-care zones. In total, 243 genera were detected in the facility of which 42 were found in all three care zones. The greatest diversity in the microbiome was observed in low care. The genera present in low, medium and high care were mostly associated with soil, water, and humans, respectively. The most prevalent genera in low, medium and high care were Pseudomonas, Acinetobacter, and Streptococcus, respectively. The integration of FCM and metagenomic data provided further information on the density of different species in the facility. PMID:27446009

  4. Quantification of Hyphomicrobium Populations in Activated Sludge from an Industrial Wastewater Treatment System as Determined by 16S rRNA Analysis

    PubMed Central

    Layton, A. C.; Karanth, P. N.; Lajoie, C. A.; Meyers, A. J.; Gregory, I. R.; Stapleton, R. D.; Taylor, D. E.; Sayler, G. S.

    2000-01-01

    The bacterial community structure of the activated sludge from a 25 million-gal-per-day industrial wastewater treatment plant was investigated using rRNA analysis. 16S ribosomal DNA (rDNA) libraries were created from three sludge samples taken on different dates. Partial rRNA gene sequences were obtained for 46 rDNA clones, and nearly complete 16S rRNA sequences were obtained for 18 clones. Seventeen of these clones were members of the beta subdivision, and their sequences showed high homology to sequences of known bacterial species as well as published 16S rDNA sequences from other activated sludge sources. Sixteen clones belonged to the alpha subdivision, 7 of which showed similarity to Hyphomicrobium species. This cluster was chosen for further studies due to earlier work on Hyphomicrobium sp. strain M3 isolated from this treatment plant. A nearly full-length 16S rDNA sequence was obtained from Hyphomicrobium sp. strain M3. Phylogenetic analysis revealed that Hyphomicrobium sp. strain M3 was 99% similar to Hyphomicrobium denitrificans DSM 1869T in Hyphomicrobium cluster II. Three of the cloned sequences from the activated sludge samples also grouped with those of Hyphomicrobium cluster II, with a 96% sequence similarity to that of Hyphomicrobium sp. strain M3. The other four cloned sequences from the activated sludge sample were more closely related to those of the Hyphomicrobium cluster I organisms (95 to 97% similarity). Whole-cell fluorescence hybridization of microorganisms in the activated sludge with genus-specific Hyphomicrobium probe S-G-Hypho-1241-a-A-19 enhanced the visualization of Hyphomicrobium and revealed that Hyphomicrobium appears to be abundant both on the outside of flocs and within the floc structure. Dot blot hybridization of activated sludge samples from 1995 with probes designed for Hyphomicrobium cluster I and Hyphomicrobium cluster II indicated that Hyphomicrobium cluster II-positive 16S rRNA dominated over Hyphomicrobium cluster I

  5. Phylogeny and genetic diversity of Bridgeoporus nobilissimus inferred using mitochondrial and nuclear rDNA sequences

    USGS Publications Warehouse

    Redberg, G.L.; Hibbett, D.S.; Ammirati, J.F., Jr.; Rodriguez, R.J.

    2003-01-01

    The genetic diversity and phylogeny of Bridgeoporus nobilissimus have been analyzed. DNA was extracted from spores collected from individual fruiting bodies representing six geographically distinct populations in Oregon and Washington. Spore samples collected contained low levels of bacteria, yeast and a filamentous fungal species. Using taxon-specific PCR primers, it was possible to discriminate among rDNA from bacteria, yeast, a filamentous associate and B. nobilissimus. Nuclear rDNA internal transcribed spacer (ITS) region sequences of B. nobilissimus were compared among individuals representing six populations and were found to have less than 2% variation. These sequences also were used to design dual and nested PCR primers for B. nobilissimus-specific amplification. Mitochondrial small-subunit rDNA sequences were used in a phylogenetic analysis that placed B. nobilissimus in the hymenochaetoid clade, where it was associated with Oxyporus and Schizopora.

  6. Highly specific and efficient primers for in-house multiplex PCR detection of Chlamydia trachomatis, Neisseria gonorrhoeae, Mycoplasma hominis and Ureaplasma urealyticum

    PubMed Central

    2014-01-01

    Background Although sophisticated methodologies are available, the use of endpoint polymerase chain reaction (PCR) to detect 16S rDNA genes remains a good approach for estimating the incidence and prevalence of specific infections and for monitoring infections. Considering the importance of the early diagnosis of sexually transmitted infections (STIs), the development of a sensitive and affordable method for identifying pathogens in clinical samples is needed. Highly specific and efficient primers for a multiplex polymerase chain reaction (m-PCR) system were designed in silico to detect the 16S rDNA genes of four bacteria that cause genital infections, and the PCR method was developed. Methods The Genosensor Probe Designer (GPD) (version 1.0a) software was initially used to design highly specific and efficient primers for in-house m-PCR. Single-locus PCR reactions were performed and standardised, and then primers for each locus in turn were added individually in subsequent amplifications until m-PCR was achieved. Amplicons of the expected size were obtained from each of the four bacterial gene fragments. Finally, the analytical specificity and limits of detection were tested. Results Because they did not amplify any product from non-STI tested species, the primers were specific. The detection limits for the Chlamydia trachomatis, Neisseria gonorrhoeae, Mycoplasma hominis and Ureaplasma urealyticum primer sets were 5.12 × 105, 3.9 × 103, 61.19 × 106 and 6.37 × 105 copies of a DNA template, respectively. Conclusions The methodology designed and standardised here could be applied satisfactorily for the simultaneous or individual detection of Chlamydia trachomatis, Neisseria gonorrhoeae, Mycoplasma hominis and Ureaplasma urealyticum. This method is at least as efficient as other previously described methods; however, this method is more affordable for low-income countries. PMID:24997675

  7. Absence of Mycoplasma-specific DNA sequence in brain, blood and CSF of patients with multiple sclerosis (MS): a study by PCR and real-time PCR.

    PubMed

    Casserly, Georgina; Barry, Thomas; Tourtellotte, Wallace W; Hogan, Edward L

    2007-02-15

    Mycoplasmas are the smallest of the known self-replicating organisms. They lack cell walls and are associated with numerous diseases in humans and animals. We are exploring the possibility that infection by Mycoplasma may induce the inflammatory demyelinating disease of the central nervous system (CNS) that is MS. The presence of specific Mycoplasma species DNA was sought in brain, serum and cerebrospinal fluid (CSF) of patients diagnosed with multiple sclerosis (MS) and other neurological diseases (OND) including inflammatory disorders. The MS samples from patients with active and progressive MS, as well as in remission, a variety of other neurological disease controls, including inflammatory CNS diseases such as meningitis, cryptococcal meningitis and encephalitis and other neurological disorders such as migraine were also examined. Clinical samples were provided by the National Neurological Research Specimen Bank and the Human Brain and Spinal Fluid Resource Centre, Los Angeles. Analysis was carried out by conventional PCR using Mycoplasma-specific primers (McAuliffe et al., 2005) that target the 16S rDNA gene in Mycoplasma species. The Mycoplasma-specific primers could detect 102 Mycoplasma species. In this study, 30 samples of human brain and 57 pairs of serum and CSF and were examined. No Mycoplasma-specific nucleic acid sequence was detected, and the consistent observation of an endogenous gene, human serum albumin (HSA), as a positive control documented the adequacy of the method. Real-time PCR analysis of serum and CSF was done also targeting utilizing the Mycoplasma 16S rDNA gene, and this also demonstrated the lack of Mycoplasma in these samples. The presence of Mycoplasma at extraneural sites in MS patients is now being explored. PMID:17234214

  8. Identification of N2-fixing plant- and fungus-associated Azoarcus species by PCR-based genomic fingerprints.

    PubMed Central

    Hurek, T; Wagner, B; Reinhold-Hurek, B

    1997-01-01

    Most species of the diazotrophic Proteobacteria Azoarcus spp. occur in association with grass roots, while A. tolulyticus and A. evansii are soil bacteria not associated with a plant host. To facilitate species identification and strain comparison, we developed a protocol for PCR-generated genomic fingerprints, using an automated sequencer for fragment analysis. Commonly used primers targeted to REP (repetitive extragenic palindromic) and ERIC (enterobacterial repetitive intergenic consensus) sequence elements failed to amplify fragments from the two species tested. In contrast, the BOX-PCR assay (targeted to repetitive intergenic sequence elements of Streptococcus) yielded species-specific genomic fingerprints with some strain-specific differences. PCR profiles of an additional PCR assay using primers targeted to tRNA genes (tDNA-PCR, for tRNA(IIe)) were more discriminative, allowing differentiation at species-specific (for two species) or infraspecies-specific level. Our protocol of several consecutive PCR assays consisted of 16S ribosomal DNA (rDNA)-targeted, genus-specific PCR followed by BOX- and tDNA-PCR; it enabled us to assign new diazotrophic isolates originating from fungal resting stages (sclerotia) to known species of Azoarcus. The assignment was confirmed by phylogenetic analysis of 16S rDNA sequences. Additionally, the phylogenetic distances and the lack of monophyly suggested emendment of the genus Azoarcus: the unnamed species Azoarcus groups C and D and a new group (E) of Azoarcus, which was detected in association with fungi, are likely to have the taxonomic rank of three different genera. According to its small subunit rRNA, the sclerotium-forming basidiomycete was related to the Ustilagomycetes, facultatively biotrophic parasites of plants. Since they occurred in a field which was under cultivation with rice and wheat, these fungi might serve as a niche for survival for Azoarcus in the soil and as a source for reinfection of plants. PMID:9361420

  9. Arrested development of the myxozoan parasite, Myxobolus cerebralis, in certain populations of mitochondrial 16S lineage III Tubifex tubifex.

    PubMed

    Baxa, D V; Kelley, G O; Mukkatira, K S; Beauchamp, K A; Rasmussen, C; Hedrick, R P

    2008-01-01

    Laboratory populations of Tubifex tubifex from mitochondrial (mt)16S ribosomal DNA (rDNA) lineage III were generated from single cocoons of adult worms releasing the triactinomyxon stages (TAMs) of the myxozoan parasite, Myxobolus cerebralis. Subsequent worm populations from these cocoons, referred to as clonal lines, were tested for susceptibility to infection with the myxospore stages of M. cerebralis. Development and release of TAMs occurred in five clonal lines, while four clonal lines showed immature parasitic forms that were not expelled from the worm (non-TAM producers). Oligochaetes from TAM- and non-TAM-producing clonal lines were confirmed as lineage III based on mt16S rDNA and internal transcribed spacer region 1 (ITS1) sequences, but these genes did not differentiate these phenotypes. In contrast, random amplified polymorphic DNA analyses of genomic DNA demonstrated unique banding patterns that distinguished the phenotypes. Cohabitation of parasite-exposed TAM- and non-TAM-producing phenotypes showed an overall decrease in expected TAM production compared to the same exposure dose of the TAM-producing phenotype without cohabitation. These studies suggest that differences in susceptibility to parasite infection can occur in genetically similar T. tubifex populations, and their coexistence may affect overall M. cerebralis production, a factor that may influence the severity of whirling disease in wild trout populations. PMID:17891544

  10. Arrested development of the myxozoan parasite, Myxobolus cerebralis, in certain populations of mitochondrial 16S lineage III Tubifex tubifex

    USGS Publications Warehouse

    Baxa, D.V.; Kelley, G.O.; Mukkatira, K.S.; Beauchamp, K.A.; Rasmussen, C.; Hedrick, R.P.

    2008-01-01

    Laboratory populations of Tubifex tubifex from mitochondrial (mt)16S ribosomal DNA (rDNA) lineage III were generated from single cocoons of adult worms releasing the triactinomyxon stages (TAMs) of the myxozoan parasite, Myxobolus cerebralis. Subsequent worm populations from these cocoons, referred to as clonal lines, were tested for susceptibility to infection with the myxospore stages of M. cerebralis. Development and release of TAMs occurred in five clonal lines, while four clonal lines showed immature parasitic forms that were not expelled from the worm (non-TAM producers). Oligochaetes from TAM- and non-TAM-producing clonal lines were confirmed as lineage III based on mt16S rDNA and internal transcribed spacer region 1 (ITS1) sequences, but these genes did not differentiate these phenotypes. In contrast, random amplified polymorphic DNA analyses of genomic DNA demonstrated unique banding patterns that distinguished the phenotypes. Cohabitation of parasite-exposed TAM- and non-TAM-producing phenotypes showed an overall decrease in expected TAM production compared to the same exposure dose of the TAM-producing phenotype without cohabitation. These studies suggest that differences in susceptibility to parasite infection can occur in genetically similar T. tubifex populations, and their coexistence may affect overall M. cerebralis production, a factor that may influence the severity of whirling disease in wild trout populations. ?? 2007 Springer-Verlag.

  11. Phylogenetic analyses of Chlamydia psittaci strains from birds based on 16S rRNA gene sequence.

    PubMed Central

    Takahashi, T; Masuda, M; Tsuruno, T; Mori, Y; Takashima, I; Hiramune, T; Kikuchi, N

    1997-01-01

    The nucleotide sequences of 16S ribosomal DNA (rDNA) were determined for 39 strains of Chlamydia psittaci (34 from birds and 5 from mammals) and for 4 Chlamydia pecorum strains. The sequences were compared phylogenetically with the gene sequences of nine Chlamydia strains (covering four species of the genus) retrieved from nucleotide databases. In the neighbor-joining tree, C. psittaci strains were more closely related to each other than to the other Chlamydia species, although a feline pneumonitis strain was distinct (983 to 98.6% similarity to other strains) and appeared to form the deepest subline within the species of C. psittaci (bootstrap value, 99%). The other strains of C. psittaci exhibiting similarity values of more than 99% were branched into several subgroups. Two pigeon strains and one turkey strain formed a distinct clade recovered in 97% of the bootstrapped trees. The other pigeon strains seemed to be distinct from the strains from psittacine birds, with 88% of bootstrap value. In the cluster of psittacine strains, three parakeet strains and an ovine abortion strain exhibited a specific association (level of sequence similarity, 99.9% or more; bootstrap value, 95%). These suggest that at least four groups of strains exist within the species C. psittaci. The 16S rDNA sequence is a valuable phylogenetic marker for the taxonomy of chlamydiae, and its analysis is a reliable tool for identification of the organisms. PMID:9350757

  12. Comparison of gull-specific assays targeting 16S rRNA gene of Catellicoccus marimammalium and Streptococcus spp.

    EPA Science Inventory

    Gulls have been implicated as a source of fecal contamination in inland and coastal waters. Only one gull-specific assay is currently available (i.e., gull2 qPCR assay). This assay is based on the 16S rRNA gene of Catellicocclls marimammalium and has showed a high level of host-s...

  13. Validation of a quantitative PCR assay for detection and quantification of 'Candidatus Xenohaliotis californiensis'.

    PubMed

    Friedman, Carolyn S; Wight, Nate; Crosson, Lisa M; White, Samuel J; Strenge, Robyn M

    2014-04-01

    Withering syndrome (WS), a serious disease affecting abalone Haliotis spp., is caused by infection from an intracellular Rickettsia-like organism (WS-RLO). Diagnosis of the disease currently relies on a combination of histological examination and molecular methods (in situ hybridization, standard PCR, and sequence analysis). However, these techniques only provide a semi-quantitative assessment of bacterial load. We created a real-time quantitative PCR (qPCR) assay to specifically identify and enumerate bacterial loads of WS-RLO in abalone tissue, fecal, and seawater samples based on 16S rDNA gene copy numbers. The qPCR assay designed to detect DNA of the WS-RLO was validated according to standards set by the World Organisation for Animal Health. Standard curves derived from purified plasmid dilutions were linear across 7 logs of concentration, and efficiencies ranged from 90.2 to 97.4%. The limit of detection was 3 gene copies per reaction. Diagnostic sensitivity was 100% and specificity was 99.8%. The qPCR assay was robust, as evidenced by its high level of repeatability and reproducibility. This study has shown for the first time that WS-RLO DNA can be detected and quantified in abalone tissue, fecal, and seawater samples. The ability to detect and quantify RLO gene copies in a variety of materials will enable us to better understand transmission dynamics in both farmed and natural environments. PMID:24695238

  14. The 16S ribosomal RNA mutation database (16SMDB).

    PubMed Central

    Triman, K L

    1996-01-01

    The 16S ribosomal RNA mutation database (16SMDB) provides a list of mutated positions in 16S ribosomal RNA from Escherichia coli and the identity of each alteration. Information provided for each mutation includes: (i) a brief description of the phenotype(s) associated with each mutation; (ii) whether a mutant phenotype has been detected by in vivo or in vitro methods; (iii) relevant literature citations. The database is available via ftp and on the World Wide Web. PMID:8594570

  15. PCR-based assessment of shellfish traceability and sustainability in international Mediterranean seafood markets.

    PubMed

    Galal-Khallaf, Asmaa; Ardura, Alba; Borrell, Yaisel J; Garcia-Vazquez, Eva

    2016-07-01

    Two mitochondrial markers (cytochrome oxidase COI and 16S rDNA) were employed for species identification of commercial shellfish from two Mediterranean countries. New COI Barcodes were generated for six species: Pleoticus robustus, Metapenaeopsis barbata, Parapenaeus fissuroides, Hymenopenaeus debilis, Metapenaeus affinis and Sepia aculeata. Biodiversity of the seafood species analyzed was greater in Egypt, with nine crustacean and two cephalopod species found compared with only three crustaceans and three cephalopods in Spain. In total, 17.2% and 15.2% products were mislabeled in Egypt and Spain, respectively. Population decline is a problem for some of the substitute species. Others were exotic and/or invasive in exporters' regions. This study offers the first comparable study of shellfish traceability in these Mediterranean markets. The PCR-based method used in this study proved to be reliable, effective and, therefore, could be employed for routine seafood analysis. PMID:26920298

  16. Intragenomic heterogeneity of the 16S rRNA gene in strain UFO1 caused by a 100-bp insertion in helix 6

    SciTech Connect

    Allison E. Ray; Stephanie A. Connon; Peter P. Sheridan; Jeremy Gilbreath; Malcolm S. Shields; Deborah T. Newby; Yoshiko Fujita; Timothy S. Magnuson

    2010-06-01

    The determination of variation in 16S rRNA gene sequences is perhaps the most common method for assessing microbial community diversity. However, the occurrence of multiple copies of 16S rRNA genes within some organisms can bias estimates of microbial diversity. During phylogenetic characterization of a metal-transforming, fermentative bacterium (strain UFO1) isolated from the Field Research Center (FRC) in Oak Ridge, TN, we detected an apparent 16S rRNA pseudogene. The putative 16S rRNA pseudogene was first detected in clone libraries constructed with 16S rRNA genes amplified from UFO1 genomic DNA. Sequencing revealed two distinct 16S rRNA genes, with one differing from the other by a 100 bp insert near the 5’ end. Ribosomal RNA was extracted from strain UFO1 and analyzed by RT-qPCR with insert and non-insert specific primers; however, only the non-insert 16S rRNA sequence was expressed. Reverse-transcribed rRNA from strain UFO1 was also used to construct a cDNA library. Of 190 clones screened by PCR, none contained the 16S rRNA gene with the 100 bp insert. Examination of GenBank 16S rRNA gene sequences revealed that the same insert sequence was present in other clones, including those from an environmental library constructed from FRC enrichments. These findings demonstrate the existence of widely disparate copies of the 16S rRNA gene in the same species and a putative 16S rRNA pseudogene, which may confound 16S rRNA-based methods for assessments of microbial diversity in environmental samples.

  17. 16S rRNA pyrosequencing-based investigation of the bacterial community in nukadoko, a pickling bed of fermented rice bran.

    PubMed

    Sakamoto, Naoshige; Tanaka, Shigemitsu; Sonomoto, Kenji; Nakayama, Jiro

    2011-01-01

    Nukadoko is a naturally fermented rice bran mash traditionally used for pickling vegetables in Japan; its refreshment and fermentation cycles sometimes continue for many years. Here, we investigated the structure and dynamics of the bacterial community in nukadoko by conducting pyrosequencing and quantitative polymerase chain reaction (PCR) analyses of 16S ribosomal RNA genes (rDNA). Of the 16 different samples studied, 13 showed Lactobacillus-dominated microbiota, suggesting that aged nukadoko samples tend to realize a niche, favorable Lactobacillus species. The lactic acid bacterial community of each of the 16 samples was classified into 3 types according to the presence or absence of 2 predominant species, Lactobacillus namurensis and Lactobacillus acetotolerans. The dynamics of the bacterial community during fermentation and the subsequent ripening process were examined using a laboratory model of nukadoko inoculated with an aged nukadoko sample (inoculated model). Lb. namurensis grew rapidly in the first 2 days, accompanied with a rapid decrease in pH and an increase in lactate levels, while Lb. acetotolerans grew with a longer doubling time and slow acidification during the 20 days after inoculation. On the other hand, spontaneous fermentation of the nukadoko model prepared from fresh rice bran without the nukadoko inoculation (inoculant-free model), showed the growth of some non-Lactobacillus species such as staphylococci and bacilli within the first 10 days; thereafter, Lb. namurensis was dominant, while Lb. acetotolerans was not detected during the 20-day experimental period. These results suggest that the naturally established Lactobacillus community in aged nukadoko is effectively involved in the biocontrol of the microbial community of nukadoko during the refreshment and fermentation cycles. PMID:21084126

  18. Phylogenetic relationships of land plants using mitochondrial small-subunit rDNA sequences.

    PubMed

    Duff, R J; Nickrent, D L

    1999-03-01

    Phylogenetic relationships among embryophytes (tracheophytes, mosses, liverworts, and hornworts) were examined using 21 newly generated mitochondrial small-subunit (19S) rDNA sequences. The "core" 19S rDNA contained more phylogenetically informative sites and lower homoplasy than either nuclear 18S or plastid 16S rDNA. Results of phylogenetic analyses using parsimony (MP) and likelihood (ML) were generally congruent. Using MP, two trees were obtained that resolved either liverworts or hornworts as the basal land plant clade. The optimal ML tree showed hornworts as basal. That topology was not statistically different from the two MP trees, thus both appear to be equally viable evolutionary hypotheses. High bootstrap support was obtained for the majority of higher level embryophyte clades named in a recent morphologically based classification, e.g., Tracheophyta, Euphyllophytina, Lycophytina, and Spermatophytata. Strong support was also obtained for the following monophyletic groups: hornworts, liverworts, mosses, lycopsids, leptosporangiate and eusporangiate ferns, gymnosperms and angiosperms. This molecular analysis supported a sister relationship between Equisetum and leptosporangiate ferns and a monophyletic gymnosperms sister to angiosperms. The topologies of deeper clades were affected by taxon inclusion (particularly hornworts) as demonstrated by jackknife analyses. This study represents the first use of mitochondrial 19S rDNA for phylogenetic purposes and it appears well-suited for examining intermediate to deep evolutionary relationships among embryophytes. PMID:10077500

  19. Specific multiplex analysis of pathogens using a direct 16S rRNA hybridization in microarray system.

    PubMed

    Hwang, Byeong Hee; Shin, Hwa Hui; Seo, Jeong Hyun; Cha, Hyung Joon

    2012-06-01

    For the rapid multiplex analysis of pathogens, 16S rRNAs from cell lysates were directly applied onto a DNA microarray at room temperature (RT) for RNA-DNA hybridization. To eliminate the labeling step, seven fluorescent-labeled detector probes were cohybridized with 16S rRNA targets and adjacent specific capture probes. We found that eight pathogens were successfully discriminated by the 16S rRNA-based direct method, which showed greater specificity than the polymerase chain reaction (PCR)-labeled method due to chaperone and distance effects. A new specificity criterion for a perfect match between RNA and DNA was suggested to be 21-41% dissimilarity using correlation analysis between the mismatch and the sequence according to the guanine-cytosine (GC) percentage or the distribution of mismatches. Six categories of food matrix (egg, meat, milk, rice, vegetable, and mixed) were also tested, and the target pathogen was successfully discriminated within statistically significant levels. Finally, we found that the intrinsic abundance of 16S rRNA molecules successfully substituted PCR-based amplification with a low limit of detection of 10-10(3) cells mL(-1) and a high quantitative linear correlation. Collectively, our suggested 16S rRNA-based direct method enables the highly sensitive, specific, and quantitative analysis of selected pathogens at RT within 2 h, even in food samples. PMID:22551354

  20. FUNGAL-SPECIFIC PCR PRIMERS DEVELOPED FOR ANALYSIS OF THE ITS REGION OF ENVIRONMENTAL DNA EXTRACTS

    EPA Science Inventory

    Background The Internal Transcribed Spacer (ITS) regions of fungal ribosomal DNA (rDNA) are highly variable sequences of great importance in distinguishing fungal species by PCR analysis. Previously published PCR primers available for amplifying these sequences from environmenta...

  1. Phylogenetic analysis of the Listeria monocytogenes based on sequencing of 16S rRNA and hlyA genes.

    PubMed

    Soni, Dharmendra Kumar; Dubey, Suresh Kumar

    2014-12-01

    The discrimination between Listeria monocytogenes and Listeria species has been detected. The 16S rRNA and hlyA were PCR amplified with set of oligonucleotide primers with flank 1,500 and 456 bp fragments, respectively. Based on the differences in 16S rRNA and hlyA genes, a total 80 isolates from different environmental, food and clinical samples confirmed it to be L. monocytogenes. The 16S rRNA sequence similarity suggested that the isolates were similar to the previously reported ones from different habitats by others. The phylogenetic interrelationships of the genus Listeria were investigated by sequencing of 16S rRNA and hlyA gene. The 16S rRNA sequence indicated that genus Listeria is comprised of following closely related but distinct lines of descent, one is the L. monocytogenes species group (including L. innocua, L. ivanovii, L. seeligeri and L. welshimeri) and other, the species L. grayi, L. rocourtiae and L. fleischmannii. The phylogenetic tree based on hlyA gene sequence clearly differentiates between the L. monocytogenes, L. ivanovii and L. seeligeri. In the present study, we identified 80 isolates of L. monocytogenes originating from different clinical, food and environmental samples based on 16S rRNA and hlyA gene sequence similarity. PMID:25205124

  2. Comparison of Gull Feces-specific Assays Targeting the 16S rRNA Gene of Catellicoccus Marimammalium and Streptococcus spp.

    EPA Science Inventory

    Two novel gull-specific qPCR assays were developed using 16S rRNA gene sequences from gull fecal clone libraries: a SYBR-green-based assay targeting Streptococcus spp. (i.e., gull3) and a TaqMan qPCR assay targeting Catellicoccus marimammalium (i.e., gull4). The main objectives ...

  3. Copy Number of the Transposon, Pokey, in rDNA Is Positively Correlated with rDNA Copy Number in Daphnia obtusa

    PubMed Central

    LeRiche, Kaitlynn; Eagle, Shannon H. C.; Crease, Teresa J.

    2014-01-01

    Pokey is a class II DNA transposon that inserts into 28S ribosomal RNA (rRNA) genes and other genomic regions of species in the subgenus, Daphnia. Two divergent lineages, PokeyA and PokeyB have been identified. Recombination between misaligned rRNA genes changes their number and the number of Pokey elements. We used quantitative PCR (qPCR) to estimate rRNA gene and Pokey number in isolates from natural populations of Daphnia obtusa, and in clonally-propagated mutation accumulation lines (MAL) initiated from a single D. obtusa female. The change in direction and magnitude of Pokey and rRNA gene number did not show a consistent pattern across ∼87 generations in the MAL; however, Pokey and rRNA gene number changed in concert. PokeyA and 28S gene number were positively correlated in the isolates from both natural populations and the MAL. PokeyB number was much lower than PokeyA in both MAL and natural population isolates, and showed no correlation with 28S gene number. Preliminary analysis did not detect PokeyB outside rDNA in any isolates and detected only 0 to 4 copies of PokeyA outside rDNA indicating that Pokey may be primarily an rDNA element in D. obtusa. The recombination rate in this species is high and the average size of the rDNA locus is about twice as large as that in other Daphnia species such as D. pulicaria and D. pulex, which may have facilitated expansion of PokeyA to much higher numbers in D. obtusa rDNA than these other species. PMID:25490398

  4. Copy number of the transposon, Pokey, in rDNA is positively correlated with rDNA copy number in Daphnia obtuse [corrected].

    PubMed

    LeRiche, Kaitlynn; Eagle, Shannon H C; Crease, Teresa J

    2014-01-01

    Pokey is a class II DNA transposon that inserts into 28S ribosomal RNA (rRNA) genes and other genomic regions of species in the subgenus, Daphnia. Two divergent lineages, PokeyA and PokeyB have been identified. Recombination between misaligned rRNA genes changes their number and the number of Pokey elements. We used quantitative PCR (qPCR) to estimate rRNA gene and Pokey number in isolates from natural populations of Daphnia obtusa, and in clonally-propagated mutation accumulation lines (MAL) initiated from a single D. obtusa female. The change in direction and magnitude of Pokey and rRNA gene number did not show a consistent pattern across ∼ 87 generations in the MAL; however, Pokey and rRNA gene number changed in concert. PokeyA and 28S gene number were positively correlated in the isolates from both natural populations and the MAL. PokeyB number was much lower than PokeyA in both MAL and natural population isolates, and showed no correlation with 28S gene number. Preliminary analysis did not detect PokeyB outside rDNA in any isolates and detected only 0 to 4 copies of PokeyA outside rDNA indicating that Pokey may be primarily an rDNA element in D. obtusa. The recombination rate in this species is high and the average size of the rDNA locus is about twice as large as that in other Daphnia species such as D. pulicaria and D. pulex, which may have facilitated expansion of PokeyA to much higher numbers in D. obtusa rDNA than these other species. PMID:25490398

  5. Assessing the Fecal Microbiota: An Optimized Ion Torrent 16S rRNA Gene-Based Analysis Protocol

    PubMed Central

    Foroni, Elena; Duranti, Sabrina; Turroni, Francesca; Lugli, Gabriele Andrea; Sanchez, Borja; Martín, Rebeca; Gueimonde, Miguel; van Sinderen, Douwe; Margolles, Abelardo; Ventura, Marco

    2013-01-01

    Assessing the distribution of 16S rRNA gene sequences within a biological sample represents the current state-of-the-art for determination of human gut microbiota composition. Advances in dissecting the microbial biodiversity of this ecosystem have very much been dependent on the development of novel high-throughput DNA sequencing technologies, like the Ion Torrent. However, the precise representation of this bacterial community may be affected by the protocols used for DNA extraction as well as by the PCR primers employed in the amplification reaction. Here, we describe an optimized protocol for 16S rRNA gene-based profiling of the fecal microbiota. PMID:23869230

  6. Molecular analysis of complete ssu to lsu rdna sequence in the harmful dinoflagellate alexandrium tamarense (korean isolate, HY970328M)

    NASA Astrophysics Data System (ADS)

    Ki, Jang-Seu; Han, Myung-Soo

    2005-09-01

    New PCR primers (N=18) were designed for the isolation of complete SSU to LSU rDNA sequences from the dinoflagellate Alexandrium tamarense. Standard PCR, employing each primer set selected for amplifications of less than 1.5 kb, successfully amplified the expected rDNA regions of A. tamarense (Korean isolate, HY970328M). Complete SSU, LSU rDNAs and ITS sequences, including 5.8S rDNA, were recorded at 1,800 bp, 520 bp and 3,393 bp, respectively. The LSU rDNA sequence was the first report in Alexandrium genus. No intron was found in the LSU rRNA coding region. Twelve D-domains within the LSU rDNA were put together into 1,879 bp (44.4% G+C), and cores into 1514 bp (42.8% G+C). The core sequence was significantly different (0.0867 of genetic distance, 91% sequence similarity) in comparison with Prorocentrum micans (GenBank access. no. X16108). The D2 region was the longest in length (300 bp) and highly variable among the 12 D-domains. In a phylogenetic analysis using complete LSU rDNA sequences of a variety of phytoplankton, A tamarense was clearly separated with high resolution against other species. The result suggests that the sequence may resolve the taxonomic ambiguities of Alexandrium genus, particularly of the tamarensis complex.

  7. PhylOPDb: a 16S rRNA oligonucleotide probe database for prokaryotic identification

    PubMed Central

    Jaziri, Faouzi; Parisot, Nicolas; Abid, Anis; Denonfoux, Jérémie; Ribière, Céline; Gasc, Cyrielle; Boucher, Delphine; Brugère, Jean-François; Mahul, Antoine; Hill, David R.C.; Peyretaillade, Eric; Peyret, Pierre

    2014-01-01

    In recent years, high-throughput molecular tools have led to an exponential growth of available 16S rRNA gene sequences. Incorporating such data, molecular tools based on target-probe hybridization were developed to monitor microbial communities within complex environments. Unfortunately, only a few 16S rRNA gene-targeted probe collections were described. Here, we present PhylOPDb, an online resource for a comprehensive phylogenetic oligonucleotide probe database. PhylOPDb provides a convivial and easy-to-use web interface to browse both regular and explorative 16S rRNA-targeted probes. Such probes set or subset could be used to globally monitor known and unknown prokaryotic communities through various techniques including DNA microarrays, polymerase chain reaction (PCR), fluorescent in situ hybridization (FISH), targeted gene capture or in silico rapid sequence identification. PhylOPDb contains 74 003 25-mer probes targeting 2178 genera including Bacteria and Archaea. Database URL: http://g2im.u-clermont1.fr/phylopdb/ PMID:24771669

  8. Development of a PCR technique specific for Demodex injai in biological specimens.

    PubMed

    Sastre, N; Ravera, I; Ferreira, D; Altet, L; Sánchez, A; Bardagí, M; Francino, O; Ferrer, L

    2013-09-01

    The identification of Demodex injai as a second Demodex species of dog opened new questions and challenges in the understanding on the Demodex-host relationships. In this paper, we describe the development of a conventional PCR technique based on published genome sequences of D. injai from GenBank that specifically detects DNA from D. injai. This technique amplifies a 238-bp fragment corresponding to a region of the mitochondrial 16S rDNA of D. injai. The PCR was positive in DNA samples obtained from mites identified morphologically as D. injai, which served as positive controls, as well as in samples from three cases of demodicosis associated with proliferation of mites identified as D. injai. Furthermore, the PCR was positive in 2 out of 19 healthy dogs. Samples of Demodex canis and Demodex folliculorum were consistently negative. Skin samples from seven dogs with generalized demodicosis caused by D. canis were all negative in the D. injai-specific PCR, demonstrating that in generalized canine demodicosis, mite proliferation is species-specific. This technique can be a useful tool in the diagnosis and in epidemiologic and pathogenic studies. PMID:23851729

  9. [Characterization of Black and Dichothrix Cyanobacteria Based on the 16S Ribosomal RNA Gene Sequence

    NASA Technical Reports Server (NTRS)

    Ortega, Maya

    2010-01-01

    My project focuses on characterizing different cyanobacteria in thrombolitic mats found on the island of Highborn Cay, Bahamas. Thrombolites are interesting ecosystems because of the ability of bacteria in these mats to remove carbon dioxide from the atmosphere and mineralize it as calcium carbonate. In the future they may be used as models to develop carbon sequestration technologies, which could be used as part of regenerative life systems in space. These thrombolitic communities are also significant because of their similarities to early communities of life on Earth. I targeted two cyanobacteria in my research, Dichothrix spp. and whatever black is, since they are believed to be important to carbon sequestration in these thrombolitic mats. The goal of my summer research project was to molecularly identify these two cyanobacteria. DNA was isolated from each organism through mat dissections and DNA extractions. I ran Polymerase Chain Reactions (PCR) to amplify the 16S ribosomal RNA (rRNA) gene in each cyanobacteria. This specific gene is found in almost all bacteria and is highly conserved, meaning any changes in the sequence are most likely due to evolution. As a result, the 16S rRNA gene can be used for bacterial identification of different species based on the sequence of their 16S rRNA gene. Since the exact sequence of the Dichothrix gene was unknown, I designed different primers that flanked the gene based on the known sequences from other taxonomically similar cyanobacteria. Once the 16S rRNA gene was amplified, I cloned the gene into specialized Escherichia coli cells and sent the gene products for sequencing. Once the sequence is obtained, it will be added to a genetic database for future reference to and classification of other Dichothrix sp.

  10. [Cloning and sequencing of 16S rRNA gene of Phytoplasma CWB1 strain associated with cactus witches' broom].

    PubMed

    Cai, H; Li, F; Kong, B; Chen, H

    2001-12-01

    A 1.5 kb DNA fragment was amplified in DNA samples extracted from Opuntia salmiana porm showed witches'-broom symptom. The result indicates the existence of phytoplasma associated with this disease and this phytoplasma was designated as CWB1. The amplified fragment was ligated to pGEM-T easy vector and then transformed into JM109 strain of E. coli. Cloned DNA fragments were verified by PCR, restriction endonuclease (EcoRI) digestion and sequence analysis. The result revealed that the 16S rRNA gene of CWB1 consists of 1489 bp and shared 99.7% homology with Faba bean phyllody which belongs to phytoplasma 16S rII-C subgroup. So we can classify this strain into phytoplasma 16S rII-C subgroup. PMID:12552825

  11. Analysis of the bacterial diversity existing on animal hide and wool: development of a preliminary PCR-restriction fragment length polymorphism fingerprint database for identifying isolates.

    PubMed

    Chen, Yu; Gao, Hongwei; Zhang, Yanming; Deng, Mingjun; Wu, Zhenxing; Zhu, Laihua; Duan, Qing; Xu, Biao; Liang, Chengzhu; Yue, Zhiqin; Xiao, Xizhi

    2012-01-01

    Twenty-one bacterial strains were isolated from imported cattle hide and rabbit wool using two types of media, nutrient broth, and nutrient broth with serum. The bacteria identified were Brevibacillus laterosporus, Leclercia adecarboxylata, Peptococcus niger, Bacillus circulans, Raoultella ornithinolytica, Bacillus subtilis, Bacillus cereus, Bacillus thermobacillus, Bacillus choshinensis, Bacillus sphaericus, Acinetobacter haemolyticus, Sphingomonas paucimobilis, Bacillus thuringiensis, Staphylococcus intermedius, Mycobacteria, Moraxella, Klebsiella pneumoniae, Ralstonia pickettii, Staphylococcus chromogenes, Comamonas testosteroni, and Cupriavidus pauculus. The 16s rDNA gene of each bacterium was amplified using the universal primers 27f and 1492r. The amplicons were digested with AvaI, BamHI, BgII, DraI, EcoRI, EcoRV, HindIII, HinfI, HpaI, PstI, SmaI, TaqII, XbaI, XmaI, AluI, XhoI, and PvuI individually. A specific fingerprint from the PCR-restriction fragment length polymorphism method based on 16s rDNA was obtained for each bacterium. The results showed that the method developed was useful not only for bacterial identification but also for the etiological investigation of pathogens in imported animal hair and wool. PMID:23451394

  12. Assessment of Helminth Biodiversity in Wild Rats Using 18S rDNA Based Metagenomics

    PubMed Central

    Tsai, Isheng J.; Palomares-Rius, Juan Emilio; Yoshida, Ayako; Ogura, Yoshitoshi; Hayashi, Tetsuya; Maruyama, Haruhiko; Kikuchi, Taisei

    2014-01-01

    Parasite diversity has important implications in several research fields including ecology, evolutionary biology and epidemiology. Wide-ranging analysis has been restricted because of the difficult, highly specialised and time-consuming processes involved in parasite identification. In this study, we assessed parasite diversity in wild rats using 18S rDNA-based metagenomics. 18S rDNA PCR products were sequenced using an Illumina MiSeq sequencer and the analysis of the sequences using the QIIME software successfully classified them into several parasite groups. The comparison of the results with those obtained using standard methods including microscopic observation of helminth parasites in the rat intestines and PCR amplification/sequencing of 18S rDNA from isolated single worms suggests that this new technique is reliable and useful to investigate parasite diversity. PMID:25340824

  13. Molecular identification of adulteration in mutton based on mitochondrial 16S rRNA gene.

    PubMed

    Xu, Jia; Zhao, Wei; Zhu, Mengru; Wen, Yuanju; Xie, Tao; He, Xiaoqian; Zhang, Yongfeng; Cao, Suizhong; Niu, Lili; Zhang, Hongping; Zhong, Tao

    2016-01-01

    The aim of this study is to set up a protocol for identification of the adulteration in mutton based on mitochondrial 16S rRNA gene. The multiplex polymerase chain reaction (multi-PCR) assay was carried out to trace the impure DNA in mutton. A universal primer pair yielded an approximate 610 bp fragment in mutton, pork, duck, chicken, horse and cat meats. The amplicons of multi-PCR assay represented the species-specific products, which could be discriminated by the size ranging from 106 bp to 532 bp. Subsequently, the authentication of each fragment was also confirmed by sequencing. Random analyses of adulterants with various meats yielded the identical results to their components, showing the suitability of the multi-PCR assay for tracing of adulterant meats with high-accuracy and precision. This assay was sensitive to detect the species-specific DNA in different proportional mixtures of mutton and duck/pork (9.1%-90.9%). In conclusion, this multi-PCR assay successfully discriminated the double-, triple-, quadruple-, and quintuple-mixtures containing variant counterparts. This method will be particularly useful in the detection of mutton adulteration in processed foods further. PMID:24739005

  14. International interlaboratory study comparing single organism 16S rRNA gene sequencing data: Beyond consensus sequence comparisons.

    PubMed

    Olson, Nathan D; Lund, Steven P; Zook, Justin M; Rojas-Cornejo, Fabiola; Beck, Brian; Foy, Carole; Huggett, Jim; Whale, Alexandra S; Sui, Zhiwei; Baoutina, Anna; Dobeson, Michael; Partis, Lina; Morrow, Jayne B

    2015-03-01

    This study presents the results from an interlaboratory sequencing study for which we developed a novel high-resolution method for comparing data from different sequencing platforms for a multi-copy, paralogous gene. The combination of PCR amplification and 16S ribosomal RNA gene (16S rRNA) sequencing has revolutionized bacteriology by enabling rapid identification, frequently without the need for culture. To assess variability between laboratories in sequencing 16S rRNA, six laboratories sequenced the gene encoding the 16S rRNA from Escherichia coli O157:H7 strain EDL933 and Listeria monocytogenes serovar 4b strain NCTC11994. Participants performed sequencing methods and protocols available in their laboratories: Sanger sequencing, Roche 454 pyrosequencing(®), or Ion Torrent PGM(®). The sequencing data were evaluated on three levels: (1) identity of biologically conserved position, (2) ratio of 16S rRNA gene copies featuring identified variants, and (3) the collection of variant combinations in a set of 16S rRNA gene copies. The same set of biologically conserved positions was identified for each sequencing method. Analytical methods using Bayesian and maximum likelihood statistics were developed to estimate variant copy ratios, which describe the ratio of nucleotides at each identified biologically variable position, as well as the likely set of variant combinations present in 16S rRNA gene copies. Our results indicate that estimated variant copy ratios at biologically variable positions were only reproducible for high throughput sequencing methods. Furthermore, the likely variant combination set was only reproducible with increased sequencing depth and longer read lengths. We also demonstrate novel methods for evaluating variable positions when comparing multi-copy gene sequence data from multiple laboratories generated using multiple sequencing technologies. PMID:27077030

  15. Isolation and characterization of a novel chlorpyrifos degrading flavobacterium species EMBS0145 by 16S rRNA gene sequencing.

    PubMed

    Amareshwari, P; Bhatia, Mayuri; Venkatesh, K; Roja Rani, A; Ravi, G V; Bhakt, Priyanka; Bandaru, Srinivas; Yadav, Mukesh; Nayarisseri, Anuraj; Nair, Achuthsankar S

    2015-03-01

    Indiscriminate application of pesticides like chlorpyrifos, diazinon, or malathion contaminate the soil in addition has being unsafe often it has raised severe health concerns. Conversely, microorganisms like Trichoderma, Aspergillus and Bacteria like Rhizobium Bacillus, Azotobacter, Flavobacterium etc have evolved that are endowed with degradation of pesticides aforementioned to non-toxic products. The current study pitches into identification of a novel species of Flavobacterium bacteria capable to degrade the Organophosphorous pesticides. The bacterium was isolated from agricultural soil collected from Guntur District, Andhra Pradesh, India. The samples were serially diluted and the aliquots were incubated for a suitable time following which the suspected colony was subjected to 16S rDNA sequencing. The sequence thus obtained was aligned pairwise against Flavobacterium species, which resulted in identification of novel specie of Flavobacterium later named as EMBS0145, the sequence of which was deposited in in GenBank with accession number JN794045. PMID:25248957

  16. 16S ribosomal RNA methylation: emerging resistance mechanism against aminoglycosides.

    PubMed

    Doi, Yohei; Arakawa, Yoshichika

    2007-07-01

    Methylation of 16S ribosomal RNA (rRNA) has recently emerged as a new mechanism of resistance against aminoglycosides among gram-negative pathogens belonging to the family Enterobacteriaceae and glucose-nonfermentative microbes, including Pseudomonas aeruginosa and Acinetobacter species. This event is mediated by a newly recognized group of 16S rRNA methylases, which share modest similarity to those produced by aminoglycoside-producing actinomycetes. Their presence confers a high level of resistance to all parenterally administered aminoglycosides that are currently in clinical use. The responsible genes are mostly located on transposons within transferable plasmids, which provides them with the potential to spread horizontally and may in part explain the already worldwide distribution of this novel resistance mechanism. Some of these organisms have been found to coproduce extended-spectrum beta-lactamases or metallo-beta-lactamases, contributing to their multidrug-resistant phenotypes. A 2-tiered approach, consisting of disk diffusion tests followed by confirmation with polymerase chain reaction, is recommended for detection of 16S rRNA methylase-mediated resistance. PMID:17554708

  17. PhytoREF: a reference database of the plastidial 16S rRNA gene of photosynthetic eukaryotes with curated taxonomy.

    PubMed

    Decelle, Johan; Romac, Sarah; Stern, Rowena F; Bendif, El Mahdi; Zingone, Adriana; Audic, Stéphane; Guiry, Michael D; Guillou, Laure; Tessier, Désiré; Le Gall, Florence; Gourvil, Priscillia; Dos Santos, Adriana L; Probert, Ian; Vaulot, Daniel; de Vargas, Colomban; Christen, Richard

    2015-11-01

    Photosynthetic eukaryotes have a critical role as the main producers in most ecosystems of the biosphere. The ongoing environmental metabarcoding revolution opens the perspective for holistic ecosystems biological studies of these organisms, in particular the unicellular microalgae that often lack distinctive morphological characters and have complex life cycles. To interpret environmental sequences, metabarcoding necessarily relies on taxonomically curated databases containing reference sequences of the targeted gene (or barcode) from identified organisms. To date, no such reference framework exists for photosynthetic eukaryotes. In this study, we built the PhytoREF database that contains 6490 plastidial 16S rDNA reference sequences that originate from a large diversity of eukaryotes representing all known major photosynthetic lineages. We compiled 3333 amplicon sequences available from public databases and 879 sequences extracted from plastidial genomes, and generated 411 novel sequences from cultured marine microalgal strains belonging to different eukaryotic lineages. A total of 1867 environmental Sanger 16S rDNA sequences were also included in the database. Stringent quality filtering and a phylogeny-based taxonomic classification were applied for each 16S rDNA sequence. The database mainly focuses on marine microalgae, but sequences from land plants (representing half of the PhytoREF sequences) and freshwater taxa were also included to broaden the applicability of PhytoREF to different aquatic and terrestrial habitats. PhytoREF, accessible via a web interface (http://phytoref.fr), is a new resource in molecular ecology to foster the discovery, assessment and monitoring of the diversity of photosynthetic eukaryotes using high-throughput sequencing. PMID:25740460

  18. Dynamics of R1 and R2 elements in the rDNA locus of Drosophila simulans.

    PubMed Central

    Pérez-González, C E; Eickbush, T H

    2001-01-01

    The mobile elements R1 and R2 insert specifically into the rRNA gene locus (rDNA locus) of arthropods, a locus known to undergo concerted evolution, the recombinational processes that preserve the sequence homogeneity of all repeats. To monitor how rapidly individual R1 and R2 insertions are turned over in the rDNA locus by these processes, we have taken advantage of the many 5' truncation variants that are generated during the target-primed reverse transcription mechanism used by these non-LTR retrotransposons for their integration. A simple PCR assay was designed to reveal the pattern of the 5' variants present in the rDNA loci of individual X chromosomes in a population of Drosophila simulans. Each rDNA locus in this population was found to have a large, unique collection of 5' variants. Each variant was present at low copy number, usually one copy per chromosome, and was seldom distributed to other chromosomes in the population. The failure of these variants to spread to other units in the same rDNA locus suggests a strong recombinational bias against R1 and R2 that results in the individual copies of these elements being rapidly lost from the rDNA locus. This bias suggests a significantly higher frequency of R1 and R2 retrotransposition than we have previously suggested. PMID:11514447

  19. Quantitative Detection of Clostridium perfringens in the Broiler Fowl Gastrointestinal Tract by Real-Time PCR

    PubMed Central

    Wise, Mark G.; Siragusa, Gregory R.

    2005-01-01

    Strains of Clostridium perfringens are a frequent cause of food-borne disease and gas gangrene and are also associated with necrotic enteritis in chickens. To detect and quantify the levels of C. perfringens in the chicken gastrointestinal tract, a quantitative real-time PCR assay utilizing a fluorogenic, hydrolysis-type probe was developed and utilized to assay material retrieved from the broiler chicken cecum and ileum. Primers and probe were selected following an alignment of 16S rDNA sequences from members of cluster I of the genus Clostridium, and proved to be specific for C. perfringens. The assay could detect approximately 50 fg of C. perfringens genomic DNA and approximately 20 cells in pure culture. Measurements of the analytical sensitivity determined with spiked intestinal contents indicated that the consistent limit of detection with ileal samples was approximately 102 CFU/g of ileal material, but only about 104 CFU/g of cecal samples. The decreased sensitivity with the cecal samples was due to the presence of an unidentified chemical PCR inhibitor(s) in the cecal DNA purifications. The assay was utilized to rapidly detect and quantify C. perfringens levels in the gut tract of broiler chickens reared without supplementary growth-promoting antibiotics that manifested symptoms of necrotic enteritis. The results illustrated that quantitative real-time PCR correlates well with quantification via standard plate counts in samples taken from the ileal region of the gastrointestinal tract. PMID:16000804

  20. Homology of the 3' terminal sequences of the 18S rRNA of Bombyx mori and the 16S rRNA of Escherchia coli.

    PubMed Central

    Samols, D R; Hagenbuchle, O; Gage, L P

    1979-01-01

    The terminal 220 base pairs (bp) of the gene for 18S rRNA and 18 bp of the adjoining spacer rDNA of the silkworm Bombyx mori have been sequenced. Comparison with the sequence of the 16S rRNA gene of Escherichia coli has shown that a region including 45 bp of the B. mori sequence at the 3' end is remarkably homologous with the 3' terminal E. coli sequence. Other homologies occur in the terminal regions of the 18S and 16S rRNAs, including a perfectly conserved stretch of 13 bp within a longer homology located 150--200 bp from the 3' termini. These homologies are the most extensive so far reported between prokaryotic and eukaryotic genomic DNA. Images PMID:390496

  1. Report of an immunocompetent case with disseminated infection due to Nocardia otitidiscaviarum: Identification by 16S rRNA gene sequencing.

    PubMed

    Eren, Esma; Ulu-Kilic, Aysegul; Atalay, Altay; Demiraslan, Hayati; Parkan, Omur; Koc, Nedret

    2016-03-01

    Nocardia otitidiscaviarum belongs to the agents of opportunistic infections seen in immunocompromised patients, but may occur rarely in immunocompetent patients. In this report we described a case of a previously healthy 69-year-old woman with cerebral and retroperitoneal abscess due to Nocardia otitidiscaviarum. The patient was admitted to hospital because of loss of strength in her right arm and leg. Nocardia spp. was isolated from the abscess material. The intracranial lesions were drained by stereotactic craniotomy. The large abscess located around the left kidney was drained and microscopic examination of aspirated material showed Nocardia spp. For species identification, 16S rRNA gene sequencing was carried out and was 100% concordant with Nocardia otitidiscaviarum. Use of 16S rDNA gene sequencing for identification permits detection of rare aetiologic agents that cause brain abscesses. PMID:27031902

  2. [Analysis of 5S rDNA changes in synthetic allopolyploids Triticum x Aegilops].

    PubMed

    Shcherban', A B; Sergeeva, E M; Badaeva, E D; Salina, E A

    2008-01-01

    By the example of three synthetic allopolyploids: Aegilops sharonensis x Ae. umbellulata (2n =28), Triticum urartu x Ae. tauschii (2n =28), T. dicoccoides x Ae. tauschii (2n =42) the 5S rDNA changes at the early stage of allopolyploidization were investigated. Using fluorescent in situ hybridization (FISH), the quantitative changes affecting the separate loci of one of the parental genomes were revealed in plants of S3 generation of each hybrid combination. Souther hybridization with genomic DNA of allopolyploid T. urartu x Ae. tauschii (TMU38 x TQ27) revealed lower intensity of the fragments from Ae. tauschii compared with the T. urartu fragments. It may be confirmation of the reduction of signal on 1D chromosome that was revealed in this hybrid using FISH. Both appearance of a new 5S rDNA fragments and full disappearance of fragments from parental species were not showed by Southern hybridization, as well as PCR-analysis of 5-15 plants of S2-S3 generations. The changes were not found under comparison of primary structure of nine 5S rDNA sequences of allopolyploid TMU38 x TQ27 with analogous sequences from parental species genomes. The observable similarity by FISH results of one of the studied synthetic allopolyploids with natural allopolyploid of similar genome composition indicates the early formation of unique for each allopolyploid 5S rDNA organization. PMID:18856060

  3. Enhanced radiative Auger emission from lithiumlike 16S13+

    NASA Astrophysics Data System (ADS)

    Bernstein, E. M.; Clark, M. W.; Oglesby, C. S.; Tanis, J. A.; Graham, W. G.; McFarland, R. H.; Morgan, T. J.; Johnson, B. M.; Jones, K. W.

    1990-03-01

    The radiative Auger emission (RAE) from 0.94-6.25-MeV/u 16S13+ (lithiumlike) projectiles excited in collisions with He target atoms has been measured. For these highly stripped ions the intensity of RAE photons relative to Kα x-ray emission is enhanced by about a factor of five compared with theoretical calculations and an earlier experimental measurement for S ions with few electron vacancies. The enhancement of RAE for S13+ is qualitatively similar to results reported previously for lithiumlike 23V20+; however, some differences between S and V are evident.

  4. Bacterial communities in two Antarctic ice cores analyzed by 16S rRNA gene sequencing analysis

    NASA Astrophysics Data System (ADS)

    Segawa, Takahiro; Ushida, Kazunari; Narita, Hideki; Kanda, Hiroshi; Kohshima, Shiro

    2010-08-01

    Antarctic ice cores could preserve ancient airborne microorganisms. We examined bacteria in two Antarctic ice core samples, an interglacial age sample from Mizuho Base and a glacial age sample from the Yamato Mountains, by 16S rRNA gene sequencing analysis. Bacterial density, the number of bacterial OTUs and Simpson’s diversity index was larger in the Mizuho sample than in the Yamato sample. The 16S rDNA clone library from the Mizuho sample was dominated by the phylum Firmicutes, while the large part of that from the Yamato sample was composed of the Gamma proteobacteria group. Major sources of these identified bacteria estimated from their database records also differed between the samples: in the Mizuho sample bacterial species recorded from animals were higher than that of the Yamato sample, while in the Yamato sample bacteria from aquatic and snow-ice environments were higher than that of the Mizuho sample. The results suggest that these bacteria were past airborne bacteria that would vary in density, diversity and species composition depending on global environmental change. Our results imply that bacteria in Antarctic ice cores could be used as new environmental markers for past environmental studies.

  5. Rapid detection of mutations associated with resistance to erythromycin in Campylobacter jejuni/coli by PCR and line probe assay.

    PubMed

    Niwa, H; Chuma, T; Okamoto, K; Itoh, K

    2001-10-01

    Mutation of 23S rDNA is one of the mechanisms of erythromycin resistance. PCR and line probe assay (PCR-LiPA) with ten oligonucleotide probes were developed to detect the mutations associated with macrolide resistance at positions of 2072, 2073 and 2074 in 23S rDNA of Campylobacter jejuni/coli. A2074-->G mutation was detected in 12 of 25 isolates, which were resistant to erythromycin. No other mutations in 23S rDNA were detected. The rest of the strains were susceptible to erythromycin and no mutation in 23S rDNA was detected. Six laboratory induced erythromycin resistant mutants had no mutations in 23S rDNA. PCR-LiPA is a useful and rapid method to detect mutations in 23S rDNA associated with erythromycin resistance in C. jejuni/coli. PMID:11691569

  6. Ovine pedomics: the first study of the ovine foot 16S rRNA-based microbiome

    PubMed Central

    Calvo-Bado, Leo A; Oakley, Brian B; Dowd, Scot E; Green, Laura E; Medley, Graham F; Ul-Hassan, Atiya; Bateman, Vicky; Gaze, William; Witcomb, Luci; Grogono-Thomas, Rose; Kaler, Jasmeet; Russell, Claire L; Wellington, Elizabeth MH

    2011-01-01

    We report the first study of the bacterial microbiome of ovine interdigital skin based on 16S rRNA by pyrosequencing and conventional cloning with Sanger-sequencing. Three flocks were selected, one a flock with no signs of footrot or interdigital dermatitis, a second flock with interdigital dermatitis alone and a third flock with both interdigital dermatitis and footrot. The sheep were classified as having either healthy interdigital skin (H) and interdigital dermatitis (ID) or virulent footrot (VFR). The ovine interdigital skin bacterial community varied significantly by flock and clinical condition. The diversity and richness of operational taxonomic units was greater in tissue from sheep with ID than H or VFR-affected sheep. Actinobacteria, Bacteriodetes, Firmicutes and Proteobacteria were the most abundant phyla comprising 25 genera. Peptostreptococcus, Corynebacterium and Staphylococcus were associated with H, ID and VFR, respectively. Sequences of Dichelobacter nodosus, the causal agent of ovine footrot, were not amplified because of mismatches in the 16S rRNA universal forward primer (27F). A specific real-time PCR assay was used to demonstrate the presence of D. nodosus, which was detected in all samples including the flock with no signs of ID or VFR. Sheep with ID had significantly higher numbers of D. nodosus (104–109 cells per g tissue) than those with H or VFR feet. PMID:21430786

  7. Ovine pedomics: the first study of the ovine foot 16S rRNA-based microbiome.

    PubMed

    Calvo-Bado, Leo A; Oakley, Brian B; Dowd, Scot E; Green, Laura E; Medley, Graham F; Ul-Hassan, Atiya; Bateman, Vicky; Gaze, William; Witcomb, Luci; Grogono-Thomas, Rose; Kaler, Jasmeet; Russell, Claire L; Wellington, Elizabeth M H

    2011-09-01

    We report the first study of the bacterial microbiome of ovine interdigital skin based on 16S rRNA by pyrosequencing and conventional cloning with Sanger-sequencing. Three flocks were selected, one a flock with no signs of footrot or interdigital dermatitis, a second flock with interdigital dermatitis alone and a third flock with both interdigital dermatitis and footrot. The sheep were classified as having either healthy interdigital skin (H) and interdigital dermatitis (ID) or virulent footrot (VFR). The ovine interdigital skin bacterial community varied significantly by flock and clinical condition. The diversity and richness of operational taxonomic units was greater in tissue from sheep with ID than H or VFR-affected sheep. Actinobacteria, Bacteriodetes, Firmicutes and Proteobacteria were the most abundant phyla comprising 25 genera. Peptostreptococcus, Corynebacterium and Staphylococcus were associated with H, ID and VFR, respectively. Sequences of Dichelobacter nodosus, the causal agent of ovine footrot, were not amplified because of mismatches in the 16S rRNA universal forward primer (27F). A specific real-time PCR assay was used to demonstrate the presence of D. nodosus, which was detected in all samples including the flock with no signs of ID or VFR. Sheep with ID had significantly higher numbers of D. nodosus (10(4)-10(9) cells per g tissue) than those with H or VFR feet. PMID:21430786

  8. Preliminary study on mitochondrial 16S rRNA gene sequences and phylogeny of flatfishes (Pleuronectiformes)

    NASA Astrophysics Data System (ADS)

    You, Feng; Liu, Jing; Zhang, Peijun; Xiang, Jianhai

    2005-09-01

    A 605 bp section of mitochondrial 16S rRNA gene from Paralichthys olivaceus, Pseudorhombus cinnamomeus, Psetta maxima and Kareius bicoloratus, which represent 3 families of Order Pleuronectiformes was amplified by PCR and sequenced to show the molecular systematics of Pleuronectiformes for comparison with related gene sequences of other 6 flatfish downloaded from GenBank. Phylogenetic analysis based on genetic distance from related gene sequences of 10 flatfish showed that this method was ideal to explore the relationship between species, genera and families. Phylogenetic trees set-up is based on neighbor-joining, maximum parsimony and maximum likelihood methods that accords to the general rule of Pleuronectiformes evolution. But they also resulted in some confusion. Unlike data from morphological characters, P. olivaceus clustered with K. bicoloratus, but P. cinnamomeus did not cluster with P. olivaceus, which is worth further studying.

  9. Intra-Genomic Heterogeneity in 16S rRNA Genes in Strictly Anaerobic Clinical Isolates from Periodontal Abscesses

    PubMed Central

    Chen, Jiazhen; Miao, Xinyu; Xu, Meng; He, Junlin; Xie, Yi; Wu, Xingwen; Chen, Gang; Yu, Liying; Zhang, Wenhong

    2015-01-01

    Background Members of the genera Prevotella, Veillonella and Fusobacterium are the predominant culturable obligate anaerobic bacteria isolated from periodontal abscesses. When determining the cumulative number of clinical anaerobic isolates from periodontal abscesses, ambiguous or overlapping signals were frequently encountered in 16S rRNA gene sequencing chromatograms, resulting in ambiguous identifications. With the exception of the genus Veillonella, the high intra-chromosomal heterogeneity of rrs genes has not been reported. Methods The 16S rRNA genes of 138 clinical, strictly anaerobic isolates and one reference strain were directly sequenced, and the chromatograms were carefully examined. Gene cloning was performed for 22 typical isolates with doublet sequencing signals for the 16S rRNA genes, and four copies of the rrs-ITS genes of 9 Prevotella intermedia isolates were separately amplified by PCR, sequenced and compared. Five conserved housekeeping genes, hsp60, recA, dnaJ, gyrB1 and rpoB from 89 clinical isolates of Prevotella were also amplified by PCR and sequenced for identification and phylogenetic analysis along with 18 Prevotella reference strains. Results Heterogeneity of 16S rRNA genes was apparent in clinical, strictly anaerobic oral bacteria, particularly in the genera Prevotella and Veillonella. One hundred out of 138 anaerobic strains (72%) had intragenomic nucleotide polymorphisms (SNPs) in multiple locations, and 13 strains (9.4%) had intragenomic insertions or deletions in the 16S rRNA gene. In the genera Prevotella and Veillonella, 75% (67/89) and 100% (19/19) of the strains had SNPs in the 16S rRNA gene, respectively. Gene cloning and separate amplifications of four copies of the rrs-ITS genes confirmed that 2 to 4 heterogeneous 16S rRNA copies existed. Conclusion Sequence alignment of five housekeeping genes revealed that intra-species nucleotide similarities were very high in the genera Prevotella, ranging from 94.3–100%. However, the

  10. Detection of Alicyclobacillus spp. in Fruit Juice by Combination of Immunomagnetic Separation and a SYBR Green I Real-Time PCR Assay.

    PubMed

    Cai, Rui; Wang, Zhouli; Yuan, Yahong; Liu, Bin; Wang, Ling; Yue, Tianli

    2015-01-01

    An approach based on immunomagnetic separation (IMS) and SYBR Green I real-time PCR (real-time PCR) with species-specific primers and melting curve analysis was proposed as a rapid and effective method for detecting Alicyclobacillus spp. in fruit juices. Specific primers targeting the 16S rDNA sequences of Alicyclobacillus spp. were designed and then confirmed by the amplification of DNA extracted from standard strains and isolates. Spiked samples containing known amounts of target bacteria were used to obtain standard curves; the correlation coefficient was greater than 0.986 and the real-time PCR amplification efficiencies were 98.9%- 101.8%. The detection limit of the testing system was 2.8×101 CFU/mL. The coefficient of variation for intra-assay and inter-assay variability were all within the acceptable limit of 5%. Besides, the performance of the IMS-real-time PCR assay was further investigated by detecting naturally contaminated kiwi fruit juice; the sensitivity, specificity and accuracy were 91.7%, 95.9% and 95.3%, respectively. The established IMS-real-time PCR procedure provides a new method for identification and quantitative detection of Alicyclobacillus spp. in fruit juice. PMID:26488469

  11. Molecular detection of the clostridia in an anaerobic biohydrogen fermentation system by hydrogenase mRNA-targeted reverse transcription-PCR.

    PubMed

    Chang, Jui-Jen; Chen, Wei-En; Shih, Shiou-Yun; Yu, Sian-Jhong; Lay, Jiunn-Jyi; Wen, Fu-Shyan; Huang, Chieh-Chen

    2006-05-01

    Molecular biological approaches were developed to monitor the potential biohydrogen-producing clostridia in an anaerobic semisolid fermentation system that used brewery yeast waste as the fermentation substrate. The denaturing gradient gel electrophoresis with 16S rDNA gene-targeted polymerase chain reaction (PCR) analysis was employed to confirm the existence of clostridia in the system. Remarkably, reproducible nucleotide sequences of clostridia were obtained from different hydrogen production stages by using hydrogenase gene-targeted reverse transcription (RT)-PCR. These RNA-based information suggested that the predominant hydrogen-producing strains possess either a specific Clostridium pasteurianum-like or a specific Clostridium saccharobutylicum-like hydrogenase sequence. Comparison of the hydrogenase gene-targeted sequence profiles between PCR and RT-PCR revealed that the specific C. pasteurianum-like hydrogenase harboring bacterial strains were dominant in both mRNA and bacterial population level. On the other hand, the specific C. saccharobutylicum-like hydrogenase harboring strains expressed high level of hydrogenase mRNA but may not be dominant in population. Furthermore, quantitative real-time RT-PCR analysis showed the expression pattern of the clostridial hydrogenase mRNA and may serve as an activity index for the system. PMID:16217655

  12. Detection of Alicyclobacillus spp. in Fruit Juice by Combination of Immunomagnetic Separation and a SYBR Green I Real-Time PCR Assay

    PubMed Central

    Yuan, Yahong; Liu, Bin; Wang, Ling; Yue, Tianli

    2015-01-01

    An approach based on immunomagnetic separation (IMS) and SYBR Green I real-time PCR (real-time PCR) with species-specific primers and melting curve analysis was proposed as a rapid and effective method for detecting Alicyclobacillus spp. in fruit juices. Specific primers targeting the 16S rDNA sequences of Alicyclobacillus spp. were designed and then confirmed by the amplification of DNA extracted from standard strains and isolates. Spiked samples containing known amounts of target bacteria were used to obtain standard curves; the correlation coefficient was greater than 0.986 and the real-time PCR amplification efficiencies were 98.9%- 101.8%. The detection limit of the testing system was 2.8×101 CFU/mL. The coefficient of variation for intra-assay and inter-assay variability were all within the acceptable limit of 5%. Besides, the performance of the IMS-real-time PCR assay was further investigated by detecting naturally contaminated kiwi fruit juice; the sensitivity, specificity and accuracy were 91.7%, 95.9% and 95.3%, respectively. The established IMS-real-time PCR procedure provides a new method for identification and quantitative detection of Alicyclobacillus spp. in fruit juice. PMID:26488469

  13. A multiplex nested PCR assay for simultaneous detection of Corchorus golden mosaic virus and a phytoplasma in white jute (Corchorus capsularis L.).

    PubMed

    Biswas, C; Dey, P; Satpathy, S

    2013-05-01

    A multiplex nested PCR assay was developed by optimizing reaction components and reaction cycling parameters for simultaneous detection of Corchorus golden mosaic virus (CoGMV) and a phytoplasma (Group 16Sr V-C) causing little leaf and bunchy top in white jute (Corchorus capsularis). Three sets of specific primers viz. a CoGMV specific (DNA-A region) primer, a 16S rDNA universal primer pair P1/P7 and nested primer pair R16F2n/R2 for phytoplasmas were used. The concentrations of the PCR components such as primers, MgCl2 , Taq DNA polymerase, dNTPs and PCR conditions including annealing temperature and amplification cycles were examined and optimized. Expected fragments of 1 kb (CoGMV), 674 bp (phytoplasma) and 370 bp (nested R16F2n/R2) were successfully amplified by this multiplex nested PCR system ensuring simultaneous, sensitive and specific detection of the phytoplasma and the virus. The multiplex nested PCR provides a sensitive, rapid and low-cost method for simultaneous detection of jute little leaf phytoplasma and CoGMV. Based on BLASTn analyses, the phytoplasma was found to belong to the Group 16Sr V-C. PMID:23413927

  14. Strengths and Limitations of 16S rRNA Gene Amplicon Sequencing in Revealing Temporal Microbial Community Dynamics

    PubMed Central

    Poretsky, Rachel; Rodriguez-R, Luis M.; Luo, Chengwei; Tsementzi, Despina; Konstantinidis, Konstantinos T.

    2014-01-01

    This study explored the short-term planktonic microbial community structure and resilience in Lake Lanier (GA, USA) while simultaneously evaluating the technical aspects of identifying taxa via 16S rRNA gene amplicon and metagenomic sequence data. 16S rRNA gene amplicons generated from four temporally discrete samples were sequenced with 454 GS-FLX-Ti yielding ∼40,000 rRNA gene sequences from each sample and representing ∼300 observed OTUs. Replicates obtained from the same biological sample clustered together but several biases were observed, linked to either the PCR or sequencing-preparation steps. In comparisons with companion whole-community shotgun metagenome datasets, the estimated number of OTUs at each timepoint was concordant, but 1.5 times and ∼10 times as many phyla and genera, respectively, were identified in the metagenomes. Our analyses showed that the 16S rRNA gene captures broad shifts in community diversity over time, but with limited resolution and lower sensitivity compared to metagenomic data. We also identified OTUs that showed marked shifts in abundance over four close timepoints separated by perturbations and tracked these taxa in the metagenome vs. 16S rRNA amplicon data. A strong summer storm had less of an effect on community composition than did seasonal mixing, which revealed a distinct succession of organisms. This study provides insights into freshwater microbial communities and advances the approaches for assessing community diversity and dynamics in situ. PMID:24714158

  15. Emergence of 16S rRNA methylase-producing Acinetobacter baumannii and Pseudomonas aeruginosa isolates in hospitals in Vietnam

    PubMed Central

    2013-01-01

    Background 16S rRNA methylase-producing Gram-negative bacteria are highly resistant to all clinically important aminoglycosides. We analyzed clinical strains of 16S rRNA methylase-producing Acinetobactor baumannii and Pseudomonas aeruginosa obtained from clinical isolates in medical settings in Vietnam. Methods From 2008 to 2011, 101 clinical strains of A. baumannii and 15 of P. aeruginosa were isolated from patients in intensive care units (ICUs) in two medical settings in Vietnam. Antimicrobial susceptibilities were determined using the microdilution method and epidemiological analysis was performed by pulsed-field gel electrophoresis and MLST. Genes encoding the 16S rRNA methylases, OXAs and CTX-Ms were analyzed by PCR and sequence analysis. Results 16S rRNA methylase-producing Gram-negative pathogens were detected in two hospitals in Vietnam. Of the 101 clinical isolates of A. baumannii and the 15 of P. aeruginosa isolated from two ICUs in these hospitals, 72 (71.3%) were highly resistant to amikacin, arbekacin and gentamicin, with MICs greater than 1,024 mg/L. The 16S rRNA methylases ArmA and RmtB were produced by 61 and 9 isolates of A. baumannii, respectively, and RmtB was produced by 2 isolates of P. aeruginosa. Moreover, 52 of the A. baumannii isolates producing 16S rRNA methylases harbored both blaOXA-23-like and blaOXA-51-like genes. Most A. baumannii isolates producing 16S rRNA methylase obtained in hospital A in Hanoi were ST91 and ST231, whereas most from hospital B in Ho Chi Minh City were ST136, ST195, and ST254. The two P. aeruginosa isolates harboring rmtB showed different patterns on PFGE, one each corresponding to ST217 and ST313. Conclusions Gram-negative bacteria producing the 16S rRNA methylases ArmA and RmtB are emerging in medical settings in Vietnam. A. baumannii isolates in northern and southern regions of Vietnam may be of different lineages. PMID:23721359

  16. 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

  17. Application of Sequence-Specific Labeled 16S rRNA Gene Oligonucleotide Probes for Genetic Profiling of Cyanobacterial Abundance and Diversity by Array Hybridization

    PubMed Central

    Rudi, Knut; Skulberg, Olav M.; Skulberg, Randi; Jakobsen, Kjetill S.

    2000-01-01

    DNA sequence information for the small-subunit rRNA gene (16S rDNA) obtained from cyanobacterial cultures was used to investigate the presence of cyanobacteria and their abundance in natural habitats. Eight planktonic communities developing in lakes characterized by relatively low algal biomass (mesotrophic) and in lakes with correspondingly high biomass (eutrophic) were selected for the study. The organismal compositions of the water samples were analyzed genetically, using multiplex sequence-specific labeling of oligonucleotide probes targeted to 16S rDNA and subsequent hybridization of the labeled probes to their respective complements spotted onto a solid support (DNA array). Ten probes were established to determine the relative abundances of the discernible cyanobacteria encountered in the selected lakes. The probes were generally specific for their targets, as determined through analyses of clone cultures. Reproducible abundance profiles were established for the lakes investigated in the subsequent analyses of natural cyanobacterial communities. The results from the genetic analyses were then compared with information obtained from standard hydrobiological and hydrochemical analyses. Qualitatively, there were relatively good correlations among the groups of organisms (Nostoc, Microcystis, and Planktothrix species) found in the different lakes. The levels of correlation were lower for the quantitative data. This may, however, be due to differences in sample processing technique. The conclusions from these comparisons are that the genetic abundance profiles may provide a foundation for separating and quantifying genetically distinct groups of cyanobacteria in their natural habitats. PMID:10966421

  18. Application of real-time PCR for detection of Lawsonia intracellularis and Brachyspira hyodysenteriae in fecal samples from pigs.

    PubMed

    Zmudzki, J; Szczotka, A; Podgórska, K; Nowak, A; Grzesiak, A; Dors, A; Pejsak, Z

    2012-01-01

    The aim of the study was to develop and validate real-time PCR method for the quantification of Lawsonia intracellularis and Brachyspira hyodysenteriae in porcine feces. Before the optimization process was performed two different extraction methods were compared to select the more efficient one. Based on the results achieved at this stage the boiling procedure was rejected and a commercially available silica-membrane based method was chosen for further analysis. The primers and the Taqman probe for B. hyodysenteriae and L. intracellularis were based on the sequence of NADH oxidase gene and 16S rDNA gene, respectively. The detection limit of the real-time PCR for suspension of feces inoculated with B. hyodysenteriae and L. intracellularis was determined to be 1.5x10(3) CFU/ml and 6.5x10(1) CFU/ml, respectively. The results of this study demonstrate that our real-time PCR is able to detect low number of B. hyodysenteriae and L. intracellularis cells which is satisfying in routine diagnosis of swine dysentery and proliferative enteropathy. Therefore, it is possible to identify both subclinically infected pigs and those representing an acute form of mentioned diseases. In summary, the quantitative real-time PCR is useful for routine diagnosis of L. intracellularis and B. hyodysenteriae. Compared to conventional PCR, the new validated quantification method based on real-time PCR is fast and with reduced risk of laboratory contamination. The novel technique is specific and even more sensitive than the previously used one. Furthermore, the new real-time PCR enables quick detection and quantification of both pathogens in fecal samples, which helps to estimate the health status of a pig herd. PMID:22844704

  19. Improved group-specific primers based on the full SILVA 16S rRNA gene reference database.

    PubMed

    Pfeiffer, Stefan; Pastar, Milica; Mitter, Birgit; Lippert, Kathrin; Hackl, Evelyn; Lojan, Paul; Oswald, Andreas; Sessitsch, Angela

    2014-08-01

    Quantitative PCR (qPCR) and community fingerprinting methods, such as the Terminal Restriction Fragment Length Polymorphism (T-RFLP) analysis,are well-suited techniques for the examination of microbial community structures. The use of phylum and class-specific primers can provide enhanced sensitivity and phylogenetic resolution as compared with domain-specific primers. To date, several phylum- and class-specific primers targeting the 16S ribosomal RNA gene have been published. However, many of these primers exhibit low discriminatory power against non-target bacteria in PCR. In this study, we evaluated the precision of certain published primers in silico and via specific PCR. We designed new qPCR and T-RFLP primer pairs (for the classes Alphaproteobacteria and Betaproteobacteria, and the phyla Bacteroidetes, Firmicutes and Actinobacteria) by combining the sequence information from a public dataset (SILVA SSU Ref 102 NR) with manual primer design. We evaluated the primer pairs via PCR using isolates of the above-mentioned groups and via screening of clone libraries from environmental soil samples and human faecal samples. As observed through theoretical and practical evaluation, the primers developed in this study showed a higher level of precision than previously published primers, thus allowing a deeper insight into microbial community dynamics. PMID:25229098

  20. Genetic mapping of the Batten disease locus (CLN3) to the interval D16S288-D16S383 by analysis of haplotypes and allelic association

    SciTech Connect

    Mitchison, H.M.; O`Rawe, A.M.; Gardiner, R.M.

    1994-07-15

    CLN3, the gene for juvenile-onset neuronal ceroid lipofuscinosis (JNCL) or Batten disease, has been localized by genetic linkage analysis to chromosome 16p between loci D16S297 and D16S57. The authors have now further refined the localization of CLN3 by haplotype analysis using two new microsatellite markers from loci D16S383 and SPN in the D16S297-D16S57 interval on a larger collaborative family resource consisting of 142 JNCL pedigrees. Crossover events in 3 maternal meioses define new flanking markers for CLN3 and localize the gene to the interval at 16p12.1-11.2 between D16S288 and D16S383, which corresponds to a genetic distance of 2.1 cM. Within this interval 4 microsatellite loci are in strong linkage disequilibrium with CLN3, and extended haplotype analysis of the associated alleles indicates that CLN3 is in closest proximity to loci D16S299 and D16S298. 6 refs., 1 fig., 2 tabs.

  1. Assessment of microbial dynamics in the Pearl River Estuary by 16S rRNA terminal restriction fragment analysis

    NASA Astrophysics Data System (ADS)

    Wu, Madeline; Song, Liansheng; Ren, Jianping; Kan, Jianjun; Qian, Pei-Yuan

    2004-10-01

    We have evaluated the feasibility of using the terminal restriction fragment length polymorphism (T-RFLP) pattern of polymerase chain reaction (PCR) amplified 16S rRNA sequences to track the changes of the free-living bacterial community for the Pearl River Estuary surface waters. The suitability of specific PCR primers, PCR bias induced by thermal cycles, and field-sampling volumes were critically evaluated in laboratory tests. We established a workable protocol and obtained TRF patterns that reflected the changes in the bacterial population. The temporal dynamics over a 24 h period were examined at one anchored station, as well as the spatial distribution pattern of the bacterial community at several stations, covering the transects along the river discharge direction and across the river plume. The TRF pattern revealed 9 dominant bacterial groups. Changes in their relative abundance reflecting the changes in the bacterial community composition were documented. Many culturable species were isolated from each field sample and a portion of the 16S rRNA gene for each species was sequenced. The species was identified based on sequence data comparison. In this region, the dominant species belong to the γ-subdivision of proteobacteria and the Bacillus/Clostridium group of Firmicutes. We also detected the wide spread distribution of Acinetobacter spp.; many of these species are known nosocomial pathogen for humans.

  2. Characteristic archaebacterial 16S rRNA oligonucleotides

    NASA Technical Reports Server (NTRS)

    McGill, T. J.; Jurka, J.; Sobieski, J. M.; Pickett, M. H.; Woese, C. R.; Fox, G. E.

    1986-01-01

    A method of analyzing 16S rRNA catalog data has been developed in which groupings at various taxonomic levels can be characterized in terms of specific "signature" oligonucleotides. This approach provides an alternative means for evaluating higher order branching possibilities and can be used to assess the phylogenetic position of isolates that are poorly placed by the usual clustering procedures. This signature approach has been applied to forty archaebacterial catalogs and every oligonucleotide with significant signature value has been identified. Sets of specific oligonucleotides were identified for every major group on a dendrogram produced by cluster analysis procedures. Signatures that would establish between group relationships were also sought and found. In the case of the Methanobacteriaceae the clustering methods suggest a specific relationship to the Methanococcaceae. This inclusion is in fact supported by six strong signature oligonucleotides. However there are also significant numbers of signature oligonucleotides supporting a specific relationship of the Methanobacteriaceae to either the Halobacteriaceae or the Methanomicrobiaceae. Thus the placement of the Methanobacteriaceae is less certain than the usual dendrograms imply. The signature approach also was used to assess the phylogenetic position of Thermoplasma acidophilum which is found to be more closely related to the methanogen/halophile Division than to the sulfur dependent Division of the archaebacteria. This does not imply however that Thermoplasma acidophilum is properly regarded as being in the methanogen/halophile Division.

  3. Metagenomic 16s rRNA investigation of microbial communities in the Black Sea estuaries in South-West of Ukraine.

    PubMed

    Bobrova, Oleksandra; Kristoffersen, Jon Bent; Oulas, Anastasis; Ivanytsia, Volodymyr

    2016-01-01

    The Black Sea estuaries represent interfaces of the sea and river environments. Microorganisms that inhabit estuarine water play an integral role in all biochemical processes that occur there and form unique ecosystems. There are many estuaries located in the Southern-Western part of Ukraine and some of them are already separated from the sea. The aim of this research was to determine the composition of microbial communities in the Khadzhibey, Dniester and Sukhyi estuaries by metagenomic 16S rDNA analysis. This study is the first complex analysis of estuarine microbiota based on isolation of total DNA from a biome that was further subjected to sequencing. DNA was extracted from water samples and sequenced on the Illumina Miseq platform using primers to the V4 variable region of the 16S rRNA gene. Computer analysis of the obtained raw sequences was done with QIIME (Quantitative Insights Into Microbial Ecology) software. As the outcome, 57970 nucleotide sequences were retrieved. Bioinformatic analysis of bacterial community in the studied samples demonstrated a high taxonomic diversity of Prokaryotes at above genus level. It was shown that majority of 16S rDNA bacterial sequences detected in the estuarine samples belonged to phyla Cyanobacteria, Proteobacteria, Bacteroidetes, Actinobacteria, Verrucomicrobia, Planctomycetes. The Khadhzibey estuary was dominated by the Proteobacteria phylum, while Dniester and Sukhyi estuaries were characterized by dominance of Cyanobacteria. The differences in bacterial populations between the Khadzhibey, Dniester and Sukhyi estuaries were demonstrated through the Beta-diversity analysis. It showed that the Khadzhibey estuary's microbial community significantly varies from the Sukhyi and Dniester estuaries. The majority of identified bacterial species is known as typical inhabitants of marine environments, however, for 2.5% of microbial population members in the studied estuaries no relatives were determined. PMID:26929931

  4. Prevalence of 16S rRNA methylase genes among β-lactamase-producing Enterobacteriaceae clinical isolates in Saudi Arabia

    PubMed Central

    Al Sheikh, Yazeed A.; Marie, Mohammed Ali M.; John, James; Krishnappa, Lakshmana Gowda; Dabwab, Khaled Homoud M.

    2014-01-01

    Background Co production of 16S rRNA methylases gene and β-Lactamase gene among Enterobacteriaceae isolates conferring resistance to both therapeutic options has serious implications for clinicians worldwide. Methods To study co existence of 16S rRNA methylases (armA, rmtA, rmtB, rmtC, rmtD, and npmA) and β-Lactamase (blaTEM-1, blaSHV-12, blaCTX-M-14) genes, we screened all phenotypic positive β-Lactamase producing enterobacteriaceae by polymerase chain reaction (PCR) targeting above genes. A total of 330 enterobacteriaceae strains were collected during study period out of that 218 isolates were identified phenotypically as β-Lactamase producers, which include 50 (22.9%) Escherichia coli; 92 (42.2%) Klebsiella pneumoniae, 44 (20.2%), Citrobactor freundii and 32 (14.7%) Enterobacter spp. Results Among this 218, only 188 isolates harbored the resistant gene for β-Lactamase production. Major β-Lactamase producing isolates were bla TEM-1 type. 122 (56 %) isolates were found to produce any one of the 16S rRNA methylase genes. A total of 116 isolates co produced β-Lactamase and at least one 16S rRNA methylases gene Co production of armA gene was found in 26 isolates with rmtB and in 4 isolates with rmtC. The rmtA and rmtD genes were not detected in any of the tested isolates. Six isolates were positive for a 16S rRNA methylase gene alone. Conclusion β-Lactamase producing isolates appears to coexist with 16S rRNA methylase predominantly armA and rmtB genes in the same isolate. We conclude the major β-Lactamase and 16S rRNA methylases co-producer was K. pneumoniae followed by E. coli. We suggest further work on evaluating other β-lactamases types and novel antibiotic resistance mechanisms among Enterobacteriaceae. PMID:25005152

  5. Automated RFLP pattern comparison and similarity coefficient calculation for rapid delineation of new and distinct phytoplasma 16S rDNA subgroup lineages

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Phytoplasmas are insect-borne, phloem-inhabiting, cell wall-less bacteria that cause numerous diseases in several hundred plant species. In adaptation to transkingdom parasitism in diverse plant and insect hosts, phytoplasma evolution has given rise to widely divergent lineages. Since phytoplasmas...

  6. Analysis of 16S rDNA and Metagenomic Sequences Revealed Microbial Community and Host-Specific Sequences of Canadian Geese Feces

    EPA Science Inventory

    There is an increasing concern regarding the public health risks associated with waterfowl fecal pollution as a result of the increase in geese populations (Branta canadensis) in or near U.S. and Canadian recreational waters. Currently, there are no methods that can be used to de...

  7. Bacterial Diversity Analysis during the Fermentation Processing of Traditional Chinese Yellow Rice Wine Revealed by 16S rDNA 454 Pyrosequencing.

    PubMed

    Fang, Ruo-si; Dong, Ya-chen; Chen, Feng; Chen, Qi-he

    2015-10-01

    Rice wine is a traditional Chinese fermented alcohol drink. Spontaneous fermentation with the use of the Chinese starter and wheat Qu lead to the growth of various microorganisms during the complete brewing process. It's of great importance to fully understand the composition of bacteria diversity in rice wine in order to improve the quality and solve safety problems. In this study, a more comprehensive bacterial description was shown with the use of bacteria diversity analysis, which enabled us to have a better understanding. Rarefaction, rank abundance, alpha Diversity, beta diversity and principal coordinates analysis simplified their complex bacteria components and provide us theoretical foundation for further investigation. It has been found bacteria diversity is more abundant at mid-term and later stage of brewing process. Bacteria community analysis reveals there is a potential safety hazard existing in the fermentation, since most of the sequence reads are assigned to Enterobacter (7900 at most) and Pantoea (7336 at most), followed by Staphylococcus (2796 at most) and Pseudomonas (1681 at most). Lactic acid bacteria are rare throughout the fermentation process which is not in accordance with other reports. This work may offer us an opportunity to investigate micro ecological fermentation system in food industry. PMID:26409170

  8. A survey of bacterial diversity from successive life stages of black soldier fly (Diptera: Stratiomyidae) by using 16S rDNA pyrosequencing.

    PubMed

    Zheng, Longyu; Crippen, Tawni L; Singh, Baneshwar; Tarone, Aaron M; Dowd, Scot; Yu, Ziniu; Wood, Thomas K; Tomberlin, Jeffery K

    2013-05-01

    Sustainable methods for managing waste associated with people and animals have been proposed in the past. Black soldier fly, Hermetia illucens (L.), larvae represent one of the more promising methods. Larvae reduce dry matter, bacteria, offensive odor, and house fly populations. Prepupae can be used as feedstuff for livestock. However, it is not known if such a method results in the proliferation of potential pathogens. Although some bacterial species have been cultured and identified from black soldier fly, a true appreciation of fly associated bacterial diversity is not known. Such information is needed to understand pathogen colonization on decomposing animal and plant waste in the presence of black soldier fly larvae as well as develop research strategies for maximizing the use of this fly to reduce waste without risking environmental harm. Using 454 sequencing, we surveyed bacterial diversity associated with successive life stages of the black soldier fly reared on plant material. Bacteria diversity classified (99.8%) across all life stages spanned six bacterial phyla with > or = 80% bootstrap support. Bacteroidetes and Proteobacteria were the most dominant phyla associated with the black soldier fly accounting for two-thirds of the fauna identified. Many of these bacteria would go undetected because of their inability to be cultured. PMID:23802462

  9. A survey of bacterial diversity from successive life stages of black soldier fly (Diptera: Stratiomyidae) by using 16S rDNA pyrosequencing

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Black soldier fly (BSF), Hermetia illucens (L.), larvae represent a sustainable method for reducing animal and plant wastes. Larvae reduce dry matter, bacteria, offensive odor, and house fly populations. The prepupae can be self-harvested and used as feedstuff for livestock and poultry. While som...

  10. Letter to the Editor: Diagnostic Criteria in Urological Diseases do not Always Match with Findings by Extended Culture Techniques and Metagenomic Sequencing of 16S rDNA

    PubMed Central

    Smelov, Vitaly; Naber, Kurt; Bjerklund Johansen, Truls E.

    2016-01-01

    Some diseases of the urinary tract are defined by the presence of microorganisms while others are defined by their absence. The underlying idea has always been that urine from healthy subjects is sterile and a negative urine culture has usually been taken as discriminative for an infection to be absent. Several disorders with symptoms that resemble infections are regarded as separate entities based on the exclusion of bacterial growth such as overactive neurogenic bladder and pelvic pain syndromes. During the recent years two paradigmata related to the role of bacteria in urological disease classification have changed completely. Firstly, bacteriuria does not necessarily mean an infection, and secondly, if extended sets of culture media for identification of fastidious and anaerobic bacteria or culture-independent metagenomic sequencing (MGS) is applied, a broad range of even non-culturable bacteria has been detected in the ”sterile” bladder urine in healthy individuals. The aim of this editorial is to initiate a discussion to redefine the criteria for urinary tract infections and non-infectious urological disorders with similar symptoms. Clinical studies, in which extended sets of culture media and MGS are integrated, are needed to clarify the pathogenesis of urological disorders where bacteria may play a role. The pure detection of bacteria in the urine does not by itself prove an infectious etiology of a specific disorder. It is important to avoid that results of new technologies lead to unnecessary antibiotic consumption with unwanted collateral damage and adverse events. PMID:27006726

  11. EFFECT OF DIFFERENT REGIONS OF AMPLIFIED 16S RDNA ON A PERFORMANCE OF A MULTIPLEXED, BEAD-BASED METHOD FOR ANALYSIS OF DNA SEQUENCES IN ENVIRONMENTAL SAMPLES.

    EPA Science Inventory

    Using a bead-based method for multiplexed analysis of community DNA, the dynamics of aquatic microbial communities can be assessed. Capture probes, specific for a genus or species of bacteria, are attached to the surface of uniquely labeled, microscopic polystyrene beads. Primers...

  12. Gut bacteria of the Pacific coast wireworm, Limonius canus, inferred from 16s rDNA sequences and their implications for control.

    Technology Transfer Automated Retrieval System (TEKTRAN)

    A multitude of bacteria have been isolated from the guts of several insect species. Some of these have been modified to interfere with the development of the host insect or interfere with the development and transmission of plant and animal pathogens transmitted by the host insect. A survey of the g...

  13. High-density universal 16S rRNA microarray analysis revealsbroader diversity than typical clone library when sampling theenvironment

    SciTech Connect

    DeSantis, Todd Z.; Brodie, Eoin L.; Moberg, Jordan P.; Zubieta,Ingrid X.; Piceno, Yvette M.; Andersen, Gary L.

    2006-06-15

    Molecular approaches aimed at detection of a broad-range ofprokaryotes in the environment routinely rely upon classifyingheterogeneous 16S rRNA genes amplified by PCR using primers with broadspecificity. The general method of sampling and categorizing DNA has beento clone then sequence the PCR products. However, the number of clonesrequired to adequately catalogue the majority of taxa in a sample isunwieldy. Alternatively, hybridizing target sequences to a universal 16SrRNA gene microarray may provide a more rapid and comprehensive view ofprokaryotic community composition. This study investigated the breadthand accuracy of a microarray in detecting diverse 16S rRNA gene sequencetypes compared to clone-and-sequencing using three environmental samples:urban aerosol, subsurface soil and subsurface water. PCR productsgenerated from universal 16S rRNA gene-targeted primers were classifiedusing either the clone-and-sequence method or by hybridization to a novelhigh-density microarray of 297,851 probes complementary to 842prokaryotic sub-families. The three clone libraries comprised 1,391high-quality sequences. Approximately 8 percent of the clones could notbe placed into a known sub-family and were considered novel. Themicroarray results confirmed the majority of clone-detected sub-familiesand additionally demonstrated greater amplicon diversity extending intophyla not observed by the cloning method. Sequences matching OTUs withinthe phyla Nitrospira, Planctomycetes, and TM7, which were uniquelydetected by the array, were verified with specific primers and subsequentamplicon sequencing. Sub-family richness detected by the arraycorresponded well with non-parametric richness predictions extrapolatedfrom clone libraries except in the water community where clone-basedrichness predictions were greatly exceeded. It was concluded thatalthough the microarray is unreliable inidentifying novel prokaryotictaxa, it reveals greater diversity in environmental samples thansequencing a

  14. Phylogenetic analysis and PCR detection of Clostridium chauvoei, Clostridium haemolyticum, Clostridium novyi types A and B, and Clostridium septicum based on the flagellin gene.

    PubMed

    Sasaki, Yoshimasa; Kojima, Akemi; Aoki, Hiroshi; Ogikubo, Yasuaki; Takikawa, Noriyasu; Tamura, Yutaka

    2002-05-01

    The flagellin genes (fliC) of Clostridium chauvoei, Clostridium haemolyticum, Clostridium novyi types A and B, and Clostridium septicum were analysed by PCR amplification and DNA sequencing. The five Clostridium species have at least two copies of the flagellin gene (fliC) arranged in tandem on the chromosome. The deduced N- and C-terminal aminoacid sequences of the flagellin proteins (FliCs) of these clostridia are well conserved but their central region aminoacid sequences are not. Phylogenic analysis based on the N-terminal aminoacid sequence of the FliC protein revealed that these clostridia, which belong to Clostridium 16S rDNA phylogenic cluster I (), are more closely related to Bacillus subtilis than to Clostridium difficile, which belongs to the cluster XI. Moreover, a multiplex polymerase reaction (PCR) system based on the fliC sequence was developed to rapidly identify C. chauvoei, C. haemolyticum, C. novyi types A and B, and C. septicum. PCR of each Clostridium amplified a species-specific band. The multiplex PCR system may be useful for rapid identification of pathogenic clostridia. PMID:11900959

  15. 16S rRNA Gene Sequence-Based Identification of Bacteria in Automatically Incubated Blood Culture Materials from Tropical Sub-Saharan Africa

    PubMed Central

    Schwarz, Norbert Georg; Hahn, Andreas; Boahen, Kennedy; Sarpong, Nimako; Adu-Sarkodie, Yaw; Halbgewachs, Eva; Marks, Florian; von Kalckreuth, Vera; Poppert, Sven; Loderstaedt, Ulrike; May, Jürgen; Hagen, Ralf Matthias

    2015-01-01

    Background The quality of microbiological diagnostic procedures depends on pre-analytic conditions. We compared the results of 16S rRNA gene PCR and sequencing from automatically incubated blood culture materials from tropical Ghana with the results of cultural growth after automated incubation. Methods Real-time 16S rRNA gene PCR and subsequent sequencing were applied to 1500 retained blood culture samples of Ghanaian patients admitted to a hospital with an unknown febrile illness after enrichment by automated culture. Results Out of all 1500 samples, 191 were culture-positive and 98 isolates were considered etiologically relevant. Out of the 191 culture-positive samples, 16S rRNA gene PCR and sequencing led to concordant results in 65 cases at species level and an additional 62 cases at genus level. PCR was positive in further 360 out of 1309 culture-negative samples, sequencing results of which suggested etiologically relevant pathogen detections in 62 instances, detections of uncertain relevance in 50 instances, and DNA contamination due to sample preparation in 248 instances. In two instances, PCR failed to detect contaminants from the skin flora that were culturally detectable. Pre-analytical errors caused many Enterobacteriaceae to be missed by culture. Conclusions Potentially correctable pre-analytical conditions and not the fastidious nature of the bacteria caused most of the discrepancies. Although 16S rRNA gene PCR and sequencing in addition to culture led to an increase in detections of presumably etiologically relevant blood culture pathogens, the application of this procedure to samples from the tropics was hampered by a high contamination rate. Careful interpretation of diagnostic results is required. PMID:26270631

  16. Leuconostoc pseudomesenteroides WCFur3 partial 16S rRNA gene

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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...

  17. 16S classifier: a tool for fast and accurate taxonomic classification of 16S rRNA hypervariable regions in metagenomic datasets.

    PubMed

    Chaudhary, Nikhil; Sharma, Ashok K; Agarwal, Piyush; Gupta, Ankit; Sharma, Vineet K

    2015-01-01

    The diversity of microbial species in a metagenomic study is commonly assessed using 16S rRNA gene sequencing. With the rapid developments in genome sequencing technologies, the focus has shifted towards the sequencing of hypervariable regions of 16S rRNA gene instead of full length gene sequencing. Therefore, 16S Classifier is developed using a machine learning method, Random Forest, for faster and accurate taxonomic classification of short hypervariable regions of 16S rRNA sequence. It displayed precision values of up to 0.91 on training datasets and the precision values of up to 0.98 on the test dataset. On real metagenomic datasets, it showed up to 99.7% accuracy at the phylum level and up to 99.0% accuracy at the genus level. 16S Classifier is available freely at http://metagenomics.iiserb.ac.in/16Sclassifier and http://metabiosys.iiserb.ac.in/16Sclassifier. PMID:25646627

  18. Quantifying Microbial Diversity: Morphotypes, 16S rRNA Genes, and Carotenoids of Oxygenic Phototrophs in Microbial Mats

    PubMed Central

    Nübel, Ulrich; Garcia-Pichel, Ferran; Kühl, Michael; Muyzer, Gerard

    1999-01-01

    We quantified the diversity of oxygenic phototrophic microorganisms present in eight hypersaline microbial mats on the basis of three cultivation-independent approaches. Morphological diversity was studied by microscopy. The diversity of carotenoids was examined by extraction from mat samples and high-pressure liquid chromatography analysis. The diversity of 16S rRNA genes from oxygenic phototrophic microorganisms was investigated by extraction of total DNA from mat samples, amplification of 16S rRNA gene segments from cyanobacteria and plastids of eukaryotic algae by phylum-specific PCR, and sequence-dependent separation of amplification products by denaturing-gradient gel electrophoresis. A numerical approach was introduced to correct for crowding the results of chromatographic and electrophoretic analyses. Diversity estimates typically varied up to twofold among mats. The congruence of richness estimates and Shannon-Weaver indices based on numbers and proportional abundances of unique morphotypes, 16S rRNA genes, and carotenoids unveiled the underlying diversity of oxygenic phototrophic microorganisms in the eight mat communities studied. PMID:9925563

  19. Flow Cytometry-assisted Cloning of Specific Sequence Motifs fromComplex 16S ribosomal RNA Gene Libraries.

    SciTech Connect

    Nielsen, J.L.; Schramm, A.; Bernhard, A.E.; van den Engh, G.J.; Stahl, D.A.

    2004-07-21

    A flow cytometry method was developed for rapid screeningand recovery of cloned DNA containing common sequence motifs. Thisapproach, termed fluorescence-activated cell sorting-assisted cloning,was used to recover sequences affiliated with a unique lineage within theBacteroidetes not abundant in a clone library of environmental 16S rRNAgenes. Retrieval and sequence analysis of phylogenetically informativegenes has become a standard cultivation-independent technique toinvestigate microbial diversity in nature (7, 18). Genes encoding the 16SrRNA, because of the relative ease of their selective amplification, havebeen most frequently employed for general diversity surveys (16).Environmental studies have also focused on specific subpopulationsaffiliated with a phylogenetic group or identified by genes encodingspecific metabolic functions (e.g., ammonia oxidation, sulfaterespiration, and nitrate reduction) (8,15,20). However, specificpopulations may be of low abundance (1,23), or the genes encodingspecific metabolic functions may be insufficiently conserved to providepriming sites for general PCR amplification. Three general approacheshave been used to obtain 16S rRNA sequence information from low-abundancepopulations: screening hundreds to thousands of clones in a general 16SrRNA gene library (21), flow cytometric sorting of a subpopulation ofenvironmentally derived cells labeled by fluorescent in situhybridization (FISH) (27), or selective PCR amplification using primersspecific for the subpopulation (2,23). While the first approach is simplytime-consuming and tedious, the second has been restricted to fairlylarge and strongly fluorescent cells from aquatic samples (5, 27). Thethird approach often generates fragments of only a few hundred bases dueto the limited number of specific priming sites. Partial sequenceinformation often degrades analysis, obscuring or distorting thephylogenetic placement of the new sequences (11, 20). A more robustcharacterization of environ

  20. Aminoglycoside Resistance: The Emergence of Acquired 16S Ribosomal RNA Methyltransferases.

    PubMed

    Doi, Yohei; Wachino, Jun-Ichi; Arakawa, Yoshichika

    2016-06-01

    Aminoglycoside-producing Actinobacteria are known to protect themselves from their own aminoglycoside metabolites by producing 16S ribosomal RNA methyltransferase (16S-RMTase), which prevents them from binding to the 16S rRNA targets. Ten acquired 16S-RMTases have been reported from gram-negative pathogens. Most of them posttranscriptionally methylate residue G1405 of 16S rRNA resulting in high-level resistance to gentamicin, tobramycin, amikacin, and plazomicin. Strains that produce 16S-RMTase are frequently multidrug-resistant or even extensively drug-resistant. Although the direct clinical impact of high-level aminoglycoside resistance resulting from production of 16S-RMTase is yet to be determined, ongoing spread of this mechanism will further limit treatment options for multidrug-resistant and extensively drug-resistant gram-negative infections. PMID:27208771

  1. Phylogeny of All Recognized Species of Ammonia Oxidizers Based on Comparative 16S rRNA and amoA Sequence Analysis: Implications for Molecular Diversity Surveys

    PubMed Central

    Purkhold, Ulrike; Pommerening-Röser, Andreas; Juretschko, Stefan; Schmid, Markus C.; Koops, Hans-Peter; Wagner, Michael

    2000-01-01

    The current perception of evolutionary relationships and the natural diversity of ammonia-oxidizing bacteria (AOB) is mainly based on comparative sequence analyses of their genes encoding the 16S rRNA and the active site polypeptide of the ammonia monooxygenase (AmoA). However, only partial 16S rRNA sequences are available for many AOB species and most AOB have not yet been analyzed on the amoA level. In this study, the 16S rDNA sequence data of 10 Nitrosomonas species and Nitrosococcus mobilis were completed. Furthermore, previously unavailable 16S rRNA sequences were determined for three Nitrosomonas sp. isolates and for the gamma-subclass proteobacterium Nitrosococcus halophilus. These data were used to revaluate the specificities of published oligonucleotide primers and probes for AOB. In addition, partial amoA sequences of 17 AOB, including the above-mentioned 15 AOB, were obtained. Comparative phylogenetic analyses suggested similar but not identical evolutionary relationships of AOB by using 16S rRNA and AmoA as marker molecules, respectively. The presented 16S rRNA and amoA and AmoA sequence data from all recognized AOB species significantly extend the currently used molecular classification schemes for AOB and now provide a more robust phylogenetic framework for molecular diversity inventories of AOB. For 16S rRNA-independent evaluation of AOB species-level diversity in environmental samples, amoA and AmoA sequence similarity threshold values were determined which can be used to tentatively identify novel species based on cloned amoA sequences. Subsequently, 122 amoA sequences were obtained from 11 nitrifying wastewater treatment plants. Phylogenetic analyses of the molecular isolates showed that in all but two plants only nitrosomonads could be detected. Although several of the obtained amoA sequences were only relatively distantly related to known AOB, none of these sequences unequivocally suggested the existence of previously unrecognized species in the

  2. Metagenomic and near full-length 16S rRNA sequence data in support of the phylogenetic analysis of the rumen bacterial community in steers.

    PubMed

    Myer, Phillip R; Kim, MinSeok; Freetly, Harvey C; Smith, Timothy P L

    2016-09-01

    Amplicon sequencing utilizing next-generation platforms has significantly transformed how research is conducted, specifically microbial ecology. However, primer and sequencing platform biases can confound or change the way scientists interpret these data. The Pacific Biosciences RSII instrument may also preferentially load smaller fragments, which may also be a function of PCR product exhaustion during sequencing. To further examine theses biases, data is provided from 16S rRNA rumen community analyses. Specifically, data from the relative phylum-level abundances for the ruminal bacterial community are provided to determine between-sample variability. Direct sequencing of metagenomic DNA was conducted to circumvent primer-associated biases in 16S rRNA reads and rarefaction curves were generated to demonstrate adequate coverage of each amplicon. PCR products were also subjected to reduced amplification and pooling to reduce the likelihood of PCR product exhaustion during sequencing on the Pacific Biosciences platform. The taxonomic profiles for the relative phylum-level and genus-level abundance of rumen microbiota as a function of PCR pooling for sequencing on the Pacific Biosciences RSII platform were provided. For more information, see "Evaluation of 16S rRNA amplicon sequencing using two next-generation sequencing technologies for phylogenetic analysis of the rumen bacterial community in steers" P.R. Myer, M. Kim, H.C. Freetly, T.P.L. Smith (2016) [1]. PMID:27508263

  3. Phylogenetic diversity of bacterial symbionts of Solemya hosts based on comparative sequence analysis of 16S rRNA genes.

    PubMed Central

    Krueger, D M; Cavanaugh, C M

    1997-01-01

    The bacterial endosymbionts of two species of the bivalve genus Solemya from the Pacific Ocean, Solemya terraeregina and Solemya pusilla, were characterized. Prokaryotic cells resembling gram-negative bacteria were observed in the gills of both host species by transmission electron microscopy. The ultrastructure of the symbiosis in both host species is remarkably similar to that of all previously described Solemya spp. By using sequence data from 16S rRNA, the identity and evolutionary origins of the S. terraeregina and S. pusilla symbionts were also determined. Direct sequencing of PCR-amplified products from host gill DNA with primers specific for Bacteria 16S rRNA genes gave a single, unambiguous sequence for each of the two symbiont species. In situ hybridization with symbiont-specific oligonucleotide probes confirmed that these gene sequences belong to the bacteria residing in the hosts gills. Phylogenetic analyses of the 16S rRNA gene sequences by both distance and parsimony methods identify the S. terraeregina and S. pusilla symbionts as members of the gamma subdivision of the Proteobacteria. In contrast to symbionts of other bivalve families, which appear to be monophyletic, the S. terraeregina and S. pusilla symbionts share a more recent common ancestry with bacteria associating endosymbiotically with bivalves of the superfamily Lucinacea than with other Solemya symbionts (host species S. velum, S. occidentalis, and S. reidi). Overall, the 16S rRNA gene sequence data suggest that the symbionts of Solemya hosts represent at least two distinct bacterial lineages within the gamma-Proteobacteria. While it is increasingly clear that all extant species of Solemya live in symbiosis with specific bacteria, the associations appear to have multiple evolutionary origins. PMID:8979342

  4. 16S rRNA phylogenetic analysis of the bacterial endosymbionts associated with cytoplasmic incompatibility in insects.

    PubMed Central

    O'Neill, S L; Giordano, R; Colbert, A M; Karr, T L; Robertson, H M

    1992-01-01

    Bacterial endosymbionts of insects have long been implicated in the phenomenon of cytoplasmic incompatibility, in which certain crosses between symbiont-infected individuals lead to embryonic death or sex ratio distortion. The taxonomic position of these bacteria has, however, not been known with any certainty. Similarly, the relatedness of the bacteria infecting various insect hosts has been unclear. The inability to grow these bacteria on defined cell-free medium has been the major factor underlying these uncertainties. We circumvented this problem by selective PCR amplification and subsequent sequencing of the symbiont 16S rRNA genes directly from infected insect tissue. Maximum parsimony analysis of these sequences indicates that the symbionts belong in the alpha-subdivision of the Proteobacteria, where they are most closely related to the Rickettsia and their relatives. They are all closely related to each other and are assigned to the type species Wolbachia pipientis. Lack of congruence between the phylogeny of the symbionts and their insect hosts suggest that horizontal transfer of symbionts between insect species may occur. Comparison of the sequences for W. pipientis and for Wolbachia persica, an endosymbiont of ticks, shows that the genus Wolbachia is polyphyletic. A PCR assay based on 16S primers was designed for the detection of W. pipientis in insect tissue, and initial screening of insects indicates that cytoplasmic incompatibility may be a more general phenomenon in insects than is currently recognized. Images PMID:1557375

  5. CATCh, an ensemble classifier for chimera detection in 16S rRNA sequencing studies.

    PubMed

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

    2015-03-01

    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

  6. Primer and platform effects on 16S rRNA tag sequencing

    SciTech Connect

    Tremblay, Julien; Singh, Kanwar; Fern, Alison; Kirton, Edward S.; He, Shaomei; Woyke, Tanja; Lee, Janey; Chen, Feng; Dangl, Jeffery L.; Tringe, Susannah G.

    2015-08-04

    Sequencing of 16S rRNA gene tags is a popular method for profiling and comparing microbial communities. The protocols and methods used, however, vary considerably with regard to amplification primers, sequencing primers, sequencing technologies; as well as quality filtering and clustering. How results are affected by these choices, and whether data produced with different protocols can be meaningfully compared, is often unknown. Here we compare results obtained using three different amplification primer sets (targeting V4, V6–V8, and V7–V8) and two sequencing technologies (454 pyrosequencing and Illumina MiSeq) using DNA from a mock community containing a known number of species as well as complex environmental samples whose PCR-independent profiles were estimated using shotgun sequencing. We find that paired-end MiSeq reads produce higher quality data and enabled the use of more aggressive quality control parameters over 454, resulting in a higher retention rate of high quality reads for downstream data analysis. While primer choice considerably influences quantitative abundance estimations, sequencing platform has relatively minor effects when matched primers are used. In conclusion, beta diversity metrics are surprisingly robust to both primer and sequencing platform biases.

  7. Primer and platform effects on 16S rRNA tag sequencing

    PubMed Central

    Tremblay, Julien; Singh, Kanwar; Fern, Alison; Kirton, Edward S.; He, Shaomei; Woyke, Tanja; Lee, Janey; Chen, Feng; Dangl, Jeffery L.; Tringe, Susannah G.

    2015-01-01

    Sequencing of 16S rRNA gene tags is a popular method for profiling and comparing microbial communities. The protocols and methods used, however, vary considerably with regard to amplification primers, sequencing primers, sequencing technologies; as well as quality filtering and clustering. How results are affected by these choices, and whether data produced with different protocols can be meaningfully compared, is often unknown. Here we compare results obtained using three different amplification primer sets (targeting V4, V6–V8, and V7–V8) and two sequencing technologies (454 pyrosequencing and Illumina MiSeq) using DNA from a mock community containing a known number of species as well as complex environmental samples whose PCR-independent profiles were estimated using shotgun sequencing. We find that paired-end MiSeq reads produce higher quality data and enabled the use of more aggressive quality control parameters over 454, resulting in a higher retention rate of high quality reads for downstream data analysis. While primer choice considerably influences quantitative abundance estimations, sequencing platform has relatively minor effects when matched primers are used. Beta diversity metrics are surprisingly robust to both primer and sequencing platform biases. PMID:26300854

  8. Primer and platform effects on 16S rRNA tag sequencing

    DOE PAGESBeta

    Tremblay, Julien; Singh, Kanwar; Fern, Alison; Kirton, Edward S.; He, Shaomei; Woyke, Tanja; Lee, Janey; Chen, Feng; Dangl, Jeffery L.; Tringe, Susannah G.

    2015-08-04

    Sequencing of 16S rRNA gene tags is a popular method for profiling and comparing microbial communities. The protocols and methods used, however, vary considerably with regard to amplification primers, sequencing primers, sequencing technologies; as well as quality filtering and clustering. How results are affected by these choices, and whether data produced with different protocols can be meaningfully compared, is often unknown. Here we compare results obtained using three different amplification primer sets (targeting V4, V6–V8, and V7–V8) and two sequencing technologies (454 pyrosequencing and Illumina MiSeq) using DNA from a mock community containing a known number of species as wellmore » as complex environmental samples whose PCR-independent profiles were estimated using shotgun sequencing. We find that paired-end MiSeq reads produce higher quality data and enabled the use of more aggressive quality control parameters over 454, resulting in a higher retention rate of high quality reads for downstream data analysis. While primer choice considerably influences quantitative abundance estimations, sequencing platform has relatively minor effects when matched primers are used. In conclusion, beta diversity metrics are surprisingly robust to both primer and sequencing platform biases.« less

  9. When fathers are instant losers: homogenization of rDNA loci in recently formed Cardamine × schulzii trigenomic allopolyploid.

    PubMed

    Zozomová-Lihová, Judita; Mandáková, Terezie; Kovaříková, Alena; Mühlhausen, Andreas; Mummenhoff, Klaus; Lysak, Martin A; Kovařík, Aleš

    2014-09-01

    Recently formed allopolyploids represent an excellent system to study the impacts of hybridization and genomic duplication on genome structure and evolution. Here we explored the 35SrRNA genes (rDNA) in the Cardamine × schulzii allohexaploid that was formed by two subsequent hybridization events within the past c. 150 yr. The rDNA loci were analyzed by cloning, next generation sequencing (NGS), RT-PCR and FISH methods. The primary C. × insueta triploid hybrid derived from C. rivularis (♀) and C. amara (♂) had gene ratios highly skewed towards maternal sequences. Similarly, C. × schulzii, originating from the secondary hybridization event involving C. × insueta (♀) and C. pratensis (♂), showed a reduction in paternal rDNA homeologs despite an excess of chromosomes inherited from C. pratensis. We also identified novel rDNA loci in C. × schulzii, suggesting that lost loci might be slowly reinstalled by translocation (but not recombination) of genes from partner genomes. Prevalent clonal propagation of allopolyploids, C. × insueta and C. × schulzii, indicates that concerted evolution of rDNA may occur in the absence of extensive meiotic cycles. Adoption of NGS in rDNA variant analysis is highly informative for deciphering the evolutionary histories of allopolyploid species with ongoing homogenization processes. PMID:24916080

  10. Selective Phylogenetic Analysis Targeted at 16S rRNA Genes of Thermophiles and Hyperthermophiles in Deep-Subsurface Geothermal Environments

    PubMed Central

    Kimura, Hiroyuki; Sugihara, Maki; Kato, Kenji; Hanada, Satoshi

    2006-01-01

    Deep-subsurface samples obtained by deep drilling are likely to be contaminated with mesophilic microorganisms in the drilling fluid, and this could affect determination of the community structure of the geothermal microflora using 16S rRNA gene clone library analysis. To eliminate possible contamination by PCR-amplified 16S rRNA genes from mesophiles, a combined thermal denaturation and enzyme digestion method, based on a strong correlation between the G+C content of the 16S rRNA gene and the optimum growth temperatures of most known prokaryotic cultures, was used prior to clone library construction. To validate this technique, hot spring fluid (76°C) and river water (14°C) were used to mimic a deep-subsurface sample contaminated with drilling fluid. After DNA extraction and PCR amplification of the 16S rRNA genes from individual samples separately, the amplified products from river water were observed to be denatured at 82°C and completely digested by exonuclease I (Exo I), while the amplified products from hot spring fluid remained intact after denaturation at 84°C and enzyme digestion with Exo I. DNAs extracted from the two samples were mixed and used as a template for amplification of the 16S rRNA genes. The amplified rRNA genes were denatured at 84°C and digested with Exo I before clone library construction. The results indicated that the 16S rRNA gene sequences from the river water were almost completely eliminated, whereas those from the hot spring fluid remained. PMID:16391020

  11. Nature of polymorphisms in 16S-23S rRNA gene intergenic transcribed spacer fingerprinting of Bacillus and related genera.

    PubMed

    Daffonchio, Daniele; Cherif, Ameur; Brusetti, Lorenzo; Rizzi, Aurora; Mora, Diego; Boudabous, Abdellatif; Borin, Sara

    2003-09-01

    The intergenic transcribed spacers (ITS) between the 16S and 23S rRNA genetic loci are frequently used in PCR fingerprinting to discriminate bacterial strains at the species and intraspecies levels. We investigated the molecular nature of polymorphisms in ITS-PCR fingerprinting of low-G+C-content spore-forming bacteria belonging to the genera Bacillus, Brevibacillus, Geobacillus, and Paenibacillus: We found that besides the polymorphisms in the homoduplex fragments amplified by PCR, heteroduplex products formed during PCR between amplicons from different ribosomal operons, with or without tRNA genes in the ITS, contribute to the interstrain variability in ITS-PCR fingerprinting patterns obtained in polyacrylamide-based gel matrices. The heteroduplex nature of the discriminating bands was demonstrated by fragment separation in denaturing polyacrylamide gels, by capillary electrophoresis, and by cloning, sequencing, and recombination of purified short and tRNA gene-containing long ITS. We also found that heteroduplex product formation is enhanced by increasing the number of PCR cycles. Homoduplex-heteroduplex polymorphisms (HHP) in a conserved region, such as the 16S and 23S rRNA gene ITS, allowed discrimination of closely related strains and species undistinguishable by other methods, indicating that ITS-HHP analysis is an easy and reproducible additional tool for strain typing. PMID:12957895

  12. Identification of a 5S rDNA spacer type specific Triticum urartu and wheats containing the T. urartu genome.

    PubMed

    Allaby, R G; Brown, T A

    2000-04-01

    A PCR system was designed to amplify 5S spacer rDNA specifically from homeologous chromosome 1 in a variety of species representative of the Aegilops and Triticum genera. Two polymerase chain reaction (PCR) primer combinations were used, one of which appears to be apomorphic in nature and specific to chromosome 1A in Triticum urartu and tetraploid and hexaploid wheats containing the AA genome donated by T. urartu. The value of studying single repeat types to investigate the molecular evolution of 5S-rDNA arrays is considered. PMID:10791812

  13. Bacterial diversity analysis of Huanglongbing pathogen-infected citrus, using PhyloChip and 16S rRNA gene clone library sequencing

    SciTech Connect

    Shankar Sagaram, U.; DeAngelis, K.M.; Trivedi, P.; Andersen, G.L.; Lu, S.-E.; Wang, N.

    2009-03-01

    The bacterial diversity associated with citrus leaf midribs was characterized 1 from citrus groves that contained the Huanglongbing (HLB) pathogen, which has yet to be cultivated in vitro. We employed a combination of high-density phylogenetic 16S rDNA microarray and 16S rDNA clone library sequencing to determine the microbial community composition of symptomatic and asymptomatic citrus midribs. Our results revealed that citrus leaf midribs can support a diversity of microbes. PhyloChip analysis indicated that 47 orders of bacteria from 15 phyla were present in the citrus leaf midribs while 20 orders from phyla were observed with the cloning and sequencing method. PhyloChip arrays indicated that nine taxa were significantly more abundant in symptomatic midribs compared to asymptomatic midribs. Candidatus Liberibacter asiaticus (Las) was detected at a very low level in asymptomatic plants, but was over 200 times more abundant in symptomatic plants. The PhyloChip analysis was further verified by sequencing 16S rDNA clone libraries, which indicated the dominance of Las in symptomatic leaves. These data implicate Las as the pathogen responsible for HLB disease. Citrus is the most important commercial fruit crop in Florida. In recent years, citrus Huanglongbing (HLB), also called citrus greening, has severely affected Florida's citrus production and hence has drawn an enormous amount of attention. HLB is one of the most devastating diseases of citrus (6,13), characterized by blotchy mottling with green islands on leaves, as well as stunting, fruit decline, and small, lopsided fruits with poor coloration. The disease tends to be associated with a phloem-limited fastidious {alpha}-proteobacterium given a provisional Candidatus status (Candidatus Liberobacter spp. later changed to Candidatus Liberibacter spp.) in nomenclature (18,25,34). Previous studies indicate that HLB infection causes disorder in the phloem and severely impairs the translocation of assimilates in host

  14. Use of PCR-DHPLC with fluorescence detection for the characterization of the bacterial diversity during cassava (Manihot esculenta Crantz) fermentation.

    PubMed

    Kodama, C S; Cuadros-Orellana, S; Bandeira, C H M M; Graças, D A; Santos, A S; Silva, A

    2014-01-01

    Denaturing high-performance liquid chromatography (DHPLC) has been described as a suitable method to study DNA polymorphisms. Here, cassava (Manihot esculenta Crantz) fermentation liquor was examined using DHPLC analysis to characterize the bacterial diversity during the fermentation process. GC-clamped amplicons corresponding to a variable region of the bacterial community 16S rDNA were synthesized using polymerase chain reaction (PCR) and then resolved on a base-composition basis using preparative DHPLC. Eluate fractions were collected at random and used as a source of whole community DNA that could be used to determine the bacterial diversity. As a first approach, GC-clamps were removed from the eluted DNA fragments using PCR to avoid the possible bias these clamps could cause during the construction of clone libraries. As a second approach, a clone library of each eluate sample was constructed, preserving the GC-clamps of the DNA fragments. The first approach generated 132 bacterial rDNA sequences with an average size of 200 bp, 45% of which had similarity to unculturable or non-classified bacteria. The second approach produced 194 sequences identified as Proteobacteria (48%), uncultured or non-classified environmental bacteria (40%) and Firmicutes (12%). We detected a remarkably greater bacterial diversity using the first approach than the second approach. The DHPLC-PCR method allowed for the fast and non-laborious detection of a vast bacterial diversity that was associated with cassava fermentation, and we conclude that it is a promising alternative for the characterization of the overall microbial diversity in complex samples. PMID:24634228

  15. Bacteroides isolated from four mammalian hosts lack host-specific 16S rRNA gene phylogeny and carbon and nitrogen utilization patterns*

    PubMed Central

    Atherly, Todd; Ziemer, Cherie J

    2014-01-01

    One-hundred-and-three isolates of Bacteroides ovatus,B. thetaiotaomicron, and B. xylanisolvens were recovered from cow, goat, human, and pig fecal enrichments with cellulose or xylan/pectin. Isolates were compared using 16S rRNA gene sequencing, repetitive sequence-based polymerase chain reaction (rep-PCR), and phenotypic microarrays. Analysis of 16S rRNA gene sequences revealed high sequence identity in these Bacteroides; with distinct phylogenetic groupings by bacterial species but not host origin. Phenotypic microarray analysis demonstrated these Bacteroides shared the ability to utilize many of the same carbon substrates, without differences due to species or host origin, indicative of their broad carbohydrate fermentation abilities. Limited nitrogen substrates were utilized; in addition to ammonia, guanine, and xanthine, purine derivatives were utilized by most isolates followed by a few amino sugars. Only rep-PCR analysis demonstrated host-specific patterns, indicating that genomic changes due to coevolution with host did not occur by mutation in the 16S rRNA gene or by a gain or loss of carbohydrate utilization genes within these Bacteroides. This is the first report to indicate that host-associated genomic differences are outside of 16S rRNA gene and carbohydrate utilization genes and suggest conservation of specific bacterial species with the same functionality across mammalian hosts for this Bacteroidetes clade. PMID:24532571

  16. Analysis of 5S rDNA arrays in Arabidopsis thaliana: physical mapping and chromosome-specific polymorphisms.

    PubMed

    Cloix, C; Tutois, S; Mathieu, O; Cuvillier, C; Espagnol, M C; Picard, G; Tourmente, S

    2000-05-01

    A physical map of a pericentromeric region of chromosome 5 containing a 5S rDNA locus and spanning approximately 1000 kb was established using the CIC YAC clones. Three 5S rDNA arrays were resolved in this YAC contig by PFGE analysis and we have mapped different types of sequences between these three blocks. 5S rDNA units from each of these three arrays of chromosome 5, and from chromosomes 3 and 4, were isolated by PCR. A total of 38 new DNA sequences were obtained. Two types of 5S rDNA repeated units exist: the major variant with 0.5-kb repeats and one with short repeats (251 bp) only detected on YAC 11A3 from chromosome 3. Although the 38 sequences displayed noticeable heterogeneity, we were able to group them according to their 5S array origin. The presence of 5S array-specific variants was confirmed with the restriction polymorphism study of all the YACs carrying 5S units. PMID:10810091

  17. Development and evaluation of immunochromatography to detect Gram-negative bacteria producing ArmA 16S rRNA methylase responsible for aminoglycoside resistance.

    PubMed

    Oshiro, Satoshi; Tada, Tatsuya; Kameoka, Yousuke; Suzuki, Kazuo; Ohmagari, Norio; Miyoshi-Akiyama, Tohru; Kirikae, Teruo

    2015-11-01

    Rapid and reliable detection of aminoglycoside-resistant bacteria is an important infection-control measure and a crucial aspect of antimicrobial chemotherapy. The enzyme 16S rRNA methylase has been shown to mediate aminoglycoside resistance in bacteria. This study describes a newly developed immunochromatographic assay using novel monoclonal antibodies (mAbs) that recognize ArmA 16S rRNA methylase. Epitope mapping showed that these mAbs recognized amino acids 1-93 of ArmA, which consists of 257 amino acids. Evaluation of the assay using ArmA producing and non-producing bacterial species, as well as bacteria producing other types of 16S rRNA methylases, indicated that immunochromatographic detection of the ArmA-type 16S rRNA methylase was fully consistent with PCR analysis for armA genes, with all immunochromatographically positive strains being resistant to aminoglycosides (MIC≥128μg/mL). The detection limit of the assay was 12ng ArmA. These findings indicate that this assay can be used for the rapid and reliable detection of the production of ArmA 16S rRNA methylase by Gram-negative bacteria, including Acinetobacter baumannii and Escherichia coli. PMID:26381663

  18. Multiplex real-time PCR for detection, identification and quantification of 'Candidatus Liberibacter solanacearum' in potato plants with zebra chip.

    PubMed

    Li, Wenbin; Abad, Jorge A; French-Monar, Ronald D; Rascoe, John; Wen, Aimin; Gudmestad, Neil C; Secor, Gary A; Lee, Ing-Ming; Duan, Yongping; Levy, Laurene

    2009-07-01

    The new Liberibacter species, 'Candidatus Liberibacter solanacearum' (Lso) recently associated with potato/tomato psyllid-transmitted diseases in tomato and capsicum in New Zealand, was found to be consistently associated with a newly emerging potato zebra chip (ZC) disease in Texas and other southwestern states in the USA. A species-specific primer LsoF was developed for both quantitative real-time PCR (qPCR) and conventional PCR (cPCR) to detect and quantify Lso in infected samples. In multiplex qPCR, a plant cytochrome oxidase (COX)-based probe-primer set was used as a positive internal control for host plants, which could be used to reliably access the DNA extraction quality and to normalize qPCR data for accurate quantification of the bacterial populations in environment samples. Neither the qPCR nor the cPCR using the primer and/or probe sets with LsoF reacted with other Liberibacter species infecting citrus or other potato pathogens. The low detection limit of the multiplex qPCR was about 20 copies of the target 16S rDNA templates per reaction for field samples. Lso was readily detected and quantified in various tissues of ZC-affected potato plants collected from fields in Texas. A thorough but uneven colonization of Lso was revealed in various tissues of potato plants. The highest Lso populations were about 3x10(8) genomes/g tissue in the root, which were 3-order higher than those in the above-ground tissues of potato plants. The Lso bacterial populations were normally distributed across the ZC-affected potato plants collected from fields in Texas, with 60% of ZC-affected potato plants harboring an average Lso population from 10(5) to 10(6) genomes/g tissue, 4% of plants hosting above 10(7) Lso genomes/g tissue, and 8% of plants holding below 10(3) Lso genomes/g tissue. The rapid, sensitive, specific and reliable multiplex qPCR showed its potential to become a powerful tool for early detection and quantification of the new Liberibacter species associated

  19. Extraction of DNA from orange juice, and detection of bacterium Candidatus Liberibacter asiaticus by real-time PCR.

    PubMed

    Bai, Jinhe; Baldwin, Elizabeth; Liao, Hui-Ling; Zhao, Wei; Kostenyuk, Igor; Burns, Jacqueline; Irey, Mike

    2013-10-01

    Orange juice processed from Huanglongbing (HLB) affected fruit is often associated with bitter taste and/or off-flavor. HLB disease in Florida is associated with Candidatus Liberibacter asiaticus (CLas), a phloem-limited bacterium. The current standard to confirm CLas for citrus trees is to take samples from midribs of leaves, which are rich in phloem tissues, and use a quantitative real-time polymerase chain reaction (qPCR) test to detect the 16S rDNA gene of CLas. It is extremely difficult to detect CLas in orange juice because of the low CLas population, high sugar and pectin concentration, low pH, and possible existence of an inhibitor to DNA amplification. The objective of this research was to improve extraction of DNA from orange juice and detection of CLas by qPCR. Homogenization using a sonicator increased DNA yield by 86% in comparison to mortar and pestle extraction. It is difficult to separate DNA from pectin; however, DNA was successfully extracted by treating the juice with pectinase. Application of an elution column successfully removed the unidentified inhibitor to DNA amplification. This work provided a protocol to extract DNA from whole orange juice and detect CLas in HLB-affected fruit. PMID:24047134

  20. Hemi-Nested PCR and RFLP Methodologies for Identifying Blood Meals of the Chagas Disease Vector, Triatoma infestans

    PubMed Central

    Roellig, Dawn M.; Gomez-Puerta, Luis A.; Mead, Daniel G.; Pinto, Jesus; Ancca-Juarez, Jenny; Calderon, Maritza; Bern, Caryn; Gilman, Robert H.; Cama, Vitaliano A.

    2013-01-01

    Trypanosoma cruzi, the etiologic agent of Chagas disease, is transmitted by hematophagous reduviid bugs within the subfamily Triatominae. These vectors take blood meals from a wide range of hosts, and their feeding behaviors have been used to investigate the ecology and epidemiology of T. cruzi. In this study we describe two PCR-based methodologies that amplify a fragment of the 16S mitochondrial rDNA, aimed to improve the identification of blood meal sources for Triatoma infestans: a.- Sequence analyses of two heminested PCRs that allow the identification of mammalian and avian species, and b.- restriction fragment length polymorphism (RFLP) analysis from the mammalian PCR to identify and differentiate multi-host blood meals. Findings from both methodologies indicate that host DNA could be detected and the host species identified in samples from laboratory reared and field collected triatomines. The implications of this study are two-fold. First, these methods can be used in areas where the fauna diversity and feeding behavior of the triatomines are unknown. Secondly, the RFLP method led to the identification of multi-host DNA from T. infestans gut contents, enhancing the information provided by this assay. These tools are important contributions for ecological and epidemiological studies of vector-borne diseases. PMID:24040328

  1. New Degenerate Cytophaga-Flexibacter-Bacteroides-Specific 16S Ribosomal DNA-Targeted Oligonucleotide Probes Reveal High Bacterial Diversity in River Taff Epilithon

    PubMed Central

    O’Sullivan, Louise A.; Weightman, Andrew J.; Fry, John C.

    2002-01-01

    River microbial communities play an important role in global nutrient cycles, and aggregated bacteria such as those in epilithic biofilms may be major contributors. In this study the bacterial diversity of River Taff epilithon in South Wales was investigated. A 16S ribosomal DNA (rDNA) clone library was constructed and analyzed by partial sequencing of 76 of 347 clones and hybridization with taxon-specific probes. The epilithon was found to be very diverse, with an estimated 59.6% of the bacterial populations not accounted for by these clones. Members of the Cytophaga-Flexibacter-Bacteroides division (CFBs) were most abundant in the library, representing 25% of clones, followed by members of the α subdivision of the division Proteobacteria (α-Proteobacteria), γ-Proteobacteria, gram-positive bacteria, Cyanobacteria, β-Proteobacteria, δ-Proteobacteria, and the Prosthecobacter group. This study concentrated on the epilithic CFB populations, and a new set of degenerate 16S rDNA probes was developed to enhance their detection, namely, CFB560, CFB562, and CFB376. The commonly used probe CF319a/b may frequently lead to the underestimation of CFB populations in environmental studies, because it does not fully detect members of the division. CFB560 had exact matches to 95.6% of CFBs listed in the Ribosomal Database Project (release 8.0) small-subunit phylogenetic trees, compared to 60% for CF319a/b. The CFB probes detected 66 of 347 epilithon TAF clones, and 60 of these were partially sequenced. They affiliated with the RDP-designated groups Cytophaga, Sphingobacterium, Lewinella, and Cytophaga aurantiaca. CFB560 and CF319a/b detected 94% (62 of 66) and 48.5% (32 of 66) of clones, respectively, and therefore CFB560 is recommended for future use. Probe design in this study illustrated that multiple degenerate positions can greatly increase target range without adversely effecting specificity or experimental performance. PMID:11772628

  2. Development and Evaluation of a Quality-Controlled Ribosomal Sequence Database for 16S Ribosomal DNA-Based Identification of Staphylococcus Species

    PubMed Central

    Becker, Karsten; Harmsen, Dag; Mellmann, Alexander; Meier, Christian; Schumann, Peter; Peters, Georg; von Eiff, Christof

    2004-01-01

    To establish an improved ribosomal gene sequence database as part of the Ribosomal Differentiation of Microorganisms (RIDOM) project and to overcome the drawbacks of phenotypic identification systems and publicly accessible sequence databases, both strands of the 5′ end of the 16S ribosomal DNA (rDNA) of 81 type and reference strains comprising all validly described staphylococcal (sub)species were sequenced. Assuming a normal distribution for pairwise distances of all unique staphylococcal sequences and choosing a reporting criterion of ≥98.7% similarity for a “distinct species,” a statistical error probability of 1.0% was calculated. To evaluate this database, a 16S rDNA fragment (corresponding to Escherichia coli positions 54 to 510) of 55 clinical Staphylococcus isolates (including those of the small-colony variant phenotype) were sequenced and analyzed by the RIDOM approach. Of these isolates, 54 (98.2%) had a similarity score above the proposed threshold using RIDOM; 48 (87.3%) of the sequences gave a perfect match, whereas 83.6% were found by searching National Center for Biotechnology Information (NCBI) database entries. In contrast to RIDOM, which showed four ambiguities at the species level (mainly concerning Staphylococcus intermedius versus Staphylococcus delphini), the NCBI database search yielded 18 taxon-related ambiguities and showed numerous matches exhibiting redundant or unspecified entries. Comparing molecular results with those of biochemical procedures, ID 32 Staph (bioMérieux, Marcy I'Etoile, France) and VITEK 2 (bioMérieux) failed to identify 13 (23.6%) and 19 (34.5%) isolates, respectively, due to incorrect identification and/or categorization below acceptable values. In contrast to phenotypic methods and the NCBI database, the novel high-quality RIDOM sequence database provides excellent identification of staphylococci, including rarely isolated species and phenotypic variants. PMID:15528685

  3. A PCR-based diagnostic assay for the detection of Roseovarius crassostreae in Crassostrea virginica affected by juvenile oyster disease (JOD)

    USGS Publications Warehouse

    Maloy, A.P.; Barber, B.J.; Boettcher, K.J.

    2005-01-01

    We have developed a PCR-assay for the diagnosis of juvenile oyster disease (JOD) based on the detection of Roseovarius crassostreae directly from affected oysters. Species-specific primers are used to amplify the 16S-23S rDNA internal transcribed spacer (ITS) of R. crassostreae, and confirmation of product identity is accomplished by restriction enzyme analysis. No false positives were obtained with either closely related bacterial species or from other DNAs present in oyster samples. The assay has the potential to detect as few as 10 cells of R. crassostreae per oyster when samples are taken from the inner valve surfaces of the animal. Inclusion of material from soft body surfaces is not necessary, and may reduce sensitivity approximately 10-fold. In a JOD-affected population, a positive PCR result was obtained from all oysters from which these bacteria were subsequently cultured. The assay also detected the presence of R. crassostreae in 2 oysters from which no R. crassostreae isolates were recovered. No R. crassostreae was detected by either PCR or bacteriology in oysters from a population that was not exhibiting JOD-signs. This assay is expected to advance regional disease management efforts and provide valuable insights into the disease process and epizootiology of JOD. ?? Inter-Research 2005.

  4. Using PCR-based detection and genotyping to trace Streptococcus salivarius meningitis outbreak strain to oral flora of radiology physician assistant.

    PubMed

    Srinivasan, Velusamy; Gertz, Robert E; Shewmaker, Patricia L; Patrick, Sarah; Chitnis, Amit S; O'Connell, Heather; Benowitz, Isaac; Patel, Priti; Guh, Alice Y; Noble-Wang, Judith; Turabelidze, George; Beall, Bernard

    2012-01-01

    We recently investigated three cases of bacterial meningitis that were reported from a midwestern radiology clinic where facemasks were not worn during spinal injection of contrast agent during myelography procedures. Using pulsed field gel electrophoresis we linked a case strain of S. salivarius to an oral specimen of a radiology physician assistant (RPA). We also used a real-time PCR assay to detect S. salivarius DNA within a culture-negative cerebrospinal fluid (CSF) specimen. Here we extend this investigation through using a nested PCR/sequencing strategy to link the culture-negative CSF specimen to the case strain. We also provide validation of the real-time PCR assay used, demonstrating that it is not solely specific for Streptococcus salivarius, but is also highly sensitive for detection of the closely related oral species Streptococcus vestibularis. Through using multilocus sequence typing and 16S rDNA sequencing we further strengthen the link between the CSF case isolate and the RPA carriage isolate. We also demonstrate that the newly characterized strains from this study are distinct from previously characterized S. salivarius strains associated with carriage and meningitis. PMID:22384169

  5. Using PCR-Based Detection and Genotyping to Trace Streptococcus salivarius Meningitis Outbreak Strain to Oral Flora of Radiology Physician Assistant

    PubMed Central

    Srinivasan, Velusamy; Gertz Jr., Robert E.; Shewmaker, Patricia L.; Patrick, Sarah; Chitnis, Amit S.; O'Connell, Heather; Benowitz, Isaac; Patel, Priti; Guh, Alice Y.; Noble-Wang, Judith; Turabelidze, George; Beall, Bernard

    2012-01-01

    We recently investigated three cases of bacterial meningitis that were reported from a midwestern radiology clinic where facemasks were not worn during spinal injection of contrast agent during myelography procedures. Using pulsed field gel electrophoresis we linked a case strain of S. salivarius to an oral specimen of a radiology physician assistant (RPA). We also used a real-time PCR assay to detect S. salivarius DNA within a culture-negative cerebrospinal fluid (CSF) specimen. Here we extend this investigation through using a nested PCR/sequencing strategy to link the culture-negative CSF specimen to the case strain. We also provide validation of the real-time PCR assay used, demonstrating that it is not solely specific for Streptococcus salivarius, but is also highly sensitive for detection of the closely related oral species Streptococcus vestibularis. Through using multilocus sequence typing and 16S rDNA sequencing we further strengthen the link between the CSF case isolate and the RPA carriage isolate. We also demonstrate that the newly characterized strains from this study are distinct from previously characterized S. salivarius strains associated with carriage and meningitis. PMID:22384169

  6. Investigation of the spectral properties of LED-based MR16s for general illumination

    NASA Astrophysics Data System (ADS)

    Brown, David F.; Nicol, David B.; Payne, Adam; Ferguson, Ian T.

    2004-10-01

    The spectral properties of commercially available LED-based and halogen MR16s were investigated. The measurements taken include TLF (Total Luminous Flux), CCT (Correlated Color Temperature), CRI (Color Rendering Index), angular variation of CCT, and luminous efficacy. The halogen MR16s were used as a baseline for comparison with LED-based MR16s. It is shown at this time that LED-based MR16s are not suitable as a direct replacement for existing alternatives due to high initial cost, low power efficiency, poor CRIs, and undesirable CCTs.

  7. Loop-mediated isothermal amplification assay for 16S rRNA methylase genes in Gram-negative bacteria.

    PubMed

    Nagasawa, Mitsuaki; Kaku, Mitsuo; Kamachi, Kazunari; Shibayama, Keigo; Arakawa, Yoshichika; Yamaguchi, Keizo; Ishii, Yoshikazu

    2014-10-01

    Using the loop-mediated isothermal amplification (LAMP) method, we developed a rapid assay for detection of 16S rRNA methylase genes (rmtA, rmtB, and armA), and investigated 16S rRNA methylase-producing strains among clinical isolates. Primer Explorer V3 software was used to design the LAMP primers. LAMP primers were prepared for each gene, including two outer primers (F3 and B3), two inner primers (FIP and BIP), and two loop primers (LF and LB). Detection was performed with the Loopamp DNA amplification kit. For all three genes (rmtA, rmtB, and armA), 10(2) copies/tube could be detected with a reaction time of 60 min. When nine bacterial species (65 strains saved in National Institute of Infectious Diseases) were tested, which had been confirmed to possess rmtA, rmtB, or armA by PCR and DNA sequencing, the genes were detected correctly in these bacteria with no false negative or false positive results. Among 8447 clinical isolates isolated at 36 medical institutions, the LAMP method was conducted for 191 strains that were resistant to aminoglycosides based on the results of antimicrobial susceptibility tests. Eight strains were found to produce 16S rRNA methylase (0.09%), with rmtB being identified in three strains (0.06%) of 4929 isolates of Enterobacteriaceae, rmtA in three strains (0.10%) of 3284 isolates of Pseudomonas aeruginosa, and armA in two strains (0.85%) of 234 isolates of Acinetobacter spp. At present, the incidence of strains possessing 16S rRNA methylase genes is very low in Japan. However, when Gram-negative bacteria showing high resistance to aminoglycosides are isolated by clinical laboratories, it seems very important to investigate the status of 16S rRNA methylase gene-harboring bacilli and monitor their trends among Japanese clinical settings. PMID:25179393

  8. Comparative evaluation of rumen metagenome community using qPCR and MG-RAST.

    PubMed

    Nathani, Neelam M; Patel, Amrutlal K; Dhamannapatil, Prakash S; Kothari, Ramesh K; Singh, Krishna M; Joshi, Chaitanya G

    2013-01-01

    Microbial profiling of metagenome communities have been studied extensively using MG-RAST and other related metagenome annotation databases. Although, database based taxonomic profiling provides snapshots of the metagenome architecture, their reliability needs to be validated through more accurate methods. Here, we performed qPCR based absolute quantitation of selected rumen microbes in the liquid and solid fraction of the rumen fluid of river buffalo adapted to varying proportion of concentrate to green or dry roughages and compared with the MG-RAST based annotation of the metagenomes sequences of 16S r-DNA amplicons and high throughput shotgun sequencing. Animals were adapted to roughage-to-concentrate ratio in the proportion of 50:50, 75:25 and 100:00, respectively for six weeks. At the end of each treatment, rumen fluid was collected at 3 h post feeding. qPCR revealed that the relative abundance of Prevotella bryantii was higher, followed by the two cellulolytic bacteria Fibrobacter succinogens and Ruminococcus flavefaciens that accounted up to 1.33% and 0.78% of the total rumen bacteria, respectively. While, Selenomonas ruminantium and archaea Methanomicrobiales were lower in microbial population in the rumen of buffalo. There was no statistically significant difference between the enumerations shown by qPCR and analysis of the shotgun sequencing data by MG-RAST except for Prevotella. These results indicate the variations in abundance of different microbial species in buffalo rumen under varied feeding regimes as well as in different fractions of rumen liquor, i.e. solid and the liquid. The results also present the reliability of shotgun sequencing to describe metagenome and analysis/annotation by MG-RAST. PMID:24025701

  9. Comparative evaluation of rumen metagenome community using qPCR and MG-RAST

    PubMed Central

    2013-01-01

    Microbial profiling of metagenome communities have been studied extensively using MG-RAST and other related metagenome annotation databases. Although, database based taxonomic profiling provides snapshots of the metagenome architecture, their reliability needs to be validated through more accurate methods. Here, we performed qPCR based absolute quantitation of selected rumen microbes in the liquid and solid fraction of the rumen fluid of river buffalo adapted to varying proportion of concentrate to green or dry roughages and compared with the MG-RAST based annotation of the metagenomes sequences of 16S r-DNA amplicons and high throughput shotgun sequencing. Animals were adapted to roughage-to-concentrate ratio in the proportion of 50:50, 75:25 and 100:00, respectively for six weeks. At the end of each treatment, rumen fluid was collected at 3 h post feeding. qPCR revealed that the relative abundance of Prevotella bryantii was higher, followed by the two cellulolytic bacteria Fibrobacter succinogens and Ruminococcus flavefaciens that accounted up to 1.33% and 0.78% of the total rumen bacteria, respectively. While, Selenomonas ruminantium and archaea Methanomicrobiales were lower in microbial population in the rumen of buffalo. There was no statistically significant difference between the enumerations shown by qPCR and analysis of the shotgun sequencing data by MG-RAST except for Prevotella. These results indicate the variations in abundance of different microbial species in buffalo rumen under varied feeding regimes as well as in different fractions of rumen liquor, i.e. solid and the liquid. The results also present the reliability of shotgun sequencing to describe metagenome and analysis/annotation by MG-RAST. PMID:24025701

  10. Characterization of microbial community in nitrogen removal process of metallurgic wastewater by PCR-DGGE.

    PubMed

    Yoshie, S; Noda, N; Miyano, T; Tsuneda, S; Hirata, A; Inamori, Y

    2002-01-01

    The metallurgic wastewater generated from the processes of recovering precious metals from industrial wastes contains high concentrations of nitrogen compounds such as ammonia and nitric acid and of salts such as sodium chloride and sodium sulfate. Biological nitrogen removal from this wastewater was attempted by a circulating bioreactor system equipped with an anoxic packed bed and an aerobic fluidized bed. The anoxic packed bed of this system was found to effectively remove nitrite and nitrate from the wastewater by denitrification at a removal ratio of 97%. As a result of denitrification activity tests at various NaCl concentrations, the sludge obtained from the anoxic packed bed exhibited accumulation of nitrite at 5.0 and 8.4% NaCl concentrations, suggesting that the reduction of nitrite is the key step in the denitrification pathway under hypersaline conditions. The microbial community analysis by denaturing gradient gel electrophoresis (DGGE) of PCR-amplified 16S ribosomal DNA (rDNA) fragments revealed that the community diversity varied in accordance with water temperature, nitrate-loading rate and ionic strength. When particular major DGGE bands were excised, reamplified and directly sequenced, the dominant species in the anoxic packed bed were affiliated with the beta and gamma subclasses of the class Proteobacteria such as Alcaligenes defragrans and Pseudomonas spp., respectively. PMID:12523738

  11. PCR-DGGE analysis of denitrifying bacteria in a metallurgic wastewater treatment process.

    PubMed

    Noda, N; Yoshie, S; Miyano, T; Tsuneda, S; Hirata, A; Inamori, Y

    2002-01-01

    The wastewater generated from the processes of recovering precious metals from industrial wastes contains high concentrations of acids such as nitric acid and of salts. Biological nitrogen removal from this wastewater was attempted by using a circulating bioreactor system equipped with an anoxic packed bed or an anoxic fluidized bed and an aerobic three-phase fluidized bed. The system was found to effectively remove nitrogen from the diluted wastewater (T-N; 1,000-4,000 mg litre(-1)). The microbial population structure of activated sludge in an anoxic reactor was analyzed by denaturing gradient gel electrophoresis (DGGE) of PCR-amplified 16S ribosomal DNA (rDNA) fragments. DGGE analysis under different operating conditions demonstrated the presence of some distinguishable bands in the separation pattern, which were most likely derived from many different species constituting the microbial communities. Furthermore, the population diversity varied in accordance with the nitrate-loading rate, water temperature and reactor condition. Some major DGGE bands were excised, reamplified and directly sequenced. It was revealed that the dominant population in the anoxic reactor were affiliated with the beta subclass of the class Proteobacteria. PMID:12216646

  12. Tetrathiobacter kashmirensis Strain CA-1 16S rRNA gene complete sequence.

    Technology Transfer Automated Retrieval System (TEKTRAN)

    This study used 1326 base pair 16S rRNA gene sequence methods to confirm the identification of a bacterium as Tetrathiobacter kashmirensis. Morphological, biochemical characteristics, and fatty acid profiles are consistent with the 16S rRNA gene sequence identification of the bacterium. The isolate...

  13. Characterization of Mycobacterium leprae Genotypes in China—Identification of a New Polymorphism C251T in the 16S rRNA Gene

    PubMed Central

    You, Yuangang; Xing, Yan; Li, Huanying; Weng, Xiaoman; Wu, Nan; Liu, Shuang; Zhang, Shanshan; Zhang, Wenhong; Zhang, Ying

    2015-01-01

    Leprosy continues to be prevalent in some mountainous regions of China, and genotypes of leprosy strains endemic to the country are not known. Mycobacterium lepromatosis is a new species that was discovered in Mexico in 2008, and it remains unclear whether this species exists in China. Here, we conducted PCR- restriction fragment length polymorphism (RFLP) analysis to classify genotypes of 85 DNA samples collected from patients from 18 different provinces. All 171 DNA samples from skin biopsies of leprosy patients were tested for the presence of Mycobacterium leprae and Mycobacterium lepromatosis by amplifying the 16S rRNA gene using nested PCR, followed by DNA sequencing. The new species M. lepromatosis was not found among the 171 specimens from leprosy patients in 22 provinces in China. However, we found three SNP genotypes among 85 leprosy patients. A mutation at C251T in the 16S rRNA gene was found in 76% of the strains. We also found that the strains that showed the 16S rRNA C251T mutation belonged to SNP type 3, whereas strains without the point mutation belonged to SNP type 1. The SNP type 3 leprosy strains were observed in patients from both the inner and coastal regions of China, but the SNP type 1 strains were focused only in the coastal region. This indicated that the SNP type 3 leprosy strains were more prevalent than the SNP type 1 strains in China. In addition, the 16S rRNA gene sequence mutation at C251T also indicated a difference in the geographical distribution of the strains. To our knowledge, this is the first report of a new polymorphism in 16S rRNA gene in M. leprae in China. Our findings shed light on the prevalent genotypes and provide insight about leprosy transmission that are important for leprosy control in China. PMID:26196543

  14. Distribution of 16S rRNA methylases among different species of Gram-negative bacilli with high-level resistance to aminoglycosides.

    PubMed

    Zhou, Y; Yu, H; Guo, Q; Xu, X; Ye, X; Wu, S; Guo, Y; Wang, M

    2010-11-01

    16S rRNA methylases confer high-level resistance to most aminoglycosides in Gram-negative bacteria. Seven 16S rRNA methylase genes, armA, rmtA, rmtB, rmtC, rmtD, rmtE and npmA, have been identified since 2003. We studied the distribution of methylase genes in more than 200 aminoglycoside-resistant Gram-negative clinical isolates collected in 2007 at our hospital in Shanghai, China. 16S rRNA methylase genes were amplified by polymerase chain reaction (PCR) among 217 consecutive clinical isolates of Gram-negative bacilli resistant to gentamicin and amikacin by a disk diffusion method. 16S rRNA methylase genes were present in 97.5% (193/198) of clinical isolates highly resistant to amikacin (≥512 μg/ml), with armA and rmtB detected in 67.2 and 30.3% of strains, respectively, while no 16S rRNA methylase genes were detected in 19 strains with amikacin minimum inhibitory concentration (MIC) ≤256 μg/ml. armA or rmtB genes were detected in 100% of 104 strains of Enterobacteriaceae, and these two genes were equally represented (49 vs. 55 strains). Genes for armA or rmtB were detected in 94.7% (89/94) of Acinetobacter baumannii and Pseudomonas aeruginosa strains, and armA was predominant (84 vs. 5 strains with rmtB). No rmtA, rmtC, rmtD or npmA genes were found. Enterobacterial repetitive intergenic consensus sequence (ERIC-PCR) indicated that armA and rmtB genes were spread by both horizontal transfer and clonal dissemination. PMID:20614151

  15. Characterization of Mycobacterium leprae Genotypes in China--Identification of a New Polymorphism C251T in the 16S rRNA Gene.

    PubMed

    Yuan, Youhua; Wen, Yan; You, Yuangang; Xing, Yan; Li, Huanying; Weng, Xiaoman; Wu, Nan; Liu, Shuang; Zhang, Shanshan; Zhang, Wenhong; Zhang, Ying

    2015-01-01

    Leprosy continues to be prevalent in some mountainous regions of China, and genotypes of leprosy strains endemic to the country are not known. Mycobacterium lepromatosis is a new species that was discovered in Mexico in 2008, and it remains unclear whether this species exists in China. Here, we conducted PCR- restriction fragment length polymorphism (RFLP) analysis to classify genotypes of 85 DNA samples collected from patients from 18 different provinces. All 171 DNA samples from skin biopsies of leprosy patients were tested for the presence of Mycobacterium leprae and Mycobacterium lepromatosis by amplifying the 16S rRNA gene using nested PCR, followed by DNA sequencing. The new species M. lepromatosis was not found among the 171 specimens from leprosy patients in 22 provinces in China. However, we found three SNP genotypes among 85 leprosy patients. A mutation at C251T in the 16S rRNA gene was found in 76% of the strains. We also found that the strains that showed the 16S rRNA C251T mutation belonged to SNP type 3, whereas strains without the point mutation belonged to SNP type 1. The SNP type 3 leprosy strains were observed in patients from both the inner and coastal regions of China, but the SNP type 1 strains were focused only in the coastal region. This indicated that the SNP type 3 leprosy strains were more prevalent than the SNP type 1 strains in China. In addition, the 16S rRNA gene sequence mutation at C251T also indicated a difference in the geographical distribution of the strains. To our knowledge, this is the first report of a new polymorphism in 16S rRNA gene in M. leprae in China. Our findings shed light on the prevalent genotypes and provide insight about leprosy transmission that are important for leprosy control in China. PMID:26196543

  16. Brachyspira aalborgi Infection Diagnosed by Culture and 16S Ribosomal DNA Sequencing Using Human Colonic Biopsy Specimens

    PubMed Central

    Kraaz, Wolfgang; Pettersson, Bertil; Thunberg, Ulf; Engstrand, Lars; Fellström, Claes

    2000-01-01

    In this study we report on the isolation and characterization of the intestinal spirochete Brachyspira aalborgi using human mucosal biopsy specimens taken from the colon of a young adult male with intestinal spirochetosis. A selective medium, containing 400 μg of spectinomycin/ml and 5 μg of polymyxin/ml was used for the isolation procedure. A high degree of similarity, in terms of phenotypic properties and 16S ribosomal DNA sequence, was observed between the isolated strain, named W1, and the type strain, 513A, of B. aalborgi. A similarity of 99.7% in the nucleotide sequence was found between W1 and 513AT, based on the almost-complete gene. A short segment of the 16S rRNA gene was amplified by PCR using genetic material enriched from paraffin-embedded biopsy specimens, which were taken from the patient on two occasions. The products showed 16S rRNA gene sequences virtually identical to that of strain 513AT in the actual region. Immunohistochemistry was performed on the colonic biopsy specimens with a polyclonal antibody raised against an intestinal spirochete isolated in a previous case of human intestinal spirochetosis. The antibody reacted strongly with the spirochete on the luminal epithelium. No immune reaction was seen within or below the surface epithelium. Routine histology did not reveal signs of colitis. Electron microscopy showed spirochetes attached end-on to the colonic mucosal surface. The isolate grew poorly on a commonly used selective medium for intestinal spirochetes, which may explain previous failures to isolate B. aalborgi. PMID:11015363

  17. Distinction between intact and antibiotic-inactivated bacteria by real-time PCR after treatment with propidium monoazide.

    PubMed

    Kobayashi, Hideo; Oethinger, Margret; Tuohy, Marion J; Hall, Gerri S; Bauer, Thomas W

    2010-09-01

    One limitation to the use of the polymerase chain reaction (PCR) to identify orthopedic infections has been apparent false-positive results, possibly due to the detection of dead bacteria. We recently showed that the use of DNA-binding agent propidium monoazide (PMA) could distinguish viable from heat-inactivated bacteria, and, in this study, we investigated whether the same technique can be applied to bacteria killed by two antibiotics with distinctly different mechanisms of action, a test of greater clinical relevance than thermal inactivation. Staphylococcus aureus and S. epidermidis were inactivated by vancomycin and gentamicin and treated with PMA or left untreated before DNA extraction. The threshold cycle difference of antibiotic-treated bacteria with and without PMA pretreatment was investigated with PCR primers for the 16S rDNA and tuf genes. Our results indicated that PMA effectively inhibited detection by PCR of bacteria, which had been inactivated by either vancomycin or gentamicin. The effect was statistically significant at 24 h after treatment (C(t) difference consistently >3; p < 0.05) and after 10 days of treatment (C(t) difference >4; p < 0.01), when compared to viable cells (C(t) difference 1-2). Vancomycin had a stronger effect on the C(t) value than gentamicin, reflecting the different mechanism of action of each antibiotic. Techniques of this type may help reduce clinically false-positive PCR results caused by the detection of dead bacteria, and may be especially useful in patients who have received antibiotics, such as patients undergoing the second stage of a two-stage revision for infected arthroplasty. PMID:20186836

  18. Exploring the Bacterial Diversity of Belgian Steak Tartare Using Metagenetics and Quantitative Real-Time PCR Analysis.

    PubMed

    Delhalle, L; Korsak, N; Taminiau, B; Nezer, C; Burteau, S; Delcenserie, V; Poullet, J B; Daube, G

    2016-02-01

    Steak tartare is a popular meat dish in Belgium. It is prepared with raw minced beef and is eaten with sauce, vegetables, and spices. Because it contains raw meat, steak tartare is highly prone to bacterial spoilage. The objective of this study was to explore the diversity of bacterial flora in steak tartare in Belgium according to the source and to determine which bacteria are able to grow during shelf life. A total of 58 samples from butchers' shops, restaurants, sandwich shops, and supermarkets were collected. These samples were analyzed using 16S rDNA metagenetics, a classical microbiological technique, and quantitative real-time PCR (qPCR) targeting the Lactobacillus genus. Samples were analyzed at the beginning and at the end of their shelf life, except for those from restaurants and sandwich shops, which were analyzed only on the purchase date. Metagenetic analysis identified up to 180 bacterial species and 90 genera in some samples. But only seven bacterial species were predominant in the samples, depending on the source: Brochothrix thermosphacta, Lactobacillus algidus, Lactococcus piscium, Leuconostoc gelidum, Photobacterium kishitani, Pseudomonas spp., and Xanthomonas oryzae. With this work, an alternative method is proposed to evaluate the total flora in food samples based on the number of reads from metagenetic analysis and the results of qPCR. The degree of underestimation of aerobic plate counts at 30°C estimated with the classical microbiology method was demonstrated in comparison with the proposed culture-independent method. Compared with culture-based methods, metagenetic analysis combined with qPCR targeting Lactobacillus provides valuable information for characterizing the bacterial flora of raw meat. PMID:26818982

  19. Monitoring Precursor 16S rRNAs of Acinetobacter spp. in Activated Sludge Wastewater Treatment Systems

    PubMed Central

    Oerther, Daniel B.; Pernthaler, Jakob; Schramm, Andreas; Amann, Rudolf; Raskin, Lutgarde

    2000-01-01

    Recently, Cangelosi and Brabant used oligonucleotide probes targeting the precursor 16S rRNA of Escherichia coli to demonstrate that the levels of precursor rRNA were more sensitive to changes in growth phase than the levels of total rRNA (G. A. Cangelosi and W. H. Brabant, J. Bacteriol. 179:4457–4463, 1997). In order to measure changes in the levels of precursor rRNA in activated sludge systems, we designed oligonucleotide probes targeting the 3′ region of the precursor 16S rRNA of Acinetobacter spp. We used these probes to monitor changes in the level of precursor 16S rRNA during batch growth of Acinetobacter spp. in Luria-Bertani (LB) medium, filtered wastewater, and in lab- and full-scale wastewater treatment systems. Consistent with the previous reports for E. coli, results obtained with membrane hybridizations and fluorescence in situ hybridizations with Acinetobacter calcoaceticus grown in LB medium showed a more substantial and faster increase in precursor 16S rRNA levels compared to the increase in total 16S rRNA levels during exponential growth. Diluting an overnight culture of A. calcoaceticus grown in LB medium with filtered wastewater resulted in a pattern of precursor 16S rRNA levels that appeared to follow diauxic growth. In addition, fluorescence in situ hybridizations with oligonucleotide probes targeting total 16S rRNA and precursor 16S rRNA showed that individual cells of A. calcoaceticus expressed highly variable levels of precursor 16S rRNA when adapting from LB medium to filtered sewage. Precursor 16S rRNA levels of Acinetobacter spp. transiently increased when activated sludge was mixed with influent wastewater in lab- and full-scale wastewater treatment systems. These results suggest that Acinetobacter spp. experience a change in growth activity within wastewater treatment systems. PMID:10788395

  20. 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 ...

  1. Comparison of two poultry litter qPCR assays targeting the 16S rRNA gene of Brevibacterium sp

    EPA Science Inventory

    Chicken feces are vectors of human pathogens and are also important sources of fecal pollution in environmental waters. Consequently, methods that can detect chicken fecal pollution are needed in public health and environmental monitoring studies. In this study, we compared a pre...

  2. Variability in abundance of the Bacterial and Archaeal 16S rRNA and amoA genes in water columns of northern South China Sea

    NASA Astrophysics Data System (ADS)

    Liu, H.; Yang, C.; Chen, S.; Xie, W.; Wang, P.; Zhang, C. L.

    2014-12-01

    Recent advances in marine microbial ecology have shown that ammonia-oxidizing Archaea (AOA) are more abundant than ammonia-oxidizing bacteria (AOB), although total Bacteria are more abundant than total Archaea in marine environments. This study aimed to examine the spatial distribution and abundance of planktonic archaeal and bacterial 16S rRNA- and amoA genes in the northern South China Sea. Water samples were collected at different depths at six stations (maximum depth ranging from 1800 m to 3200 m)with four stations (B2, B3, B6, B7) located along a transect from the northeastern continental slope to the Bashi Strait and the other two (D3, D5) located southwest of this transect. Quantitative PCR of the 16S rRNA- and amoA genes was used to estimate the abundances of total Archaea, total Bacteria, and AOA and AOB, respectively. At the B series stations, the abundance of bacterial 16S rRNA gene was twofold to 36fold higher than that of the archaeal 16S rRNA gene while fivefold lower to sixfold higher at the two D stations, with both genes showing peak values slightly below sea surface (5-75 m depths) at all stations. The archaeal amoA gene had similar variations with the archaeal 16S rRNA gene, but was 1-4 orders of magnitude lower than the archaeal 16S rRNA gene at all stations. Bacterial amoA gene was below the detection at all stations. Our results also show the difference in depth profiles among these stations, which may be caused by the difference in water movement between these regions. The non-detection of bacterial amoA gene indicates that ammonia-oxidizing Archaea are the dominant group of microorganisms in nitrification of the South China Sea, which is consistent with observations in other oceans.

  3. 16S rRNA gene phylogenesis of culturable predominant bacteria from diseased Apostichopus japonicus (Holothuroidea, Echinodermata)

    NASA Astrophysics Data System (ADS)

    Ma, Haiyan; Jiang, Guoliang; Wu, Zhiqiang; Wang, Xin

    2009-06-01

    Cultured Apostichopus japonicus in China suffers from a kind of skin ulceration disease that has caused severe economic loss in recent years. The disease, pathogens of which are supposed to be bacteria by most researchers, is highly infectious and can often cause all individuals in the same culture pool to die in a very short time. The 16S rRNA gene phylogenesis of the culturable bacteria from the lesions of diseased individuals was conducted to study the biodiversity of the bacterial communities in the lesions and to identify probable pathogen(s) associated with this kind of disease. S. japonica samples were selected from a hatchery located in the eastern part of Qingdao, China. Bacterial universal primers GM5F and DS907R were used to amplify the 16S rRNA gene of bacteria colonies, and touchdown PCR was performed to amplify the target sequences. The results suggest that γ- proteobacteria (Alteromonadales and Vibrionales) of CFB group, many strains of which have been also determined as pathogens in other marine species, are the predominant bacterial genera of the diseased Apostichopus japonicus individuals.

  4. Comparative sequence analyses of the genes coding for 16S rRNA of Lactobacillus casei-related taxa.

    PubMed

    Mori, K; Yamazaki, K; Ishiyama, T; Katsumata, M; Kobayashi, K; Kawai, Y; Inoue, N; Shinano, H

    1997-01-01

    The primary structures of the 16S rRNA genes of the type strains of Lactobacillus casei and related taxa were determined by PCR DNA-sequencing methods. The sequences of Lactobacillus casei, Lactobacillus zeae, Lactobacillus paracasei, and Lactobacillus rhamnosus were different. The Knuc values ranged from 0.0040 to 0.0126. On the basis of the Knuc values and the levels of DNA-DNA relatedness among the strains of these species, the L. casei-related taxa should be classified in the following three species: L. zeae, which includes the type strains of L. zeae and L. casei; a species that includes the strains of L. paracasei and L. casei ATCC 334; and L. rhamnosus. PMID:8995801

  5. Use of Quantitative 16S Ribosomal DNA Detection for Diagnosis of Central Vascular Catheter-Associated Bacterial Infection

    PubMed Central

    Warwick, S.; Wilks, M.; Hennessy, E.; Powell-Tuck, J.; Small, M.; Sharp, J.; Millar, M. R.

    2004-01-01

    Many central vascular catheters (CVCs) are removed unnecessarily because current diagnostic methods for CVC-associated infection are unreliable. A quantitative PCR assay using primers and probe targeted to bacterial 16S ribosomal DNA was used to measure the levels of bacterial DNA in blood samples drawn through the CVC in a population of patients receiving intravenous nutrition. Bacterial DNA concentrations were raised in 16 of 16 blood samples taken during episodes of probable bacterial CVC-associated infection. Bacterial DNA concentrations were raised in 4 of 29 episodes in which bacterial CVC-associated infection was unlikely. The use of this technique has the potential to substantially reduce the unnecessary removal of CVCs. PMID:15070980

  6. Bacterial Diversity and Community Structure of Supragingival Plaques in Adults with Dental Health or Caries Revealed by 16S Pyrosequencing

    PubMed Central

    Xiao, Cuicui; Ran, Shujun; Huang, Zhengwei; Liang, Jingping

    2016-01-01

    Dental caries has a polymicrobial etiology within the complex oral microbial ecosystem. However, the overall diversity and structure of supragingival plaque microbiota in adult dental health and caries are not well understood. Here, 160 supragingival plaque samples from patients with dental health and different severities of dental caries were collected for bacterial genomic DNA extraction, pyrosequencing by amplification of the 16S rDNA V1–V3 hypervariable regions, and bioinformatic analysis. High-quality sequences (2,261,700) clustered into 10,365 operational taxonomic units (OTUs; 97% identity), representing 453 independent species belonging to 122 genera, 66 families, 34 orders, 21 classes, and 12 phyla. All groups shared 7522 OTUs, indicating the presence of a core plaque microbiome. α diversity analysis showed that the microbial diversity in healthy plaques exceeded that of dental caries, with the diversity decreasing gradually with the severity of caries. The dominant phyla of plaque microbiota included Bacteroidetes, Actinobacteria, Proteobacteria, Firmicutes, Fusobacteria, and TM7. The dominant genera included Capnocytophaga, Prevotella, Actinomyces, Corynebacterium, Neisseria, Streptococcus, Rothia, and Leptotrichia. β diversity analysis showed that the plaque microbial community structure was similar in all groups. Using LEfSe analysis, 25 differentially abundant taxa were identified as potential biomarkers. Key genera (27) that potentially contributed to the differential distributions of plaque microbiota between groups were identified by PLS-DA analysis. Finally, co-occurrence network analysis and function predictions were performed. Treatment strategies directed toward modulating microbial interactions and their functional output should be further developed. PMID:27499752

  7. Changing of the Guard: Migrating 16S and ITS Diversity Profiling from 454 Onto Alternate NGS Platforms

    PubMed Central

    McNally, R.; Johnson, M.; Kasinadhuni, N.; Tinning, M.; McGrath, K.

    2014-01-01

    The Australian Genome Research Facility (AGRF) currently operates a microbial diversity profiling service on the 454 GS-FLX platform, targeting various regions of 16S and ITS microbial genes. The recent announcement by Roche to discontinue support prompted us to investigate how this service can be transitioned to alternate NGS platforms. The objective of this study was to evaluate the accuracy and reproducibility of each platform/target combination for both artificial control samples (known gDNA pools) and natural microbial community samples (soils). Replicate PCR amplicons were generated for several microbial and fungal regions (16S and ITS), and sequenced on the GS-FLX, MiSeq and Ion Torrent PGM platforms. The resulting sequences were identified at different taxonomic levels (phylum to species) and the results for each sample, platform and target compiled. This comparison revealed distinct and reproducible differences between the same samples run on different platforms. While no platform was able to perfectly describe the proportions of the artificial pooled sample, each one showed a high level of reproducibility (< 1% variance). This suggests that PCR bias during amplification gives rise to consistent biases in resulting microbial distributions and confirms this method as a useful tool for comparing samples and monitoring changes, but demonstrate its weakness for absolute quantitation. Between platforms, the long reads of the GS-FLX gave the greatest resolution for microbial identification, followed by the MiSeq. Despite the shorter amplicon size, the ion torrent was still able to provide high resolution profiles of the microbial samples. These results show that currently, the 454 still provides higher resolution data sets for microbial diversity than other available platforms. However, as read lengths improve and error rates decrease, these alternate platforms should provide a suitable transition pathway for researchers interested in profiling microbial

  8. 16S rRNA gene-based metagenomic analysis reveals differences in bacteria-derived extracellular vesicles in the urine of pregnant and non-pregnant women

    PubMed Central

    Yoo, Jae Young; Rho, Mina; You, Young-Ah; Kwon, Eun Jin; Kim, Min-Hye; Kym, Sungmin; Jee, Young-Koo; Kim, Yoon-Keun; Kim, Young Ju

    2016-01-01

    Recent evidence has indicated that bacteria-derived extracellular vesicles (EVs) are important for host–microbe communication. The aims of the present study were to evaluate whether bacteria-derived EVs are excreted via the urinary tract and to compare the composition of bacteria-derived EVs in the urine of pregnant and non-pregnant women. Seventy-three non-pregnant and seventy-four pregnant women were enrolled from Dankook University and Ewha Womans University hospitals. DNA was extracted from urine EVs after EV isolation using the differential centrifugation method. 16S ribosomal RNA (16S rRNA) gene sequencing was performed using high-throughput 454 pyrosequencing after amplification of the V1–V3 region of the 16S rDNA. The composition of 13 taxa differed significantly between the pregnant and non-pregnant women. At the genus level, Bacillus spp. EVs were more significantly enriched in the urine of the pregnant women than in that of the non-pregnant women (45.61% vs 0.12%, respectively). However, Pseudomonas spp. EVs were more dominant in non-pregnant women than in pregnant women (13.2% vs 4.09%, respectively). Regarding the compositional difference between pregnant women with normal and preterm delivery, EVs derived from Ureaplasma spp. and the family Veillonellaceae (including Megasphaera spp.) were more abundant in the urine of preterm-delivered women than in that of women with normal deliveries. Taken together, these data showed that Bacillus spp. EVs predominate in the urine of pregnant women, whereas Pseudomonas spp. EVs predominate in the urine of non-pregnant women; this suggests that Bacillus spp. EVs might have an important role in the maintenance of pregnancy. PMID:26846451

  9. 16S rRNA gene-based metagenomic analysis reveals differences in bacteria-derived extracellular vesicles in the urine of pregnant and non-pregnant women.

    PubMed

    Yoo, Jae Young; Rho, Mina; You, Young-Ah; Kwon, Eun Jin; Kim, Min-Hye; Kym, Sungmin; Jee, Young-Koo; Kim, Yoon-Keun; Kim, Young Ju

    2016-01-01

    Recent evidence has indicated that bacteria-derived extracellular vesicles (EVs) are important for host-microbe communication. The aims of the present study were to evaluate whether bacteria-derived EVs are excreted via the urinary tract and to compare the composition of bacteria-derived EVs in the urine of pregnant and non-pregnant women. Seventy-three non-pregnant and seventy-four pregnant women were enrolled from Dankook University and Ewha Womans University hospitals. DNA was extracted from urine EVs after EV isolation using the differential centrifugation method. 16S ribosomal RNA (16S rRNA) gene sequencing was performed using high-throughput 454 pyrosequencing after amplification of the V1-V3 region of the 16S rDNA. The composition of 13 taxa differed significantly between the pregnant and non-pregnant women. At the genus level, Bacillus spp. EVs were more significantly enriched in the urine of the pregnant women than in that of the non-pregnant women (45.61% vs 0.12%, respectively). However, Pseudomonas spp. EVs were more dominant in non-pregnant women than in pregnant women (13.2% vs 4.09%, respectively). Regarding the compositional difference between pregnant women with normal and preterm delivery, EVs derived from Ureaplasma spp. and the family Veillonellaceae (including Megasphaera spp.) were more abundant in the urine of preterm-delivered women than in that of women with normal deliveries. Taken together, these data showed that Bacillus spp. EVs predominate in the urine of pregnant women, whereas Pseudomonas spp. EVs predominate in the urine of non-pregnant women; this suggests that Bacillus spp. EVs might have an important role in the maintenance of pregnancy. PMID:26846451

  10. 16S ribosomal RNA-based methods to monitor changes in the hindgut bacterial community of piglets after oral administration of Lactobacillus sobrius S1.

    PubMed

    Su, Yong; Yao, Wen; Perez-Gutierrez, Odette N; Smidt, Hauke; Zhu, Wei-Yun

    2008-04-01

    16S ribosomal RNA (rRNA) gene based PCR/denaturing gradient gel electrophoresis (DGGE) and real-time PCR were used to monitor the changes in the composition of microbiota in the hindgut of piglets after oral administration of Lactobacillus sobrius S1. Six litters of neonatal piglets were divided randomly into control group and treatment group. At 7, 9, and 11 days of age, piglets in the treatment group orally received a preparation of L. sobrius S1. At 7, 14, 21(weaning), 24, and 35 days of age, one piglet from each litter was sacrificed and digesta samples of hindgut were collected. DGGE analysis of 16S rRNA gene V6-V8 region for all bacteria showed that several populations present in the hindgut of piglets, represented by far-migrating bands, disappeared after weaning. Most of these bands corresponded to Lactobacillus spp. as revealed by sequence analysis. Quantitative real-time PCR specific for lactobacilli further demonstrated that the number of lactobacilli population tended to decrease after the piglets were weaned. Drastic changes of L. amylovorus and L. sobrius in total Lactobacillus populations were also observed in the colon of piglets around weaning, as monitored by 16S rRNA gene V2-V3 region based Lactobacillus-specific PCR-DGGE. Species-specific real-time PCR also revealed that the population of L. sobrius declined apparently in the colon of piglets after weaning. No remarkable changes in the overall microbial community in the hindgut were found between control and treatment groups. However, comparison of DGGE profiles between the two groups revealed a specific band related to Clostridium disporicum that was found in treatment group on day 14. On day 35, a specific band appeared only in the control group, representing a population most closely related to Streptococcus suis (99%). Real-time PCR showed that L. sobrius 16S rRNA gene copies in treatment group were relatively higher than in the control group (10(8.45) vs. 10(6.83)) on day 35, but no

  11. Avoidance and Potential Remedy Solutions of Chimeras in Reconstructing the Phylogeny of Aphids Using the 16S rRNA Gene of Buchnera: A Case in Lachninae (Hemiptera).

    PubMed

    Chen, Rui; Wang, Zhe; Chen, Jing; Qiao, Ge-Xia

    2015-01-01

    It is known that PCR amplification of highly homologous genes from complex DNA mixtures can generate a significant proportion of chimeric sequences. The 16S rRNA gene is not only widely used in estimating the species diversity of endosymbionts in aphids but also used to explore the co-diversification of aphids and their endosymbionts. Thus, chimeric sequences may lead to the discovery of non-existent endosymbiont species and mislead Buchnera-based phylogenetic analysis that lead to false conclusions. In this study, a high probability (6.49%) of chimeric sequence occurrence was found in the amplified 16S rRNA gene sequences of endosymbionts from aphid species in the subfamily Lachninae. These chimeras are hybrid products of multiple parent sequences from the dominant species of endosymbionts in each corresponding host. It is difficult to identify the chimeric sequences of a new or unidentified species due to the high variability of their main parent, Buchnera aphidicola, and because the chimeric sequences can confuse the phylogenetic analysis of 16S rRNA gene sequences. These chimeras present a challenge to Buchnera-based phylogenetic research in aphids. Thus, our study strongly suggests that using appropriate methods to detect chimeric 16S rRNA sequences may avoid some false conclusions in endosymbiont-based aphid research. PMID:26307984

  12. Avoidance and Potential Remedy Solutions of Chimeras in Reconstructing the Phylogeny of Aphids Using the 16S rRNA Gene of Buchnera: A Case in Lachninae (Hemiptera)

    PubMed Central

    Chen, Rui; Wang, Zhe; Chen, Jing; Qiao, Ge-Xia

    2015-01-01

    It is known that PCR amplification of highly homologous genes from complex DNA mixtures can generate a significant proportion of chimeric sequences. The 16S rRNA gene is not only widely used in estimating the species diversity of endosymbionts in aphids but also used to explore the co-diversification of aphids and their endosymbionts. Thus, chimeric sequences may lead to the discovery of non-existent endosymbiont species and mislead Buchnera-based phylogenetic analysis that lead to false conclusions. In this study, a high probability (6.49%) of chimeric sequence occurrence was found in the amplified 16S rRNA gene sequences of endosymbionts from aphid species in the subfamily Lachninae. These chimeras are hybrid products of multiple parent sequences from the dominant species of endosymbionts in each corresponding host. It is difficult to identify the chimeric sequences of a new or unidentified species due to the high variability of their main parent, Buchnera aphidicola, and because the chimeric sequences can confuse the phylogenetic analysis of 16S rRNA gene sequences. These chimeras present a challenge to Buchnera-based phylogenetic research in aphids. Thus, our study strongly suggests that using appropriate methods to detect chimeric 16S rRNA sequences may avoid some false conclusions in endosymbiont-based aphid research. PMID:26307984

  13. First Experience of a Multicenter External Quality Assessment of Molecular 16S rRNA Gene Detection in Bone and Joint Infections

    PubMed Central

    Bémer, Pascale; Valentin, Anne Sophie; Héry-Arnaud, Geneviève; Tandé, Didier; Jolivet-Gougeon, Anne; Vincent, Pascal; Kempf, Marie; Lemarié, Carole; Guinard, Jérôme; Bret, Laurent; Cognée, Anne Sophie; Gibaud, Sophie; Burucoa, Christophe; Corvec, Stéphane

    2014-01-01

    The objective of this study was to assess the performance of seven French laboratories for 16S rRNA gene detection by real-time PCR in the diagnosis of bone and joint infection (BJI) to validate a large multicenter study. External quality control (QC) was required owing to the differences in extraction procedures and the molecular equipment used in the different laboratories. Three proficiency sets were organized, including four bacterial DNA extracts and four bead mill-pretreated osteoarticular specimens. Extraction volumes, 16S rRNA gene primers, and sequencing interpretation rules were standardized. In order to assess each laboratory's ability to achieve the best results, scores were assigned, and each QC series was classified as optimal, acceptable, or to be improved. A total of 168 QCs were sent, and 160 responses were analyzed. The expected results were obtained for 93.8%, with the same proportion for extracts (75/80) and clinical specimens (75/80). For the specimens, there was no significant difference between manual and automated extraction. This QC demonstrated the ability to achieve good and homogeneous results using the same 16S rRNA gene PCR with different equipment and validates the possibility of high-quality multicenter studies using molecular diagnosis for BJI. PMID:25411177

  14. First experience of a multicenter external quality assessment of molecular 16S rRNA gene detection in bone and joint infections.

    PubMed

    Plouzeau, Chloé; Bémer, Pascale; Valentin, Anne Sophie; Héry-Arnaud, Geneviève; Tandé, Didier; Jolivet-Gougeon, Anne; Vincent, Pascal; Kempf, Marie; Lemarié, Carole; Guinard, Jérôme; Bret, Laurent; Cognée, Anne Sophie; Gibaud, Sophie; Burucoa, Christophe; Corvec, Stéphane

    2015-02-01

    The objective of this study was to assess the performance of seven French laboratories for 16S rRNA gene detection by real-time PCR in the diagnosis of bone and joint infection (BJI) to validate a large multicenter study. External quality control (QC) was required owing to the differences in extraction procedures and the molecular equipment used in the different laboratories. Three proficiency sets were organized, including four bacterial DNA extracts and four bead mill-pretreated osteoarticular specimens. Extraction volumes, 16S rRNA gene primers, and sequencing interpretation rules were standardized. In order to assess each laboratory's ability to achieve the best results, scores were assigned, and each QC series was classified as optimal, acceptable, or to be improved. A total of 168 QCs were sent, and 160 responses were analyzed. The expected results were obtained for 93.8%, with the same proportion for extracts (75/80) and clinical specimens (75/80). For the specimens, there was no significant difference between manual and automated extraction. This QC demonstrated the ability to achieve good and homogeneous results using the same 16S rRNA gene PCR with different equipment and validates the possibility of high-quality multicenter studies using molecular diagnosis for BJI. PMID:25411177

  15. Targeting single-nucleotide polymorphisms in the 16S rRNA gene to detect and differentiate Legionella pneumophila and non-Legionella pneumophila species.

    PubMed

    Zhan, Xiao-Yong; Hu, Chao-Hui; Zhu, Qing-Yi

    2016-08-01

    A PCR-based method targeting single-nucleotide polymorphisms (SNPs) in the 16S rRNA gene was developed for differential identification of Legionella pneumophila and non-Legionella pneumophila. Based on the bioinformatics analysis for 176 Legionella 16S rRNA gene fragments of 56 different Legionella species, a set of SNPs, A(628)C(629) was found to be highly specific to L. pneumophila strains. A multiplex assay was designed that was able to distinguish sites with limited sequence heterogeneity between L. pneumophila and non-L. pneumophila in the targeted 16S rRNA gene. The assay amplified a 261-bp amplicon for Legionella spp. and a set of 203- and 97-bp amplicons only specific to L. pneumophila species. Among 49 ATCC strains and 284 Legionella isolates from environmental water and clinical samples, 100 % of L. pneumophila and non-L. pneumophila strains were correctly identified and differentiated by this assay. The assay presents a more rapid, sensitive and alternative method to the currently available PCR-sequencing detection and differentiation method. PMID:27112927

  16. Phylogenetic analysis of encapsulated and non-encapsulated Trichinella species by studying the 5S rDNA tandemly repeated intergenic region.

    PubMed

    van der Giessen, J W B; Fonville, M; Briels, I; Pozio, E

    2005-09-01

    The identification of sequence regions in the genomes of pathogens which can be useful to distinguish among species and genotypes, is of great importance for epidemiological, molecular, and phylogenetic studies. The 5S ribosomal DNA intergenic spacer region has been identified as a good target to distinguish among eight Trichinella species and genotypes. The recent discovery of two non-encapsulated species in this genus, Trichinella papuae and Trichinella zimbabwensis, which can infect both mammals and reptiles, has suggested analyzing their 5S rDNA. Amplification of the tandem repeats of the 5S rDNA intergenic region of encapsulated species of Trichinella shows a 751bp fragment, whereas the three non-encapsulated species show a fragment of 800bp with T. pseudospiralis showing an additional fragment of 522bp. Although the size of the 800bp PCR fragments of T. papuae and T. zimbabwensis are similar to that of T. pseudospiralis, there are differences in the 5S rDNA intergenic regions among the three non-encapsulated species. Phylogenetic analysis of the 5S rDNA intergenic regions shows a clustering together of the three non-encapsulated Trichinella species that is well separated from the encapsulated ones. In addition, a single PCR-based method allows distinguishing non-encapsulated and encapsulated species. PMID:16076532

  17. Molecular characterization of Gastrodiscoides hominis (Platyhelminthes: Trematoda: Digenea) inferred from ITS rDNA sequence analysis.

    PubMed

    Goswami, L M; Prasad, P K; Tandon, V; Chatterjee, A

    2009-06-01

    Gastrodiscoides hominis (Digenea: Paramphistomata: Gastrodiscidae) is an amphistomid intestinal fluke of pigs causing gastrodiscoidiosis. With the use of molecular tools assisting the conventional diagnostic procedures, we aimed at finding out molecular characterization of G. hominis using PCR amplifications of rDNA ITS (1, 2) sequences. The sequences obtained (GenBank accession numbers EF027096, EF027097, EF027098, EU887294, and EU887295) were compared with available sequences of other digenean parasites, particularly those having a zoonotic potential in the northeastern region of India. The BLAST search revealed a close similarity with members of the family Paramphistomidae, showing maximum similarity with the amphistome, Homalogaster paloniae (subfamily Paramphistominae). Based on various tree construction methods, phylogeny of G. hominis is discussed. PMID:19198879

  18. Molecular characterization and Phylogenetic analysis of 16S rRNA from a new Candidatus Liberibacter strain associated with Zebra chip disease of Potato (Solanum tuberosum L.) and the potato psyllid

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The full-length 16S rRNA gene region of a new Candidatus Liberibacter strain was PCR amplified from tubers of potato plants showing Zebra Chip (ZC) disease symptoms and also from the potato psyllid [Bactericera (= Paratrioza) cockerelli Sulc], the presumptive vector of the ZC disease causal agent. ...

  19. Virtual PCR

    SciTech Connect

    Gardner, S N; Clague, D S; Vandersall, J A; Hon, G; Williams, P L

    2006-02-23

    The polymerase chain reaction (PCR) stands among the keystone technologies for analysis of biological sequence data. PCR is used to amplify DNA, to generate many copies from as little as a single template. This is essential, for example, in processing forensic DNA samples, pathogen detection in clinical or biothreat surveillance applications, and medical genotyping for diagnosis and treatment of disease. It is used in virtually every laboratory doing molecular, cellular, genetic, ecologic, forensic, or medical research. Despite its ubiquity, we lack the precise predictive capability that would enable detailed optimization of PCR reaction dynamics. In this LDRD, we proposed to develop Virtual PCR (VPCR) software, a computational method to model the kinetic, thermodynamic, and biological processes of PCR reactions. Given a successful completion, these tools will allow us to predict both the sequences and concentrations of all species that are amplified during PCR. The ability to answer the following questions will allow us both to optimize the PCR process and interpret the PCR results: What products are amplified when sequence mixtures are present, containing multiple, closely related targets and multiplexed primers, which may hybridize with sequence mismatches? What are the effects of time, temperature, and DNA concentrations on the concentrations of products? A better understanding of these issues will improve the design and interpretation of PCR reactions. The status of the VPCR project after 1.5 years of funding is consistent with the goals of the overall project which was scoped for 3 years of funding. At half way through the projected timeline of the project we have an early beta version of the VPCR code. We have begun investigating means to improve the robustness of the code, performed preliminary experiments to test the code and begun drafting manuscripts for publication. Although an experimental protocol for testing the code was developed, the preliminary

  20. Thermus thermophilus 16S rRNA is transcribed from an isolated transcription unit.

    PubMed Central

    Hartmann, R K; Erdmann, V A

    1989-01-01

    A cloned 16S rRNA gene from the extreme thermophilic eubacterium Thermus thermophilus HB8 was used to characterize the in vivo expression of the 16S rRNA genes in this organism by nuclease S1 mapping. The gene represents an isolated transcription unit encoding solely 16S rRNA. Under exponential growth conditions, transcription was initiated at a single promoter, which represents the structural equivalent of Escherichia coli rrn P2 promoters. The promoter-leader region was very similar to the E. coli rrn P2 promoter-leader segment that is responsible for antitermination. The T. thermophilus leader region was approximately 85 nucleotides shorter than its E. coli P2 counterpart. Potential processing intermediates were correlated with a proposed secondary structure of T. thermophilus pre-16S rRNA. Images PMID:2722737

  1. Scanning of 16S Ribosomal RNA for Peptide Nucleic Acid Targets.

    PubMed

    Górska, Anna; Markowska-Zagrajek, Agnieszka; Równicki, Marcin; Trylska, Joanna

    2016-08-25

    We have designed a protocol and server to aid in the search for putative binding sites in 16S rRNA that could be targeted by peptide nucleic acid oligomers. Various features of 16S rRNA were considered to score its regions as potential targets for sequence-specific binding that could result in inhibition of ribosome function. Specifically, apart from the functional importance of a particular rRNA region, we calculated its accessibility, flexibility, energetics of strand invasion by an oligomer, as well as similarity to human rRNA. To determine 16S rRNA flexibility in the ribosome context, we performed all-atom molecular dynamics simulations of the 30S subunit in explicit solvent. We proposed a few 16S RNA target sites, and one of them was tested experimentally to verify inhibition of bacterial growth by a peptide nucleic acid oligomer. PMID:27105576

  2. A Comparison between Transcriptome Sequencing and 16S Metagenomics for Detection of Bacterial Pathogens in Wildlife

    PubMed Central

    Razzauti, Maria; Galan, Maxime; Bernard, Maria; Maman, Sarah; Klopp, Christophe; Charbonnel, Nathalie; Vayssier-Taussat, Muriel; Eloit, Marc; Cosson, Jean-François

    2015-01-01

    Background Rodents are major reservoirs of pathogens responsible for numerous zoonotic diseases in humans and livestock. Assessing their microbial diversity at both the individual and population level is crucial for monitoring endemic infections and revealing microbial association patterns within reservoirs. Recently, NGS approaches have been employed to characterize microbial communities of different ecosystems. Yet, their relative efficacy has not been assessed. Here, we compared two NGS approaches, RNA-Sequencing (RNA-Seq) and 16S-metagenomics, assessing their ability to survey neglected zoonotic bacteria in rodent populations. Methodology/Principal Findings We first extracted nucleic acids from the spleens of 190 voles collected in France. RNA extracts were pooled, randomly retro-transcribed, then RNA-Seq was performed using HiSeq. Assembled bacterial sequences were assigned to the closest taxon registered in GenBank. DNA extracts were analyzed via a 16S-metagenomics approach using two sequencers: the 454 GS-FLX and the MiSeq. The V4 region of the gene coding for 16S rRNA was amplified for each sample using barcoded universal primers. Amplicons were multiplexed and processed on the distinct sequencers. The resulting datasets were de-multiplexed, and each read was processed through a pipeline to be taxonomically classified using the Ribosomal Database Project. Altogether, 45 pathogenic bacterial genera were detected. The bacteria identified by RNA-Seq were comparable to those detected by 16S-metagenomics approach processed with MiSeq (16S-MiSeq). In contrast, 21 of these pathogens went unnoticed when the 16S-metagenomics approach was processed via 454-pyrosequencing (16S-454). In addition, the 16S-metagenomics approaches revealed a high level of coinfection in bank voles. Conclusions/Significance We concluded that RNA-Seq and 16S-MiSeq are equally sensitive in detecting bacteria. Although only the 16S-MiSeq method enabled identification of bacteria in each

  3. APPLICATION OF POLYMERASE CHAIN REACTION (PCR) AND PCR BASED RESTRICTION FRAGMENT LENGTH POLYMORPHISM FOR DETECTION AND IDENTIFICATION OF DERMATOPHYTES FROM DERMATOLOGICAL SPECIMENS

    PubMed Central

    Bagyalakshmi, R; Senthilvelan, B; Therese, K L; Murugusundram, S; Madhavan, H N

    2008-01-01

    Objective: To develop and optimize polymerase chain reaction-based restriction fragment length polymorphism (PCR-RFLP) targeting 18S rDNA and internal transcribed spacer (ITS) region of fungi for rapid detection and identification of dermatophytes. Materials and Methods: Two PCR-RFLP methods targeting 18S rDNA and ITS regions of fungi were optimized using standard and laboratory isolates of dermatophytes and other fungi. Sixty-eight dermatological clinical specimens (nail clippings (56), material obtained from blisters (8), hair root (2), scraping from scaly plaque of foot (1) and skin scraping (1) collected by the dermatologist were subjected to both the optimized PCR-RFLP and conventional mycological (smear and culture) methods. Results: PCRs targeting 18S rDNA and the ITS region were sensitive to detect 10 picograms and 1 femtogram of T. rubrum DNA, respectively. PCR targeting 18S rDNA was specific for dermatophytes and subsequent RFLP identified them to species level. PCR-RFLP targeting the ITS region differentiated dermatophytes from other fungi with identification to species level. Among the 68 clinical specimens tested, both PCR-RFLP methods revealed the presence of dermatophytes in 27 cases (39.7%), whereas culture revealed the same only in 2 cases (7.40%), increasing the clinical sensitivity by 32.3%. Among 20 smear positive specimens, both PCR-RFLP methods detected dermatophytes in 12 (17.6%). Both the methods detected the presence of dermatophytes in 13 (19.11%) smear and culture negative specimens, increasing the clinical sensitivity by 36.1%. Conclusion: PCR-RFLP methods targeting 18S rDNA and the ITS regions of fungi were specific and highly sensitive for detection and speciation of dermatophytes. PMID:19967012

  4. 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.

  5. Plant rDNA database: update and new features

    PubMed Central

    Garcia, Sònia; Gálvez, Francisco; Gras, Airy; Kovařík, Aleš; Garnatje, Teresa

    2014-01-01

    The Plant rDNA database (www.plantrdnadatabase.com) is an open access online resource providing detailed information on numbers, structures and positions of 5S and 18S-5.8S-26S (35S) ribosomal DNA loci. The data have been obtained from >600 publications on plant molecular cytogenetics, mostly based on fluorescent in situ hybridization (FISH). This edition of the database contains information on 1609 species derived from 2839 records, which means an expansion of 55.76 and 94.45%, respectively. It holds the data for angiosperms, gymnosperms, bryophytes and pteridophytes available as of June 2013. Information from publications reporting data for a single rDNA (either 5S or 35S alone) and annotation regarding transcriptional activity of 35S loci now appears in the database. Preliminary analyses suggest greater variability in the number of rDNA loci in gymnosperms than in angiosperms. New applications provide ideograms of the species showing the positions of rDNA loci as well as a visual representation of their genome sizes. We have also introduced other features to boost the usability of the Web interface, such as an application for convenient data export and a new section with rDNA–FISH-related information (mostly detailing protocols and reagents). In addition, we upgraded and/or proofread tabs and links and modified the website for a more dynamic appearance. This manuscript provides a synopsis of these changes and developments. Database URL: http://www.plantrdnadatabase.com PMID:24980131

  6. Secondray structure and sequence of ITS2-rDNA of the Egyptian malaria vector Anopheles pharoensis (Theobald).

    PubMed

    Wassim, Nahla M

    2014-04-01

    Out of the twelve Anophelines present in Egypt, only five species known to be malaria vectors. Anopheles (An.) pharoensis proved to be the important vector all over Egypt, especially in the Delta. Anopheles sergenti proved to be the primary vector in the Oases of the Western Desert, An. multicolor in Faiyoum, An. stephensi in the Red Sea Coast, and An. superpictus in Sinai. Genomic DNA was isolated from single adult mosquito of An. pharoensis (Sahel Sudanese form), PCR was performed to amplify ITS2 region of rDNA using specific primers for 5.8S and 28S rDNA genes. The amplicons were purified, directly sequenced and aligned to the sequence of the same region of An. gambiae, using clustalw2. The length of ITS2-rDNA of An. pharoensis was 411bp. The GC content of the ITS2 reported 53% is consistent with spacer base composition in Anopheles species. The similarity between the two species was 52% and genetic distance was 0.46.Variable simple sequence repeats (SSRs) are found at low frequency. The secondary structure of rDNA-ITS2was predicted by MFOLD and was -192; 60 to-195.32 kilocalories/mole. PMID:24961025

  7. First report on the bacterial diversity in the distal gut of dholes (Cuon alpinus) by using 16S rRNA gene sequences analysis.

    PubMed

    Chen, Lei; Zhang, Honghai; Liu, Guangshuai; Sha, Weilai

    2016-05-01

    The aim of this study was to investigate the bacterial community in the distal gut of dholes (Cuon alpinus) based on the analysis of bacterial 16S rRNA gene sequences. Fecal samples were collected from five healthy unrelated dholes captured from Qilian Mountain in Gansu province of China. The diversity of the fecal bacteria community was investigated by constructing a polymerase chain reaction (PCR)-amplified 16S rRNA gene clone library. Bacterial 16S rRNA gene was amplified by using universal bacterial primers 27F and 1492R. A total of 275 chimera-free near full length 16S rRNA gene sequences were collected, and 78 non-redundant bacteria phylotypes (operational taxonomical units, OTUs) were identified according to the 97 % sequence similarity. Forty-two OTUs (53.8 %) showed less than 98 % sequence similarity to 16S rRNA gene sequences reported previously. Phylogenetic analysis demonstrated that dhole bacterial community comprised five different phyla, with the majority of sequences being classified within the phylum Bacteroidetes (64.7 %), followed by Firmicutes (29.8 %), Fusobacteria (4.7 %),Proteobacteria (0.4 %), and Actinobacteria (0.4 %). The only order Bacteroidales in phylum Bacteroidetes was the most abundant bacterial group in the intestinal bacterial community of dholes. Firmicutes and Bacteroidetes were the two most diverse bacterial phyla with 46.2 and 44.9 % of OTUs contained, respectively. Bacteroidales and Clostridiales were the two most diverse bacterial orders that contained 44.9 and 39.7 % of OTUs, respectively. PMID:26423781

  8. Yersinia spp. Identification Using Copy Diversity in the Chromosomal 16S rRNA Gene Sequence

    PubMed Central

    Chen, Yuhuang; Liu, Chang; Xiao, Yuchun; Li, Xu; Su, Mingming; Jing, Huaiqi; Wang, Xin

    2016-01-01

    API 20E strip test, the standard for Enterobacteriaceae identification, is not sufficient to discriminate some Yersinia species for some unstable biochemical reactions and the same biochemical profile presented in some species, e.g. Yersinia ferderiksenii and Yersinia intermedia, which need a variety of molecular biology methods as auxiliaries for identification. The 16S rRNA gene is considered a valuable tool for assigning bacterial strains to species. However, the resolution of the 16S rRNA gene may be insufficient for discrimination because of the high similarity of sequences between some species and heterogeneity within copies at the intra-genomic level. In this study, for each strain we randomly selected five 16S rRNA gene clones from 768 Yersinia strains, and collected 3,840 sequences of the 16S rRNA gene from 10 species, which were divided into 439 patterns. The similarity among the five clones of 16S rRNA gene is over 99% for most strains. Identical sequences were found in strains of different species. A phylogenetic tree was constructed using the five 16S rRNA gene sequences for each strain where the phylogenetic classifications are consistent with biochemical tests; and species that are difficult to identify by biochemical phenotype can be differentiated. Most Yersinia strains form distinct groups within each species. However Yersinia kristensenii, a heterogeneous species, clusters with some Yersinia enterocolitica and Yersinia ferderiksenii/intermedia strains, while not affecting the overall efficiency of this species classification. In conclusion, through analysis derived from integrated information from multiple 16S rRNA gene sequences, the discrimination ability of Yersinia species is improved using our method. PMID:26808495

  9. Yersinia spp. Identification Using Copy Diversity in the Chromosomal 16S rRNA Gene Sequence.

    PubMed

    Hao, Huijing; Liang, Junrong; Duan, Ran; Chen, Yuhuang; Liu, Chang; Xiao, Yuchun; Li, Xu; Su, Mingming; Jing, Huaiqi; Wang, Xin

    2016-01-01

    API 20E strip test, the standard for Enterobacteriaceae identification, is not sufficient to discriminate some Yersinia species for some unstable biochemical reactions and the same biochemical profile presented in some species, e.g. Yersinia ferderiksenii and Yersinia intermedia, which need a variety of molecular biology methods as auxiliaries for identification. The 16S rRNA gene is considered a valuable tool for assigning bacterial strains to species. However, the resolution of the 16S rRNA gene may be insufficient for discrimination because of the high similarity of sequences between some species and heterogeneity within copies at the intra-genomic level. In this study, for each strain we randomly selected five 16S rRNA gene clones from 768 Yersinia strains, and collected 3,840 sequences of the 16S rRNA gene from 10 species, which were divided into 439 patterns. The similarity among the five clones of 16S rRNA gene is over 99% for most strains. Identical sequences were found in strains of different species. A phylogenetic tree was constructed using the five 16S rRNA gene sequences for each strain where the phylogenetic classifications are consistent with biochemical tests; and species that are difficult to identify by biochemical phenotype can be differentiated. Most Yersinia strains form distinct groups within each species. However Yersinia kristensenii, a heterogeneous species, clusters with some Yersinia enterocolitica and Yersinia ferderiksenii/intermedia strains, while not affecting the overall efficiency of this species classification. In conclusion, through analysis derived from integrated information from multiple 16S rRNA gene sequences, the discrimination ability of Yersinia species is improved using our method. PMID:26808495

  10. Mutational robustness of 16S ribosomal RNA, shown by experimental horizontal gene transfer in Escherichia coli

    PubMed Central

    Kitahara, Kei; Yasutake, Yoshiaki; Miyazaki, Kentaro

    2012-01-01

    The bacterial ribosome consists of three rRNA molecules and 57 proteins and plays a crucial role in translating mRNA-encoded information into proteins. Because of the ribosome’s structural and mechanistic complexity, it is believed that each ribosomal component coevolves to maintain its function. Unlike 5S rRNA, 16S and 23S rRNAs appear to lack mutational robustness, because they form the structural core of the ribosome. However, using Escherichia coli Δ7 (null mutant of operons) as a host, we have recently shown that an active hybrid ribosome whose 16S rRNA has been specifically substituted with that from non–E. coli bacteria can be reconstituted in vivo. To investigate the mutational robustness of 16S rRNA and the structural basis for its functionality, we used a metagenomic approach to screen for 16S rRNA genes that complement the growth of E. coli Δ7. Various functional genes were obtained from the Gammaproteobacteria and Betaproteobacteria lineages. Despite the large sequence diversity (80.9–99.0% identity with E. coli 16S rRNA) of the functional 16S rRNA molecules, the doubling times (DTs) of each mutant increased only modestly with decreasing sequence identity (average increase in DT, 4.6 s per mutation). The three-dimensional structure of the 30S ribosome showed that at least 40.7% (628/1,542) of the nucleotides were variable, even at ribosomal protein-binding sites, provided that the secondary structures were properly conserved. Our results clearly demonstrate that 16S rRNA functionality largely depends on the secondary structure but not on the sequence itself. PMID:23112186

  11. Amplification of 16S ribosomal RNA genes of autotrophic ammonia-oxidizing bacteria demonstrates the ubiquity of nitrosospiras in the environment.

    PubMed

    Hiorns, W D; Hastings, R C; Head, I M; McCarthy, A J; Saunders, J R; Pickup, R W; Hall, G H

    1995-11-01

    Oligonucleotide sequences selected from the 16S rRNA genes of various species of ammonia-oxidizing bacteria were evaluated as specific PCR amplification primers and probes. The specificities of primer pairs for eubacterial, Nitrosospira and Nitrosomonas rRNA genes were established with sequence databases, and the primer pairs were used to amplify DNA from laboratory cultures and environmental samples. Eubacterial rRNA genes amplified from samples of soil and activated sludge hybridized with an oligonucleotide probe specific for Nitrosospira spp., but not with a Nitrosomonas-specific probe. Lakewater and sediment samples were analysed using a nested PCR technique in which eubacterial rRNA genes were subjected to a secondary amplification with Nitrosomonas or Nitrosospira specific primers. Again, the presence of Nitrosospira DNA, but not Nitrosomonas DNA, was detected and this was confirmed by hybridization of the amplified DNA with an internal oligonucleotide probe. Enrichments of lakewater and sediment samples, incubated for two weeks in the presence of ammonium, produced nitrite and were found to contain DNA from both Nitrosospira and Nitrosomonas as determined by nested PCR amplification and probing of 16S rRNA genes. This demonstrates that Nitrosospira spp. are widespread in the environment. The implications of the detection of Nitrosomonas DNA only after enrichment culture are discussed. PMID:8535507

  12. The Mycoplasma gallisepticum 16S-23S rRNA intergenic spacer region sequence as a novel tool for epizootiological studies.

    PubMed

    Raviv, Ziv; Callison, S; Ferguson-Noel, N; Laibinis, V; Wooten, R; Kleven, S H

    2007-06-01

    Mycoplasma gallisepticum (MG) contains two sets of rRNA genes (5S, 16S and 23S) in its genome, but only one of the two is organized in an operon cluster and contains a unique 660-nucleotide intergenic spacer region (IGSR) between the 16S and the 23S rRNA genes. We designed a polymerase chain reaction (PCR) for the specific amplification of the complete MG IGSR segment. The MG IGSR PCR was tested on 18 avian mollicute species and was confirmed as MG specific. The reaction sensitivity was demonstrated by comparing it to the well-established MG mgc2 PCR. The MG IGSR sequence was found to be highly variable (discrimination [D] index of 0.950) among a variety of MG laboratory strains, vaccine strains, and field isolates. The sequencing of the MG IGSR appears to be a valuable single-locus sequence typing (SLST) tool for MG isolate differentiation in diagnostic cases and epizootiological studies. PMID:17626483

  13. [Analysis on microbial community in biofilm coating onto solid carbon source using the PCR-DGGE technique].

    PubMed

    Xu, Ying; Qiu, Tian-Leii; Han, Mei-Lin; Li, Jun; Wang, Xu-Ming

    2013-08-01

    Polylactic acid/Poly (3-Hydroxybutyrate-co-3-Hydroxyvalerate) (PLA/PHBV) granules were used as both carbon source and biofilm carrier for nitrate removal from the contaminated water. The polymerase chain reaction-denature gradient gel electrophoresis (PCR-DGGE) was used to investigate the dynamic change of microbial community in the biofilm coating onto PLA/PHBV granules. The results showed that a decrease in microbial diversity was observed at the initial stage of reactor operation. Shannon-Wiener and Simpson diversity indexes of microbial community in the biofilm slightly changed during the stable period of the reactor running. The major microorganisms in the biofilm were Gram-negative rod bacteria including the genera of Diaphorobacter, Acidovorax, Rubrivivax, Azospira, Thermomonas and Devosia, and all of them belonged to alpha-, beta- and gamma- Proteobacteria, according to the SEM photo of biofilm and 16S rDNA sequences of the DGGE profile. Moreover, the abundance of Diaphorobacter-like genera was the highest in the solid-phase-denitrification biofilm. PMID:24191577

  14. Differentiation of Closely Related Carnobacterium Food Isolates Based on 16S-23S Ribosomal DNA Intergenic Spacer Region Polymorphism

    PubMed Central

    Kabadjova, Petia; Dousset, Xavier; Le Cam, Virginie; Prevost, Hervé

    2002-01-01

    A novel strategy for identification of Carnobacterium food isolates based on restriction fragment length polymorphism (RFLP) of PCR-amplified 16S-23S ribosomal intergenic spacer regions (ISRs) was developed. PCR amplification from all Carnobacterium strains studied always yielded three ISR amplicons, which were designated the small ISR (S-ISR), the medium ISR (M-ISR), and the large ISR (L-ISR). The lengths of these ISRs varied from one species to another. Carnobacterium divergens NCDO 2763T and C. mobile DSM 4849T generated one major S-ISR band (ca. 400 bp) and minor M-ISR and L-ISR bands (ca. 500 and ca. 600 bp, respectively). The ISRs amplified from C. gallinarum NCFB 2766T and C. piscicola NCDO 2762T were larger (S-ISR, ca. 600 bp; M-ISR, ca. 700 bp; and L-ISR, ca. 800 bp). The L-ISR contained two tDNAs coding for tRNAIle and tRNAAla genes. The M-ISR included one tRNAAla gene, and the S-ISR did not contain a tDNA gene. The RFLP scheme devised involves estimation of variable PCR product sizes together with HinfI, TaqI, and HindIII restriction analysis. Forty-two isolates yielded four unique band patterns that correctly resolved these isolates into four Carnobacterium species. This method is very suitable for rapid, low-cost identification of a wide variety of Carnobacterium species without sequencing. PMID:12406725

  15. Tax4Fun: predicting functional profiles from metagenomic 16S rRNA data

    PubMed Central

    Aßhauer, Kathrin P.; Wemheuer, Bernd; Daniel, Rolf; Meinicke, Peter

    2015-01-01

    Motivation: The characterization of phylogenetic and functional diversity is a key element in the analysis of microbial communities. Amplicon-based sequencing of marker genes, such as 16S rRNA, is a powerful tool for assessing and comparing the structure of microbial communities at a high phylogenetic resolution. Because 16S rRNA sequencing is more cost-effective than whole metagenome shotgun sequencing, marker gene analysis is frequently used for broad studies that involve a large number of different samples. However, in comparison to shotgun sequencing approaches, insights into the functional capabilities of the community get lost when restricting the analysis to taxonomic assignment of 16S rRNA data. Results: Tax4Fun is a software package that predicts the functional capabilities of microbial communities based on 16S rRNA datasets. We evaluated Tax4Fun on a range of paired metagenome/16S rRNA datasets to assess its performance. Our results indicate that Tax4Fun provides a good approximation to functional profiles obtained from metagenomic shotgun sequencing approaches. Availability and implementation: Tax4Fun is an open-source R package and applicable to output as obtained from the SILVAngs web server or the application of QIIME with a SILVA database extension. Tax4Fun is freely available for download at http://tax4fun.gobics.de/. Contact: kasshau@gwdg.de Supplementary information: Supplementary data are available at Bioinformatics online. PMID:25957349

  16. Accurate taxonomy assignments from 16S rRNA sequences produced by highly parallel pyrosequencers

    PubMed Central

    Liu, Zongzhi; DeSantis, Todd Z.; Andersen, Gary L.; Knight, Rob

    2008-01-01

    The recent introduction of massively parallel pyrosequencers allows rapid, inexpensive analysis of microbial community composition using 16S ribosomal RNA (rRNA) sequences. However, a major challenge is to design a workflow so that taxonomic information can be accurately and rapidly assigned to each read, so that the composition of each community can be linked back to likely ecological roles played by members of each species, genus, family or phylum. Here, we use three large 16S rRNA datasets to test whether taxonomic information based on the full-length sequences can be recaptured by short reads that simulate the pyrosequencer outputs. We find that different taxonomic assignment methods vary radically in their ability to recapture the taxonomic information in full-length 16S rRNA sequences: most methods are sensitive to the region of the 16S rRNA gene that is targeted for sequencing, but many combinations of methods and rRNA regions produce consistent and accurate results. To process large datasets of partial 16S rRNA sequences obtained from surveys of various microbial communities, including those from human body habitats, we recommend the use of Greengenes or RDP classifier with fragments of at least 250 bases, starting from one of the primers R357, R534, R798, F343 or F517. PMID:18723574

  17. Rapid Sanger Sequencing of the 16S rRNA Gene for Identification of Some Common Pathogens

    PubMed Central

    Zhou, Guangbiao; Shi, Xiaojun; Su, Jianhui; Chen, Guanwu; Lin, Kun

    2014-01-01

    Conventional Sanger sequencing remains time-consuming and laborious. In this study, we developed a rapid improved sequencing protocol of 16S rRNA for pathogens identification by using a new combination of SYBR Green I real-time PCR and Sanger sequencing with FTA® cards. To compare the sequencing quality of this method with conventional Sanger sequencing, 12 strains, including three kinds of strains (1 reference strain and 3 clinical strains, which were previously identified by biochemical tests), which have 4 Pseudomonas aeruginosa, 4 Staphyloccocus aureus and 4 Escherichia coli, were targeted. Additionally, to validate the sequencing results and bacteria identification, expanded specimens with 90 clinical strains, also comprised of the three kinds of strains which included 30 samples respectively, were performed as just described. The results showed that although statistical differences (P<0.05) were found in sequencing quality between the two methods, their identification results were all correct and consistent. The workload, the time consumption and the cost per batch were respectively light versus heavy, 8 h versus 11 h and $420 versus $400. In the 90 clinical strains, all of the Pseudomonas aeruginosa and Staphyloccocus aureus strains were correctly identified, but only 26.7% of the Escherichia coli strains were recognized as Escherichia coli, while 33.3% as Shigella sonnei and 40% as Shigella dysenteriae. The protocol described here is a rapid, reliable, stable and convenient method for 16S rRNA sequencing, and can be used for Pseudomonas aeruginosa and Staphyloccocus aureus identification, yet it is not completely suitable for discriminating Escherichia coli and Shigella strains. PMID:24551186

  18. Variation in rDNA locus number and position among legume species and detection of 2 linked rDNA loci in the model Medicago truncatula by FISH.

    PubMed

    Abirached-Darmency, Mona; Prado-Vivant, Emilce; Chelysheva, Liudmila; Pouthier, Thomas

    2005-06-01

    Within Fabaceae, legume species have a variable genome size, chromosome number, and ploidy level. The genome distribution of ribosomal genes, easily detectable by fluorescent in situ hybridization (FISH), is a good tool for anchoring physical and genetic comparative maps. The organisation of 45S rDNA and 5S loci was analysed by FISH in the 4 closely related species: Pisum sativum, Medicago truncatula, Medicago sativa (2 diploid taxa), and Lathyrus sativus. The 2 types of rDNA arrays displayed interspecific variation in locus number and location, but little intraspecific variation was detected. In the model legume, M. truncatula, the presence of 2 adjacent 45S rDNA loci was demonstrated, and the location of the rDNA loci was independent of the general evolution of the genome DNA. The different parameters relative to clustering of the rDNA loci in specific chromosome regions and the possible basis of rDNA instability are discussed. PMID:16121252

  19. Short communication: Evaluation of the microbiota of kefir samples using metagenetic analysis targeting the 16S and 26S ribosomal DNA fragments.

    PubMed

    Korsak, N; Taminiau, B; Leclercq, M; Nezer, C; Crevecoeur, S; Ferauche, C; Detry, E; Delcenserie, V; Daube, G

    2015-06-01

    Milk kefir is produced by fermenting milk in the presence of kefir grains. This beverage has several benefits for human health. The aim of this experiment was to analyze 5 kefir grains (and their products) using a targeted metagenetic approach. Of the 5 kefir grains analyzed, 1 was purchased in a supermarket, 2 were provided by the Ministry of Agriculture (Namur, Belgium), and 2 were provided by individuals. The metagenetic approach targeted the V1-V3 fragment of the 16S ribosomal (r)DNA for the grains and the resulting beverages at 2 levels of grain incorporation (5 and 10%) to identify the bacterial species population. In contrast, the 26S rDNA pyrosequencing was performed only on kefir grains with the aim of assessing the yeast populations. In parallel, pH measurements were performed on the kefir obtained from the kefir grains using 2 incorporation rates. Regarding the bacterial population, 16S pyrosequencing revealed the presence of 20 main bacterial species, with a dominance of the following: Lactobacillus kefiranofaciens, Lactococcus lactis ssp. cremoris, Gluconobacter frateurii, Lactobacillus kefiri, Acetobacter orientalis, and Acetobacter lovaniensis. An important difference was noticed between the kefir samples: kefir grain purchased from a supermarket (sample E) harbored a much higher proportion of several operational taxonomic units of Lactococcus lactis and Leuconostoc mesenteroides. This sample of grain was macroscopically different from the others in terms of size, apparent cohesion of the grains, structure, and texture, probably associated with a lower level of Lactobacillus kefiranofaciens. The kefir (at an incorporation rate of 5%) produced from this sample of grain was characterized by a lower pH value (4.5) than the others. The other 4 samples of kefir (5%) had pH values above 5. Comparing the kefir grain and the kefir, an increase in the population of Gluconobacter in grain sample B was observed. This was also the case for Acetobacter orientalis

  20. Genetic stock structure of walleye pollock (Theragra chalcogramma) inferred by PCR-RFLP analysis of the mitochondrial DNA and SNP analysis of nuclear DNA.

    PubMed

    Yanagimoto, Takashi; Kitamura, Toru; Kobayashi, Takanori

    2012-09-01

    Walleye pollock, Theragra chalocogramma, is one of the most important species in the North Pacific and Bering Sea ecosystems. However genetic population structuring of walleye pollock is uncertain. In the present study, genetic variation of walleye pollock collected in several spawning areas ranging from the Japan Sea to the Gulf of Alaska was investigated by DNA analysis. Three regions of the spacer control region, the ND5 and ND6 region (ND complex), and the ND1 and 16S rRNA region (rDNA complex) were amplified using the polymerase chain reaction (PCR). Restriction fragment length polymorphism (RFLP) was conducted on these PCR products and composite haplotypes were calculated. Furthermore, several nuclear DNA regions (actin, Calmodulin, S7 ribosomal protein, creatin kinase, and SypI gene) were investigated to study the stock structure of walleye pollock. It was considered that Calmodulin gene was one of good genetic marker, therefore we conducted SNP analysis for Calmodulin gene by SnaPshot kits. In RFLP analyses, there were no area-specific fragment patterns in the three regions, control region, ND complex and rDNA complex of mtDNA. However compositions of the fragment patterns for the three digested sets, control region/HinfI, rDNA complex/MspI and ND complex/MspI indicated that there are significant differences between around the Japan (Sado-Funka Bay-Wakkanai-Rausu) and the Bering Sea (Western Bering Sea-Nabarin-Atka I.-Bogoslof I). Furthermore, in the case of haplotype frequency, composition showed also significant genetic difference between two areas. Moreover, in Calmodulin analyses, haplotype compositions were changing from western area to eastern area gradually and the results of AMOVA analysis showed that there are interesting differences between western Pacific, western Bering Sea, and eastern Bering Sea. Judging from these results, it was considered that there are three populations of walleye pollock in the Northern Ocean. However, area

  1. A quantitative PCR assay for the detection of phytoplasmas causing almond brownline, peach yellow leafroll, and pear decline diseases in California

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Phytoplasmas cause several important diseases in California’s almond and fruit tree orchards. A phytoplasma associated with almond brown line (ABL) disease, peach yellow leafroll phytoplasma (PYLR-P) and pear decline phytoplasma (PD-P) are genetically related based on homology in the 16S-23S rDNA sp...

  2. Research Techniques Made Simple: Bacterial 16S Ribosomal RNA Gene Sequencing in Cutaneous Research.

    PubMed

    Jo, Jay-Hyun; Kennedy, Elizabeth A; Kong, Heidi H

    2016-03-01

    Skin serves as a protective barrier and also harbors numerous microorganisms collectively comprising the skin microbiome. As a result of recent advances in sequencing (next-generation sequencing), our understanding of microbial communities on skin has advanced substantially. In particular, the 16S ribosomal RNA gene sequencing technique has played an important role in efforts to identify the global communities of bacteria in healthy individuals and patients with various disorders in multiple topographical regions over the skin surface. Here, we describe basic principles, study design, and a workflow of 16S ribosomal RNA gene sequencing methodology, primarily for investigators who are not familiar with this approach. This article will also discuss some applications and challenges of 16S ribosomal RNA sequencing as well as directions for future development. PMID:26902128

  3. Sequence of the 16S ribosomal RNA from Halobacterium volcanii, an archaebacterium

    NASA Technical Reports Server (NTRS)

    Gupta, R.; Lanter, J. M.; Woese, C. R.

    1983-01-01

    The sequence of the 16S ribosomal RNA (rRNA) from the archaebacterium Halobacterium volcanii has been determined by DNA sequencing methods. The archaebacterial rRNA is similar to its eubacterial counterpart in secondary structure. Although it is closer in sequence to the eubacterial 16S rRNA than to the eukaryotic 16S-like rRNA, the H. volcanii sequence also shows certain points of specific similarity to its eukaryotic counterpart. Since the H. volcanii sequence is closer to both the eubacterial and the eukaryotic sequences than these two are to one another, it follows that the archaebacterial sequence resembles their common ancestral sequence more closely than does either of the other two versions.

  4. Depletion of pre-16S rRNA in starved Escherichia coli cells.

    PubMed

    Cangelosi, G A; Brabant, W H

    1997-07-01

    Specific hybridization assays for intermediates in rRNA synthesis (pre-rRNA) may become useful for monitoring the growth activity of individual microbial species in complex natural systems. This possibility depends upon the assumption that rRNA processing in microbial cells continues after growth and pre-rRNA synthesis cease, resulting in drainage of the pre-rRNA pool. This is not the case in many eukaryotic cells, but less is known about the situation in bacteria. Therefore, we used DNA probes to measure steady-state cellular pre-16S rRNA pools during growth state transitions in Escherichia coli. Pre-16S rRNA became undetectable when cells entered the stationary phase on rich medium and was replenished upon restoration of favorable growth conditions. These fluctuations were of much greater magnitude than concurrent fluctuations in the mature 16S rRNA pool. The extent of pre-16S rRNA depletion depended upon the circumstances limiting growth. It was significantly more pronounced in carbon-energy-starved cells than in nitrogen-starved cells or in cells treated with energy uncouplers. In the presence of the transcriptional inhibitor rifampin, rates of pre-16S rRNA depletion in carbon-energy-starved cells and nitrogen-starved cells were similar, suggesting that the difference between these conditions resides primarily at the level of pre-rRNA synthesis. Chloramphenicol, which inhibits the final steps in rRNA maturation, halted pre-16S rRNA depletion under all conditions. The data show that E. coli cells continue to process pre-rRNA after growth and rrn operon transcription cease, leading to drainage of the pre-rRNA pool. This supports the feasibility of using pre-rRNA-targeted probes to monitor bacterial growth in natural systems, with the caveat that patterns of pre-rRNA depletion vary with the conditions limiting growth. PMID:9226253

  5. 16S rRNA Phylogenetic Investigation of the Candidate Division “Korarchaeota”

    PubMed Central

    Auchtung, Thomas A.; Takacs-Vesbach, Cristina D.; Cavanaugh, Colleen M.

    2006-01-01

    The environmental distribution and phylogeny of “Korarchaeota,” a proposed ancient archaeal division, was investigated by using the 16S rRNA gene framework. Korarchaeota-specific primers were designed based on previously published sequences and used to screen a variety of environments. Korarchaeota 16S rRNA genes were amplified exclusively from high temperature Yellowstone National Park hot springs and a 9°N East Pacific Rise deep-sea hydrothermal vent. Phylogenetic analyses of these and all available sequences suggest that Korarchaeota exhibit a high level of endemicity. PMID:16820509

  6. Phylogenetic diversity in the genus Bacillus as seen by 16S rRNA sequencing studies

    NASA Technical Reports Server (NTRS)

    Rossler, D.; Ludwig, W.; Schleifer, K. H.; Lin, C.; McGill, T. J.; Wisotzkey, J. D.; Jurtshuk, P. Jr; Fox, G. E.

    1991-01-01

    Comparative sequence analysis of 16S ribosomal (r)RNAs or DNAs of Bacillus alvei, B. laterosporus, B. macerans, B. macquariensis, B. polymyxa and B. stearothermophilus revealed the phylogenetic diversity of the genus Bacillus. Based on the presently available data set of 16S rRNA sequences from bacilli and relatives at least four major "Bacillus clusters" can be defined: a "Bacillus subtilis cluster" including B. stearothermophilus, a "B. brevis cluster" including B. laterosporus, a "B. alvei cluster" including B. macerans, B. maquariensis and B. polymyxa and a "B. cycloheptanicus branch".

  7. PCR thermocycler

    DOEpatents

    Benett, William J.; Richards, James B.

    2003-01-01

    A sleeve-type silicon polymerase chain reaction (PCR) chamber or thermocycler having improved thermal performance. The silicon sleeve reaction chamber is improved in thermal performance by etched features therein that reduce thermal mass and increase the surface area of the sleeve for cooling. This improved thermal performance of the thermocycler enables an increase in speed and efficiency of the reaction chamber. The improvement is accomplished by providing grooves in the faces of the sleeve and a series of grooves on the interior surfaces that connect with grooves on the faces of the sleeve. The grooves can be anisotropically etched in the silicon sleeve simultaneously with formation of the chamber.

  8. PCR thermocycler

    DOEpatents

    Benett, William J.; Richards, James B.

    2005-05-17

    A sleeve-type silicon polymerase chain reaction (PCR) chamber or thermocycler having improved thermal performance. The silicon sleeve reaction chamber is improved in thermal performance by etched features therein that reduce thermal mass and increase the surface area of the sleeve for cooling. This improved thermal performance of the thermocycler enables an increase in speed and efficiency of the reaction chamber. The improvement is accomplished by providing grooves in the faces of the sleeve and a series of grooves on the interior surfaces that connect with grooves on the faces of the sleeve. The grooves can be anisotropically etched in the silicon sleeve simultaneously with formation of the chamber.

  9. Impact of Fishmeal Replacement in Diets for Gilthead Sea Bream (Sparus aurata) on the Gastrointestinal Microbiota Determined by Pyrosequencing the 16S rRNA Gene.

    PubMed

    Estruch, G; Collado, M C; Peñaranda, D S; Tomás Vidal, A; Jover Cerdá, M; Pérez Martínez, G; Martinez-Llorens, S

    2015-01-01

    Recent studies have demonstrated the impact of diet on microbiota composition, but the essential need for the optimization of production rates and costs forces farms and aquaculture production to carry out continuous dietary tests. In order to understand the effect of total fishmeal replacement by vegetable-based feed in the sea bream (Sparus aurata), the microbial composition of the stomach, foregut, midgut and hindgut was analysed using high-throughput 16S rDNA sequencing, also considering parameters of growth, survival and nutrient utilisation indices.A total of 91,539 16S rRNA filtered-sequences were analysed, with an average number of 3661.56 taxonomically assigned, high-quality sequences per sample. The dominant phyla throughout the whole gastrointestinal tract were Actinobacteria, Protebacteria and Firmicutes. A lower diversity in the stomach in comparison to the other intestinal sections was observed. The microbial composition of the Recirculating Aquaculture System was totally different to that of the sea bream gastrointestinal tract. Total fishmeal replacement had an important impact on microbial profiles but not on diversity. Streptococcus (p-value: 0.043) and Photobacterium (p-value: 0.025) were highly represented in fish fed with fishmeal and vegetable-meal diets, respectively. In the stomach samples with the vegetable diet, reads of chloroplasts and mitochondria from vegetable dietary ingredients were rather abundant. Principal Coordinate Analysis showed a clear differentiation between diets in the microbiota present in the gut, supporting the presence of specific bacterial consortia associated with the diet.Although differences in growth and nutritive parameters were not observed, a negative effect of the vegetable diet on the survival rate was determined. Further studies are required to shed more light on the relationship between the immune system and sea bream gastrointestinal tract microbiota and should consider the modulation of the microbiota to

  10. Impact of Fishmeal Replacement in Diets for Gilthead Sea Bream (Sparus aurata) on the Gastrointestinal Microbiota Determined by Pyrosequencing the 16S rRNA Gene

    PubMed Central

    Estruch, G.; Collado, M. C.; Peñaranda, D. S.; Tomás Vidal, A.; Jover Cerdá, M.; Pérez Martínez, G.; Martinez-Llorens, S.

    2015-01-01

    Recent studies have demonstrated the impact of diet on microbiota composition, but the essential need for the optimization of production rates and costs forces farms and aquaculture production to carry out continuous dietary tests. In order to understand the effect of total fishmeal replacement by vegetable-based feed in the sea bream (Sparus aurata), the microbial composition of the stomach, foregut, midgut and hindgut was analysed using high-throughput 16S rDNA sequencing, also considering parameters of growth, survival and nutrient utilisation indices.A total of 91,539 16S rRNA filtered-sequences were analysed, with an average number of 3661.56 taxonomically assigned, high-quality sequences per sample. The dominant phyla throughout the whole gastrointestinal tract were Actinobacteria, Protebacteria and Firmicutes. A lower diversity in the stomach in comparison to the other intestinal sections was observed. The microbial composition of the Recirculating Aquaculture System was totally different to that of the sea bream gastrointestinal tract. Total fishmeal replacement had an important impact on microbial profiles but not on diversity. Streptococcus (p-value: 0.043) and Photobacterium (p-value: 0.025) were highly represented in fish fed with fishmeal and vegetable-meal diets, respectively. In the stomach samples with the vegetable diet, reads of chloroplasts and mitochondria from vegetable dietary ingredients were rather abundant. Principal Coordinate Analysis showed a clear differentiation between diets in the microbiota present in the gut, supporting the presence of specific bacterial consortia associated with the diet.Although differences in growth and nutritive parameters were not observed, a negative effect of the vegetable diet on the survival rate was determined. Further studies are required to shed more light on the relationship between the immune system and sea bream gastrointestinal tract microbiota and should consider the modulation of the microbiota to

  11. Repertoire of novel sequence signatures for the detection of Candidatus Liberibacter asiaticus by quantitative real-time PCR

    PubMed Central

    2014-01-01

    Background Huanglongbing (HLB) or citrus greening is a devastating disease of citrus. The gram-negative bacterium Candidatus Liberibacter asiaticus (Las) belonging to the α-proteobacteria is responsible for HLB in North America as well as in Asia. Currently, there is no cure for this disease. Early detection and quarantine of Las-infected trees are important management strategies used to prevent HLB from invading HLB-free citrus producing regions. Quantitative real-time PCR (qRT-PCR) based molecular diagnostic assays have been routinely used in the detection and diagnosis of Las. The oligonucleotide primer pairs based on conserved genes or regions, which include 16S rDNA and the β-operon, have been widely employed in the detection of Las by qRT-PCR. The availability of whole genome sequence of Las now allows the design of primers beyond the conserved regions for the detection of Las explicitly. Results We took a complimentary approach by systematically screening the genes in a genome-wide fashion, to identify the unique signatures that are only present in Las by an exhaustive sequence based similarity search against the nucleotide sequence database. Our search resulted in 34 probable unique signatures. Furthermore, by designing the primer pair specific to the identified signatures, we showed that most of our primer sets are able to detect Las from the infected plant and psyllid materials collected from the USA and China by qRT-PCR. Overall, 18 primer pairs of the 34 are found to be highly specific to Las with no cross reactivity to the closely related species Ca. L. americanus (Lam) and Ca. L. africanus (Laf). Conclusions We have designed qRT-PCR primers based on Las specific genes. Among them, 18 are suitable for the detection of Las from Las-infected plant and psyllid samples. The repertoire of primers that we have developed and characterized in this study enhanced the qRT-PCR based molecular diagnosis of HLB. PMID:24533511

  12. Improved Bacterial 16S rRNA Gene (V4 and V4-5) and Fungal Internal Transcribed Spacer Marker Gene Primers for Microbial Community Surveys

    SciTech Connect

    Walters , William; Hyde, Embriette R.; Berg-Lyons, Donna; Ackermann, Gail; Humphrey, Greg; Parada , Alma; Gilbert, Jack A.; Jansson, Janet K.; Caporaso, Greg; Fuhrman, Jed A.; Apprill, Amy; Knight, Rob

    2015-12-22

    Designing primers for PCR-based taxonomic surveys that amplify a broad range of phylotypes in varied community samples is a difficult challenge, and the comparability of datasets amplified with varied primers requires attention. Here we examine the performance of modified 16S rRNA gene and ITS primers for archaea/bacteria and fungi, respectively, with non-aquatic samples. We moved primer barcodes to the 5’-end, allowing for a range of different 3’ primer pairings, such as the 515f/926r primer pair, which amplifies variable regions 4-5 of the 16S rRNA gene. We additionally demonstrate that modifications to the 515f/806r (variable region 4) 16S primer pair, which improves detection of Thaumarchaeota and SAR11 in marine samples, do not degrade performance on taxa already amplified effectively by the original primer set. Alterations to the fungal ITS primers did result in differential but overall improved performance compared to the original primers. In both cases, the improved primers should be widely adopted for amplicon studies.

  13. Randomly Detected Genetically Modified (GM) Maize (Zea mays L.) near a Transport Route Revealed a Fragile 45S rDNA Phenotype

    PubMed Central

    Waminal, Nomar Espinosa; Ryu, Ki Hyun; Choi, Sun-Hee; Kim, Hyun Hee

    2013-01-01

    Monitoring of genetically modified (GM) crops has been emphasized to prevent their potential effects on the environment and human health. Monitoring of the inadvertent dispersal of transgenic maize in several fields and transport routes in Korea was carried out by qualitative multiplex PCR, and molecular analyses were conducted to identify the events of the collected GM maize. Cytogenetic investigations through fluorescence in situ hybridization (FISH) of the GM maize were performed to check for possible changes in the 45S rDNA cluster because this cluster was reported to be sensitive to replication and transcription stress. Three GM maize kernels were collected from a transport route near Incheon port, Korea, and each was found to contain NK603, stacked MON863 x NK603, and stacked NK603 x MON810 inserts, respectively. Cytogenetic analysis of the GM maize containing the stacked NK603 x MON810 insert revealed two normal compact 5S rDNA signals, but the 45S rDNA showed a fragile phenotype, demonstrating a “beads-on-a-string” fragmentation pattern, which seems to be a consequence of genetic modification. Implications of the 45S rDNA cluster fragility in GM maize are also discussed. PMID:24040165

  14. Effect of gemini (alkanediyl-α,ω-bis(dimethylcetylammonium bromide)) (16-s-16, s = 4, 5, 6) surfactants on the interaction of ninhydrin with chromium-glycylphenylalanine

    NASA Astrophysics Data System (ADS)

    Kumar, Dileep; Rub, Malik Abdul; Akram, Mohd.; Kabir-ud-Din

    2014-11-01

    The effect of gemini (alkanediyl-α,ω-bis(dimethylcetylammonium bromide)) (16-s-16, s = 4, 5, 6) surfactants on the interaction of ninhydrin with chromium(III) complex of glycylphenylalanine ([Cr(III)-Gly-Phe]2+) has been investigated using UV-visible spectrophotometer at different temperatures. The order of reaction with respect to [Cr(III)-Gly-Phe]2+ is unity while it is fractional with respect to ninhydrin. Whereas, the values of rate constant (kψ) increase and leveling-off regions, like conventional single chain cetyltrimethylammonium bromide (CTAB) surfactant, were observed with geminis, later produces a third region of increasing kψ at higher gemini surfactant concentrations. This unusual third-region effect of the gemini micelles is assigned to changes in their micellar morphologies. The results obtained in micellar media were treated in terms of pseudo-phase model. The values of thermodynamic parameters (Ea, ΔH# and ΔS#) and binding constants (KA and KNin) have been evaluated.

  15. Effect of gemini (alkanediyl-α,ω-bis(dimethylcetylammonium bromide)) (16-s-16, s=4, 5, 6) surfactants on the interaction of ninhydrin with chromium-glycylphenylalanine.

    PubMed

    Kumar, Dileep; Rub, Malik Abdul; Akram, Mohd; Kabir-ud-Din

    2014-11-11

    The effect of gemini (alkanediyl-α,ω-bis(dimethylcetylammonium bromide)) (16-s-16, s=4, 5, 6) surfactants on the interaction of ninhydrin with chromium(III) complex of glycylphenylalanine ([Cr(III)-Gly-Phe]2+) has been investigated using UV-visible spectrophotometer at different temperatures. The order of reaction with respect to [Cr(III)-Gly-Phe]2+ is unity while it is fractional with respect to ninhydrin. Whereas, the values of rate constant (kψ) increase and leveling-off regions, like conventional single chain cetyltrimethylammonium bromide (CTAB) surfactant, were observed with geminis, later produces a third region of increasing kψ at higher gemini surfactant concentrations. This unusual third-region effect of the gemini micelles is assigned to changes in their micellar morphologies. The results obtained in micellar media were treated in terms of pseudo-phase model. The values of thermodynamic parameters (Ea, ΔH# and ΔS#) and binding constants (KA and KNin) have been evaluated. PMID:24878435

  16. 16S-23S ribosomal RNA spacer regions of Acetobacter europaeus and A. xylinum, tRNA genes and antitermination sequences.

    PubMed

    Sievers, M; Alonso, L; Gianotti, S; Boesch, C; Teuber, M

    1996-08-15

    The 16S-23S ribosomal RNA spacer regions of Acetobacter europaeus DSM 6160, A. xylinum NCIB 11664 and A. xylinum CL27 were amplified by PCR. Specific PCR products were obtained from each strain and their nucleotide sequences determined. The spacer region of A. europaeus comprises 768 nucleotides (nt), that of A. xylinum 778 nt and that of A. xylinum CL27 759 nt. Genes encoding tRNAIle and tRNAAla were identified. Putative antitermination sequences were found between the tRNAAla sequence and the 5'-terminus of the 23S rRNA coding sequence. The boxA element has the nucleotide sequence TGCTCTTTGATA. Based on hybridization data of digested chromosomal DNA with spacer-specific probes, the copy number of the rrn operons on the chromosome of Acetobacter strains is estimated to be four. PMID:8759788

  17. Conserved Organisation of 45S rDNA Sites and rDNA Gene Copy Number among Major Clades of Early Land Plants.

    PubMed

    Rosato, Marcela; Kovařík, Aleš; Garilleti, Ricardo; Rosselló, Josep A

    2016-01-01

    Genes encoding ribosomal RNA (rDNA) are universal key constituents of eukaryotic genomes, and the nuclear genome harbours hundreds to several thousand copies of each species. Knowledge about the number of rDNA loci and gene copy number provides information for comparative studies of organismal and molecular evolution at various phylogenetic levels. With the exception of seed plants, the range of 45S rDNA locus (encoding 18S, 5.8S and 26S rRNA) and gene copy number variation within key evolutionary plant groups is largely unknown. This is especially true for the three earliest land plant lineages Marchantiophyta (liverworts), Bryophyta (mosses), and Anthocerotophyta (hornworts). In this work, we report the extent of rDNA variation in early land plants, assessing the number of 45S rDNA loci and gene copy number in 106 species and 25 species, respectively, of mosses, liverworts and hornworts. Unexpectedly, the results show a narrow range of ribosomal locus variation (one or two 45S rDNA loci) and gene copies not present in vascular plant lineages, where a wide spectrum is recorded. Mutation analysis of whole genomic reads showed higher (3-fold) intragenomic heterogeneity of Marchantia polymorpha (Marchantiophyta) rDNA compared to Physcomitrella patens (Bryophyta) and two angiosperms (Arabidopsis thaliana and Nicotiana tomentosifomis) suggesting the presence of rDNA pseudogenes in its genome. No association between phylogenetic position, taxonomic adscription and the number of rDNA loci and gene copy number was found. Our results suggest a likely evolutionary rDNA stasis during land colonisation and diversification across 480 myr of bryophyte evolution. We hypothesise that strong selection forces may be acting against ribosomal gene locus amplification. Despite showing a predominant haploid phase and infrequent meiosis, overall rDNA homogeneity is not severely compromised in bryophytes. PMID:27622766

  18. Molecular Diagnosis of Actinomadura madurae Infection by 16S rRNA Deep Sequencing

    PubMed Central

    SenGupta, Dhruba J.; Hoogestraat, Daniel R.; Cummings, Lisa A.; Bryant, Bronwyn H.; Natividad, Catherine; Thielges, Stephanie; Monsaas, Peter W.; Chau, Mimosa; Barbee, Lindley A.; Rosenthal, Christopher; Cookson, Brad T.; Hoffman, Noah G.

    2013-01-01

    Next-generation DNA sequencing can be used to catalog individual organisms within complex, polymicrobial specimens. Here, we utilized deep sequencing of 16S rRNA to implicate Actinomadura madurae as the cause of mycetoma in a diabetic patient when culture and conventional molecular methods were overwhelmed by overgrowth of other organisms. PMID:24108607

  19. Bacterial metabarcoding by 16S rRNA gene ion torrent amplicon sequencing.

    PubMed

    Fantini, Elio; Gianese, Giulio; Giuliano, Giovanni; Fiore, Alessia

    2015-01-01

    Ion Torrent is a next generation sequencing technology based on the detection of hydrogen ions produced during DNA chain elongation; this technology allows analyzing and characterizing genomes, genes, and species. Here, we describe an Ion Torrent procedure applied to the metagenomic analysis of 16S rRNA gene amplicons to study the bacterial diversity in food and environmental samples. PMID:25343859

  20. Ovine pedomics: the first study of the ovine foot 16S rRNA-based microbiome

    Technology Transfer Automated Retrieval System (TEKTRAN)

    We report the first study of the bacterial microbiome of ovine interdigital skin based on 16S rRNA by pyrosequencing and conventional cloning with Sanger-sequencing. Ovine foot rot is an infectious, contagious disease of sheep that causes severe lameness and economic loss from decreased flock produc...

  1. Testing the potential of a ribosomal 16S marker for DNA metabarcoding of insects.

    PubMed

    Elbrecht, Vasco; Taberlet, Pierre; Dejean, Tony; Valentini, Alice; Usseglio-Polatera, Philippe; Beisel, Jean-Nicolas; Coissac, Eric; Boyer, Frederic; Leese, Florian

    2016-01-01

    Cytochrome c oxidase I (COI) is a powerful marker for DNA barcoding of animals, with good taxonomic resolution and a large reference database. However, when used for DNA metabarcoding, estimation of taxa abundances and species detection are limited due to primer bias caused by highly variable primer binding sites across the COI gene. Therefore, we explored the ability of the 16S ribosomal DNA gene as an alternative metabarcoding marker for species level assessments. Ten bulk samples, each containing equal amounts of tissue from 52 freshwater invertebrate taxa, were sequenced with the Illumina NextSeq 500 system. The 16S primers amplified three more insect species than the Folmer COI primers and amplified more equally, probably due to decreased primer bias. Estimation of biomass might be less biased with 16S than with COI, although variation in read abundances of two orders of magnitudes is still observed. According to these results, the marker choice depends on the scientific question. If the goal is to obtain a taxonomic identification at the species level, then COI is more appropriate due to established reference databases and known taxonomic resolution of this marker, knowing that a greater proportion of insects will be missed using COI Folmer primers. If the goal is to obtain a more comprehensive survey the 16S marker, which requires building a local reference database, or optimised degenerated COI primers could be more appropriate. PMID:27114891

  2. Testing the potential of a ribosomal 16S marker for DNA metabarcoding of insects

    PubMed Central

    Elbrecht, Vasco; Taberlet, Pierre; Dejean, Tony; Valentini, Alice; Usseglio-Polatera, Philippe; Beisel, Jean-Nicolas; Coissac, Eric; Boyer, Frederic

    2016-01-01

    Cytochrome c oxidase I (COI) is a powerful marker for DNA barcoding of animals, with good taxonomic resolution and a large reference database. However, when used for DNA metabarcoding, estimation of taxa abundances and species detection are limited due to primer bias caused by highly variable primer binding sites across the COI gene. Therefore, we explored the ability of the 16S ribosomal DNA gene as an alternative metabarcoding marker for species level assessments. Ten bulk samples, each containing equal amounts of tissue from 52 freshwater invertebrate taxa, were sequenced with the Illumina NextSeq 500 system. The 16S primers amplified three more insect species than the Folmer COI primers and amplified more equally, probably due to decreased primer bias. Estimation of biomass might be less biased with 16S than with COI, although variation in read abundances of two orders of magnitudes is still observed. According to these results, the marker choice depends on the scientific question. If the goal is to obtain a taxonomic identification at the species level, then COI is more appropriate due to established reference databases and known taxonomic resolution of this marker, knowing that a greater proportion of insects will be missed using COI Folmer primers. If the goal is to obtain a more comprehensive survey the 16S marker, which requires building a local reference database, or optimised degenerated COI primers could be more appropriate. PMID:27114891

  3. Problem-Based Test: Functional Analysis of Mutant 16S rRNAs

    ERIC Educational Resources Information Center

    Szeberenyi, Jozsef

    2010-01-01

    Terms to be familiar with before you start to solve the test: ribosome, ribosomal subunits, antibiotics, point mutation, 16S, 5S, and 23S rRNA, Shine-Dalgarno sequence, mRNA, tRNA, palindrome, hairpin, restriction endonuclease, fMet-tRNA, peptidyl transferase, initiation, elongation, termination of translation, expression plasmid, transformation,…

  4. 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.

  5. Diversity and distribution of 16S rRNA and phenol monooxygenase genes in the rhizosphere and endophytic bacteria isolated from PAH-contaminated sites

    NASA Astrophysics Data System (ADS)

    Peng, Anping; Liu, Juan; Ling, Wanting; Chen, Zeyou; Gao, Yanzheng

    2015-07-01

    This is the first investigation of the diversity and distribution of 16S rRNA and phenol monooxygenase (PHE) genes in endophytic and rhizosphere bacteria of plants at sites contaminated with different levels of PAHs. Ten PAHs at concentrations from 34.22 to 55.29 and 45.79 to 97.81 mg·kg-1 were measured in rhizosphere soils of Alopecurus aequalis Sobol and Oxalis corniculata L., respectively. The diversity of 16S rRNA and PHE genes in rhizosphere soils or plants changed with varying PAH pollution levels, as shown based on PCR-DGGE data. Generally, higher Shannon-Weiner indexes were found in mild or moderate contaminated areas. A total of 82 different bacterial 16S rRNA gene sequences belonging to five phyla; namely, Acfinobacteria, Proteobacteria, Chloroflexi, Cyanophyta, and Bacteroidetes, were obtained from rhizosphere soils. For the 57 identified PHE gene sequences, 18 were excised from rhizosphere bacteria and 39 from endophytic bacteria. The copy numbers of 16S rRNA and PHE genes in rhizosphere and endophytic bacteria varied from 3.83 × 103 to 2.28 × 106 and 4.17 × 102 to 1.99 × 105, respectively. The copy numbers of PHE genes in rhizosphere bacteria were significantly higher than in endophytic bacteria. Results increase our understanding of the diversity of rhizosphere and endophytic bacteria from plants grown in PAH-contaminated sites.

  6. Diversity and distribution of 16S rRNA and phenol monooxygenase genes in the rhizosphere and endophytic bacteria isolated from PAH-contaminated sites

    PubMed Central

    Peng, Anping; Liu, Juan; Ling, Wanting; Chen, Zeyou; Gao, Yanzheng

    2015-01-01

    This is the first investigation of the diversity and distribution of 16S rRNA and phenol monooxygenase (PHE) genes in endophytic and rhizosphere bacteria of plants at sites contaminated with different levels of PAHs. Ten PAHs at concentrations from 34.22 to 55.29 and 45.79 to 97.81 mg·kg−1 were measured in rhizosphere soils of Alopecurus aequalis Sobol and Oxalis corniculata L., respectively. The diversity of 16S rRNA and PHE genes in rhizosphere soils or plants changed with varying PAH pollution levels, as shown based on PCR-DGGE data. Generally, higher Shannon-Weiner indexes were found in mild or moderate contaminated areas. A total of 82 different bacterial 16S rRNA gene sequences belonging to five phyla; namely, Acfinobacteria, Proteobacteria, Chloroflexi, Cyanophyta, and Bacteroidetes, were obtained from rhizosphere soils. For the 57 identified PHE gene sequences, 18 were excised from rhizosphere bacteria and 39 from endophytic bacteria. The copy numbers of 16S rRNA and PHE genes in rhizosphere and endophytic bacteria varied from 3.83 × 103 to 2.28 × 106 and 4.17 × 102 to 1.99 × 105, respectively. The copy numbers of PHE genes in rhizosphere bacteria were significantly higher than in endophytic bacteria. Results increase our understanding of the diversity of rhizosphere and endophytic bacteria from plants grown in PAH-contaminated sites. PMID:26184609

  7. Diversity and distribution of 16S rRNA and phenol monooxygenase genes in the rhizosphere and endophytic bacteria isolated from PAH-contaminated sites.

    PubMed

    Peng, Anping; Liu, Juan; Ling, Wanting; Chen, Zeyou; Gao, Yanzheng

    2015-01-01

    This is the first investigation of the diversity and distribution of 16S rRNA and phenol monooxygenase (PHE) genes in endophytic and rhizosphere bacteria of plants at sites contaminated with different levels of PAHs. Ten PAHs at concentrations from 34.22 to 55.29 and 45.79 to 97.81 mg·kg(-1) were measured in rhizosphere soils of Alopecurus aequalis Sobol and Oxalis corniculata L., respectively. The diversity of 16S rRNA and PHE genes in rhizosphere soils or plants changed with varying PAH pollution levels, as shown based on PCR-DGGE data. Generally, higher Shannon-Weiner indexes were found in mild or moderate contaminated areas. A total of 82 different bacterial 16S rRNA gene sequences belonging to five phyla; namely, Acfinobacteria, Proteobacteria, Chloroflexi, Cyanophyta, and Bacteroidetes, were obtained from rhizosphere soils. For the 57 identified PHE gene sequences, 18 were excised from rhizosphere bacteria and 39 from endophytic bacteria. The copy numbers of 16S rRNA and PHE genes in rhizosphere and endophytic bacteria varied from 3.83 × 10(3) to 2.28 × 10(6) and 4.17 × 10(2) to 1.99 × 10(5), respectively. The copy numbers of PHE genes in rhizosphere bacteria were significantly higher than in endophytic bacteria. Results increase our understanding of the diversity of rhizosphere and endophytic bacteria from plants grown in PAH-contaminated sites. PMID:26184609

  8. Microbial Community Composition and Diversity via 16S rRNA Gene Amplicons: Evaluating the Illumina Platform

    PubMed Central

    Sinclair, Lucas; Osman, Omneya Ahmed; Bertilsson, Stefan; Eiler, Alexander

    2015-01-01

    As new sequencing technologies become cheaper and older ones disappear, laboratories switch vendors and platforms. Validating the new setups is a crucial part of conducting rigorous scientific research. Here we report on the reliability and biases of performing bacterial 16S rRNA gene amplicon paired-end sequencing on the MiSeq Illumina platform. We designed a protocol using 50 barcode pairs to run samples in parallel and coded a pipeline to process the data. Sequencing the same sediment sample in 248 replicates as well as 70 samples from alkaline soda lakes, we evaluated the performance of the method with regards to estimates of alpha and beta diversity. Using different purification and DNA quantification procedures we always found up to 5-fold differences in the yield of sequences between individually barcodes samples. Using either a one-step or a two-step PCR preparation resulted in significantly different estimates in both alpha and beta diversity. Comparing with a previous method based on 454 pyrosequencing, we found that our Illumina protocol performed in a similar manner – with the exception for evenness estimates where correspondence between the methods was low. We further quantified the data loss at every processing step eventually accumulating to 50% of the raw reads. When evaluating different OTU clustering methods, we observed a stark contrast between the results of QIIME with default settings and the more recent UPARSE algorithm when it comes to the number of OTUs generated. Still, overall trends in alpha and beta diversity corresponded highly using both clustering methods. Our procedure performed well considering the precisions of alpha and beta diversity estimates, with insignificant effects of individual barcodes. Comparative analyses suggest that 454 and Illumina sequence data can be combined if the same PCR protocol and bioinformatic workflows are used for describing patterns in richness, beta-diversity and taxonomic composition. PMID:25647581

  9. Development of a universal microarray based on the ligation detection reaction and 16S rrna gene polymorphism to target diversity of cyanobacteria.

    PubMed

    Castiglioni, Bianca; Rizzi, Ermanno; Frosini, Andrea; Sivonen, Kaarina; Rajaniemi, Pirjo; Rantala, Anne; Mugnai, Maria Angela; Ventura, Stefano; Wilmotte, Annick; Boutte, Christophe; Grubisic, Stana; Balthasart, Pierre; Consolandi, Clarissa; Bordoni, Roberta; Mezzelani, Alessandra; Battaglia, Cristina; De Bellis, Gianluca

    2004-12-01

    The cyanobacteria are photosynthetic prokaryotes of significant ecological and biotechnological interest, since they strongly contribute to primary production and are a rich source of bioactive compounds. In eutrophic fresh and brackish waters, their mass occurrences (water blooms) are often toxic and constitute a high potential risk for human health. Therefore, rapid and reliable identification of cyanobacterial species in complex environmental samples is important. Here we describe the development and validation of a microarray for the identification of cyanobacteria in aquatic environments. Our approach is based on the use of a ligation detection reaction coupled to a universal array. Probes were designed for detecting 19 cyanobacterial groups including Anabaena/Aphanizomenon, Calothrix, Cylindrospermopsis, Cylindrospermum, Gloeothece, halotolerants, Leptolyngbya, Palau Lyngbya, Microcystis, Nodularia, Nostoc, Planktothrix, Antarctic Phormidium, Prochlorococcus, Spirulina, Synechococcus, Synechocystis, Trichodesmium, and Woronichinia. These groups were identified based on an alignment of over 300 cyanobacterial 16S rRNA sequences. For validation of the microarrays, 95 samples (24 axenic strains from culture collections, 27 isolated strains, and 44 cloned fragments recovered from environmental samples) were tested. The results demonstrated a high discriminative power and sensitivity to 1 fmol of the PCR-amplified 16S rRNA gene. Accurate identification of target strains was also achieved with unbalanced mixes of PCR amplicons from different cyanobacteria and an environmental sample. Our universal array method shows great potential for rapid and reliable identification of cyanobacteria. It can be easily adapted to future development and could thus be applied both in research and environmental monitoring. PMID:15574913

  10. Rapid Origin Determination of the Northern Mauxia Shrimp (Acetes chinensis) Based on Allele Specific Polymerase Chain Reaction of Partial Mitochondrial 16S rRNA Gene

    PubMed Central

    Kang, Jung-Ha; Noh, Eun-Soo; Park, Jung-Youn; An, Chel-Min; Choi, Jung-Hwa; Kim, Jin-Koo

    2015-01-01

    Acetes chinensis is an economically important shrimp that belongs to the Sergestidae family; following fermentation, A. chinensis′ economic value, however, is low in China, and much of the catch in China is exported to Korea at a low price, thus leading to potential false labeling. For this reason, we developed a simple method to identify A. chinensis′ origin using allele-specific polymerase chain reaction (PCR). Ten single nucleotide polymorphisms (SNPs) were identified from partial (i.e., 570 bp) DNA sequence analysis of the mitochondrial 16s rRNA gene in 96 Korean and 96 Chinese individual shrimp. Among 10 SNP sites, four sites were observed in populations from both countries, and two sites located in the middle with SNP sites at their 3′-ends were used to design allele-specific primers. Among the eight internal primers, the C220F primer specific to the Chinese A. chinensis population amplified a DNA fragment of 364 bp only from that population. We were able to identify the A. chinensis population origin with 100% accuracy using multiplex PCR performed with two external primers and C220F primers. These results show that the 16S rRNA gene that is generally used for the identification of species can be used for the identification of the origin within species of A. chinensis, which is an important finding for the fair trade of the species between Korea and China. PMID:25656197

  11. Rapid Origin Determination of the Northern Mauxia Shrimp (Acetes chinensis) Based on Allele Specific Polymerase Chain Reaction of Partial Mitochondrial 16S rRNA Gene.

    PubMed

    Kang, Jung-Ha; Noh, Eun-Soo; Park, Jung-Youn; An, Chel-Min; Choi, Jung-Hwa; Kim, Jin-Koo

    2015-04-01

    Acetes chinensis is an economically important shrimp that belongs to the Sergestidae family; following fermentation, A. chinensis' economic value, however, is low in China, and much of the catch in China is exported to Korea at a low price, thus leading to potential false labeling. For this reason, we developed a simple method to identify A. chinensis' origin using allele-specific polymerase chain reaction (PCR). Ten single nucleotide polymorphisms (SNPs) were identified from partial (i.e., 570 bp) DNA sequence analysis of the mitochondrial 16s rRNA gene in 96 Korean and 96 Chinese individual shrimp. Among 10 SNP sites, four sites were observed in populations from both countries, and two sites located in the middle with SNP sites at their 3'-ends were used to design allele-specific primers. Among the eight internal primers, the C220F primer specific to the Chinese A. chinensis population amplified a DNA fragment of 364 bp only from that population. We were able to identify the A. chinensis population origin with 100% accuracy using multiplex PCR performed with two external primers and C220F primers. These results show that the 16S rRNA gene that is generally used for the identification of species can be used for the identification of the origin within species of A. chinensis, which is an important finding for the fair trade of the species between Korea and China. PMID:25656197

  12. Molecular Identification of Mycobacterium Species of Public Health Importance in Cattle in Zimbabwe by 16S rRNA Gene Sequencing

    PubMed Central

    Padya, Leah; Chin'ombe, Nyasha; Magwenzi, Marcelyn; Mbanga, Joshua; Ruhanya, Vurayai; Nziramasanga, Pasipanodya

    2015-01-01

    Mycobacterium species are naturally found in the environment as well as in domestic animals such as cattle. So far, more than 150 species of Mycobacterium, some of which are pathogenic, have been identified. Laboratory isolation, detection and identification of Mycobacterium species are therefore critical if human and animal infections are to be controlled. The objective of this study was to identify Mycobacterium species isolated in cattle in Zimbabwe using 16S ribosomal RNA gene amplification and sequencing. A total of 134 cow dung samples were collected throughout Zimbabwe and mycobacteria were isolated by culture. Only 49 culture isolates that were found to be acid-fast bacilli positive by Ziehl-Neelsen staining. The 16S rRNA gene was successfully amplified by PCR in 41 (84%) of the samples. There was no amplification in 8 (16%) of the samples. Out of the 41 samples that showed amplification, 26 (63%) had strong PCR bands and were selected for DNA sequencing. Analysis of the DNA sequences showed that 7 (27%) belonged to Mycobacterium neoaurum, 6 (23%) belonged to Mycobacterium fortuitum, 3 (12%) to Mycobacterium goodii, 2 (1%) to Mycobacterium arupense, 2 (1%) to Mycobacterium peregrinum or M. septicum and 1 isolate (0.04%) to Mycobacterium elephantis. There were 5 (19%) isolates that were non-mycobacteria and identified as Gordonia terrae, a close relative of Mycobacterium. The study therefore provided a molecular basis for detection and identification of Mycobacterium species in animals and humans. PMID:26668660

  13. Persistent spread of the rmtB 16S rRNA methyltransferase gene among Escherichia coli isolates from diseased food-producing animals in China.

    PubMed

    Xia, Jing; Sun, Jian; Cheng, Ke; Li, Liang; Fang, Liang-Xing; Zou, Meng-Ting; Liao, Xiao-Ping; Liu, Ya-Hong

    2016-05-30

    A total of 963 non-duplicate Escherichia coli strains isolated from food-producing animals between 2002 and 2012 were screened for the presence of the 16S rRNA methyltransferase genes. Among the positive isolates, resistance determinants to extended spectrum β-lactamases, plasmid-mediated quinolone resistance genes as well as floR and fosA/A3/C2 were detected using PCR analysis. These isolates were further subjected to antimicrobial susceptibility testing, molecular typing, PCR-based plasmid replicon typing and plasmid analysis. Of the 963 E. coli isolates, 173 (18.0%), 3 (0.3%) and 2 (0.2%) were rmtB-, armA- and rmtE-positive strains, respectively. All the 16S rRNA methyltransferase gene-positive isolates were multidrug resistant and over 90% of them carried one or more type of resistance gene. IncF (especially IncFII) and non-typeable plasmids played the main role in the dissemination of rmtB, followed by the IncN plasmids. Plasmids that harbored rmtB ranged in size from 20kb to 340kb EcoRI-RFLP testing of the 109 rmtB-positive plasmids from different years and different origins suggested that horizontal (among diverse animals) and vertical transfer of IncF, non-typeable and IncN-type plasmids were responsible for the spread of rmtB gene. In summary, our findings highlight that rmtB was the most prevalent 16S rRNA methyltransferase gene, which present persistent spread in food-producing animals in China and a diverse group of plasmids was responsible for rmtB dissemination. PMID:27139028

  14. PCR-BASED IDENTIFICATION OF BACTERIA IN ENDODONTIC INFECTIONS

    Technology Transfer Automated Retrieval System (TEKTRAN)

    PCR primers, that target the bacterial 16S rRNA genes (or the tuf gene for Enterococcus genus), were used to identify 10 putative bacterial pathogens in root canals with necrotic pulp. In addition, the associations of these microorganisms with symptoms and a history of diabetes mellitus were invest...

  15. PCR Primers for Metazoan Nuclear 18S and 28S Ribosomal DNA Sequences

    PubMed Central

    Machida, Ryuji J.; Knowlton, Nancy

    2012-01-01

    Background Metagenetic analyses, which amplify and sequence target marker DNA regions from environmental samples, are increasingly employed to assess the biodiversity of communities of small organisms. Using this approach, our understanding of microbial diversity has expanded greatly. In contrast, only a few studies using this approach to characterize metazoan diversity have been reported, despite the fact that many metazoan species are small and difficult to identify or are undescribed. One of the reasons for this discrepancy is the availability of universal primers for the target taxa. In microbial studies, analysis of the 16S ribosomal DNA is standard. In contrast, the best gene for metazoan metagenetics is less clear. In the present study, we have designed primers that amplify the nuclear 18S and 28S ribosomal DNA sequences of most metazoan species with the goal of providing effective approaches for metagenetic analyses of metazoan diversity in environmental samples, with a particular emphasis on marine biodiversity. Methodology/Principal Findings Conserved regions suitable for designing PCR primers were identified using 14,503 and 1,072 metazoan sequences of the nuclear 18S and 28S rDNA regions, respectively. The sequence similarity of both these newly designed and the previously reported primers to the target regions of these primers were compared for each phylum to determine the expected amplification efficacy. The nucleotide diversity of the flanking regions of the primers was also estimated for genera or higher taxonomic groups of 11 phyla to determine the variable regions within the genes. Conclusions/Significance The identified nuclear ribosomal DNA primers (five primer pairs for 18S and eleven for 28S) and the results of the nucleotide diversity analyses provide options for primer combinations for metazoan metagenetic analyses. Additionally, advantages and disadvantages of not only the 18S and 28S ribosomal DNA, but also other marker regions as targets

  16. A renaissance for the pioneering 16S rRNA gene

    SciTech Connect

    Tringe, Susannah; Hugenholtz, Philip

    2008-09-07

    Culture-independent molecular surveys using the 16S rRNA gene have become a mainstay for characterizing microbial community structure over the last quarter century. More recently this approach has been overshadowed by metagenomics, which provides a global overview of a community's functional potential rather than just an inventory of its inhabitants. However, the pioneering 16S rRNA gene is making a comeback in its own right thanks to a number of methodological advancements including higher resolution (more sequences), analysis of multiple related samples (e.g. spatial and temporal series) and improved metadata and use of metadata. The standard conclusion that microbial ecosystems are remarkably complex and diverse is now being replaced by detailed insights into microbial ecology and evolution based only on this one historically important marker gene.

  17. An intron within the 16S ribosomal RNA gene of the archaeon Pyrobaculum aerophilum

    NASA Technical Reports Server (NTRS)

    Burggraf, S.; Larsen, N.; Woese, C. R.; Stetter, K. O.

    1993-01-01

    The 16S rRNA genes of Pyrobaculum aerophilum and Pyrobaculum islandicum were amplified by the polymerase chain reaction, and the resulting products were sequenced directly. The two organisms are closely related by this measure (over 98% similar). However, they differ in that the (lone) 16S rRNA gene of Pyrobaculum aerophilum contains a 713-bp intron not seen in the corresponding gene of Pyrobaculum islandicum. To our knowledge, this is the only intron so far reported in the small subunit rRNA gene of a prokaryote. Upon excision the intron is circularized. A secondary structure model of the intron-containing rRNA suggests a splicing mechanism of the same type as that invoked for the tRNA introns of the Archaea and Eucarya and 23S rRNAs of the Archaea. The intron contains an open reading frame whose protein translation shows no certain homology with any known protein sequence.

  18. Rapid identification of Renibacterium salmoninarum using an oligonucleotide probe complementary to 16S rRNA.

    PubMed

    Mattsson, J G; Gersdorf, H; Jansson, E; Hongslo, T; Göbel, U B; Johansson, K E

    1993-02-01

    Bacterial kidney disease in salmonid fish is caused by the slow-growing Gram-positive rod, Renibacterium salmoninarum. The partial sequence of 16S rRNA from R. salmoninarum was determined and compared with published bacterial 16S rRNA sequences. From this sequence information, a 30-bases-long oligonucleotide was designed and used as a specific probe for identification of R. salmoninarum in filter hybridization experiments. Strong specific hybridization signals were observed for all strains of R. salmoninarum tested. Furthermore, no cross-hybridization could be seen against 22 other bacterial species, among them other salmonid fish pathogens. The detection limit for the probe in direct filter hybridization by the dot-blot technique was 2.5 x 10(4) bacteria. It was also possible to detect R. salmoninarum in clinical samples by direct filter hybridization. PMID:8455640

  19. Structural Analysis of Base Substitutions in Thermus thermophilus 16S rRNA Conferring Streptomycin Resistance

    PubMed Central

    Demirci, Hasan; Murphy, Frank V.; Murphy, Eileen L.; Connetti, Jacqueline L.; Dahlberg, Albert E.; Jogl, Gerwald

    2014-01-01

    Streptomycin is a bactericidal antibiotic that induces translational errors. It binds to the 30S ribosomal subunit, interacting with ribosomal protein S12 and with 16S rRNA through contacts with the phosphodiester backbone. To explore the structural basis for streptomycin resistance, we determined the X-ray crystal structures of 30S ribosomal subunits from six streptomycin-resistant mutants of Thermus thermophilus both in the apo form and in complex with streptomycin. Base substitutions at highly conserved residues in the central pseudoknot of 16S rRNA produce novel hydrogen-bonding and base-stacking interactions. These rearrangements in secondary structure produce only minor adjustments in the three-dimensional fold of the pseudoknot. These results illustrate how antibiotic resistance can occur as a result of small changes in binding site conformation. PMID:24820088

  20. The complete nucleotide sequence of a 16S ribosomal RNA gene from a blue-green alga, Anacystis nidulans.

    PubMed

    Tomioka, N; Sugiura, M

    1983-01-01

    The complete nucleotide sequence of a 16S ribosomal RNA gene from a blue-green alga, Anacystis nidulans, has been determined. Its coding region is estimated to be 1,487 base pairs long, which is nearly identical to those reported for chloroplast 16S rRNA genes and is about 4% shorter than that of the Escherichia coli gene. The 16S rRNA sequence of A. nidulans has 83% homology with that of tobacco chloroplast and 74% homology with that of E. coli. Possible stem and loop structures of A. nidulans 16S rRNA sequences resemble more closely those of chloroplast 16S rRNAs than those of E. coli 16S rRNA. These observations support the endosymbiotic theory of chloroplast origin. PMID:6412038

  1. Distinct genetic lineages of Bactrocera caudata (Insecta: Tephritidae) revealed by COI and 16S DNA sequences.

    PubMed

    Lim, Phaik-Eem; Tan, Ji; Suana, I Wayan; Eamsobhana, Praphathip; Yong, Hoi Sen

    2012-01-01

    The fruit fly Bactrocera caudata is a pest species of economic importance in Asia. Its larvae feed on the flowers of Cucurbitaceae such as Cucurbita moschata. To-date it is distinguished from related species based on morphological characters. Specimens of B. caudata from Peninsular Malaysia and Indonesia (Bali and Lombok) were analysed using the partial DNA sequences of cytochrome c oxidase subunit I (COI) and 16S rRNA genes. Both gene sequences revealed that B. caudata from Peninsular Malaysia was distinctly different from B. caudata of Bali and Lombok, without common haplotype between them. Phylogenetic analysis revealed two distinct clades, indicating distinct genetic lineage. The uncorrected 'p' distance for COI sequences between B. caudata of Malaysia-Thailand-China and B. caudata of Bali-Lombok was 5.65%, for 16S sequences from 2.76 to 2.99%, and for combined COI and 16S sequences 4.45 to 4.46%. The 'p' values are distinctly different from intraspecific 'p' distance (0-0.23%). Both the B. caudata lineages are distinctly separated from related species in the subgenus Zeugodacus - B. ascita, B. scutellata, B. ishigakiensis, B. diaphora, B. tau, B. cucurbitae, and B. depressa. Molecular phylogenetic analysis indicates that the B. caudata lineages are closely related to B. ascita sp. B, and form a clade with B. scutellata, B. ishigakiensis, B. diaphora and B. ascita sp. A. This study provides additional baseline for the phylogenetic relationships of Bactrocera fruit flies of the subgenus Zeugodacus. Both the COI and 16S genes could be useful markers for the molecular differentiation and phylogenetic analysis of tephritid fruit flies. PMID:22615962

  2. Greengenes: Chimera-checked 16S rRNA gene database and workbenchcompatible in ARB

    SciTech Connect

    DeSantis, T.Z.; Hugenholtz, P.; Larsen, N.; Rojas, M.; Brodie,E.L; Keller, K.; Huber, T.; Dalevi, D.; Hu, P.; Andersen, G.L.

    2006-02-01

    A 16S rRNA gene database (http://greengenes.lbl.gov) addresses limitations of public repositories by providing chimera-screening, standard alignments and taxonomic classification using multiple published taxonomies. It was revealed that incongruent taxonomic nomenclature exists among curators even at the phylum-level. Putative chimeras were identified in 3% of environmental sequences and 0.2% of records derived from isolates. Environmental sequences were classified into 100 phylum-level lineages within the Archaea and Bacteria.

  3. Characterization of the genus Bifidobacterium by automated ribotyping and 16S rRNA gene sequences.

    PubMed

    Sakata, Shinji; Ryu, Chun Sun; Kitahara, Maki; Sakamoto, Mitsuo; Hayashi, Hidenori; Fukuyama, Masafumi; Benno, Yoshimi

    2006-01-01

    In order to characterize the genus Bifidobacterium, ribopatterns and approximately 500 bp (Escherichia coli positions 27 to 520) of 16S rRNA gene sequences of 28 type strains and 64 reference strains of the genus Bifidobacterium were determined. Ribopatterns obtained from Bifidobacterium strains were divided into nine clusters (clusters I-IX) with a similarity of 60%. Cluster V, containing 17 species, was further subdivided into 22 subclusters with a similarity of 90%. In the genus Bifidobacterium, four groups were shown according to Miyake et al.: (i) the Bifidobacterium longum infantis-longum-suis type group, (ii) the B. catenulatum-pseudocatenulatum group, (iii) the B. gallinarum-saeculare-pullorum group, and (iv) the B. coryneforme-indicum group, which showed higher than 97% similarity of the 16S rRNA gene sequences in each group. Using ribotyping analysis, unique ribopatterns were obtained from these species, and they could be separated by cluster analysis. Ribopatterns of six B. adolescentis strains were separated into different clusters, and also showed diversity in 16S rRNA gene sequences. B. adolescentis consisted of heterogeneous strains. The nine strains of B. pseudolongum subsp. pseudolongum were divided into five subclusters. Each type strain of B. pseudolongum subsp. pseudolongum and B. pseudolongum subsp. globosum and two intermediate groups, which were suggested by Yaeshima et al., consisted of individual clusters. B. animalis subsp. animalis and B. animalis subsp. lactis could not be separated by ribotyping using Eco RI. We conclude that ribotyping is able to provide another characteristic of Bifidobacterium strains in addition to 16S rRNA gene sequence phylogenetic analysis, and this information suggests that ribotyping analysis is a useful tool for the characterization of Bifidobacterium species in combination with other techniques for taxonomic characterization. PMID:16428867

  4. Distinct Genetic Lineages of Bactrocera caudata (Insecta: Tephritidae) Revealed by COI and 16S DNA Sequences

    PubMed Central

    Lim, Phaik-Eem; Tan, Ji; Suana, I. Wayan; Eamsobhana, Praphathip; Yong, Hoi Sen

    2012-01-01

    The fruit fly Bactrocera caudata is a pest species of economic importance in Asia. Its larvae feed on the flowers of Cucurbitaceae such as Cucurbita moschata. To-date it is distinguished from related species based on morphological characters. Specimens of B. caudata from Peninsular Malaysia and Indonesia (Bali and Lombok) were analysed using the partial DNA sequences of cytochrome c oxidase subunit I (COI) and 16S rRNA genes. Both gene sequences revealed that B. caudata from Peninsular Malaysia was distinctly different from B. caudata of Bali and Lombok, without common haplotype between them. Phylogenetic analysis revealed two distinct clades, indicating distinct genetic lineage. The uncorrected ‘p’ distance for COI sequences between B. caudata of Malaysia-Thailand-China and B. caudata of Bali-Lombok was 5.65%, for 16S sequences from 2.76 to 2.99%, and for combined COI and 16S sequences 4.45 to 4.46%. The ‘p’ values are distinctly different from intraspecific ‘p’ distance (0–0.23%). Both the B. caudata lineages are distinctly separated from related species in the subgenus Zeugodacus – B. ascita, B. scutellata, B. ishigakiensis, B. diaphora, B. tau, B. cucurbitae, and B. depressa. Molecular phylogenetic analysis indicates that the B. caudata lineages are closely related to B. ascita sp. B, and form a clade with B. scutellata, B. ishigakiensis, B. diaphora and B. ascita sp. A. This study provides additional baseline for the phylogenetic relationships of Bactrocera fruit flies of the subgenus Zeugodacus. Both the COI and 16S genes could be useful markers for the molecular differentiation and phylogenetic analysis of tephritid fruit flies. PMID:22615962

  5. Use of mitochondrial RNA genes for the differentiation of four Trichinella species by multiplex PCR amplification.

    PubMed

    Blaga, R; Fu, BaoQuan; Le Rhun, D; Le Naour, E; Heckman, A; Zocevic, A; Liu, MingYuan; Boireau, P

    2009-06-01

    Until now, four species of the Trichinella genus have been identified in Europe: Trichinella spiralis, T. nativa, T. britovi and T. pseudospiralis. The aim of this work was to establish a sound polymerase chain reaction (PCR)-based method to differentiate these four species using mitochondrial rDNA as a reliable genetic marker and to evaluate the sensitivity of this method. Full-length DNA sequences coding for the small and large mitochondrial rRNA (mt-rrnS and mt-rrnL) of the four species are described. A multiplex PCR was designed and successfully tested on 24 European isolates. As few as one larva, or 100 pg of genomic DNA was detected, providing equivalent sensitivity to previously described PCR methods. The PCR-based method of mitochondrial rDNA amplification was thereby established as a sensitive and reproductive diagnostic method for the four European Trichinella species. PMID:19389269

  6. Intrinsic challenges in ancient microbiome reconstruction using 16S rRNA gene amplification

    PubMed Central

    Ziesemer, Kirsten A.; Mann, Allison E.; Sankaranarayanan, Krithivasan; Schroeder, Hannes; Ozga, Andrew T.; Brandt, Bernd W.; Zaura, Egija; Waters-Rist, Andrea; Hoogland, Menno; Salazar-García, Domingo C.; Aldenderfer, Mark; Speller, Camilla; Hendy, Jessica; Weston, Darlene A.; MacDonald, Sandy J.; Thomas, Gavin H.; Collins, Matthew J.; Lewis, Cecil M.; Hofman, Corinne; Warinner, Christina

    2015-01-01

    To date, characterization of ancient oral (dental calculus) and gut (coprolite) microbiota has been primarily accomplished through a metataxonomic approach involving targeted amplification of one or more variable regions in the 16S rRNA gene. Specifically, the V3 region (E. coli 341–534) of this gene has been suggested as an excellent candidate for ancient DNA amplification and microbial community reconstruction. However, in practice this metataxonomic approach often produces highly skewed taxonomic frequency data. In this study, we use non-targeted (shotgun metagenomics) sequencing methods to better understand skewed microbial profiles observed in four ancient dental calculus specimens previously analyzed by amplicon sequencing. Through comparisons of microbial taxonomic counts from paired amplicon (V3 U341F/534R) and shotgun sequencing datasets, we demonstrate that extensive length polymorphisms in the V3 region are a consistent and major cause of differential amplification leading to taxonomic bias in ancient microbiome reconstructions based on amplicon sequencing. We conclude that systematic amplification bias confounds attempts to accurately reconstruct microbiome taxonomic profiles from 16S rRNA V3 amplicon data generated using universal primers. Because in silico analysis indicates that alternative 16S rRNA hypervariable regions will present similar challenges, we advocate for the use of a shotgun metagenomics approach in ancient microbiome reconstructions. PMID:26563586

  7. Interpreting 16S metagenomic data without clustering to achieve sub-OTU resolution.

    PubMed

    Tikhonov, Mikhail; Leach, Robert W; Wingreen, Ned S

    2015-01-01

    The standard approach to analyzing 16S tag sequence data, which relies on clustering reads by sequence similarity into Operational Taxonomic Units (OTUs), underexploits the accuracy of modern sequencing technology. We present a clustering-free approach to multi-sample Illumina data sets that can identify independent bacterial subpopulations regardless of the similarity of their 16S tag sequences. Using published data from a longitudinal time-series study of human tongue microbiota, we are able to resolve within standard 97% similarity OTUs up to 20 distinct subpopulations, all ecologically distinct but with 16S tags differing by as little as one nucleotide (99.2% similarity). A comparative analysis of oral communities of two cohabiting individuals reveals that most such subpopulations are shared between the two communities at 100% sequence identity, and that dynamical similarity between subpopulations in one host is strongly predictive of dynamical similarity between the same subpopulations in the other host. Our method can also be applied to samples collected in cross-sectional studies and can be used with the 454 sequencing platform. We discuss how the sub-OTU resolution of our approach can provide new insight into factors shaping community assembly. PMID:25012900

  8. Analysis of the microbiome: Advantages of whole genome shotgun versus 16S amplicon sequencing.

    PubMed

    Ranjan, Ravi; Rani, Asha; Metwally, Ahmed; McGee, Halvor S; Perkins, David L

    2016-01-22

    The human microbiome has emerged as a major player in regulating human health and disease. Translational studies of the microbiome have the potential to indicate clinical applications such as fecal transplants and probiotics. However, one major issue is accurate identification of microbes constituting the microbiota. Studies of the microbiome have frequently utilized sequencing of the conserved 16S ribosomal RNA (rRNA) gene. We present a comparative study of an alternative approach using whole genome shotgun sequencing (WGS). In the present study, we analyzed the human fecal microbiome compiling a total of 194.1 × 10(6) reads from a single sample using multiple sequencing methods and platforms. Specifically, after establishing the reproducibility of our methods with extensive multiplexing, we compared: 1) The 16S rRNA amplicon versus the WGS method, 2) the Illumina HiSeq versus MiSeq platforms, 3) the analysis of reads versus de novo assembled contigs, and 4) the effect of shorter versus longer reads. Our study demonstrates that whole genome shotgun sequencing has multiple advantages compared with the 16S amplicon method including enhanced detection of bacterial species, increased detection of diversity and increased prediction of genes. In addition, increased length, either due to longer reads or the assembly of contigs, improved the accuracy of species detection. PMID:26718401

  9. Oligodeoxynucleotide probes for Campylobacter fetus and Campylobacter hyointestinalis based on 16S rRNA sequences.

    PubMed Central

    Wesley, I V; Wesley, R D; Cardella, M; Dewhirst, F E; Paster, B J

    1991-01-01

    Deoxyoligonucleotide probes were constructed for the identification of Campylobacter fetus and Campylobacter hyointestinalis based on 16S rRNA sequence data. Probes were targeted to hypervariable regions of 16S rRNA. Specificity of oligonucleotide probes was tested in a colony blot assay with type strains of 15 Campylobacter and Arcobacter species as well as in a slot blot format using genomic DNA extracted from field strains of C. fetus and C. hyointestinalis. Two oligonucleotides were constructed for C. fetus that hybridized with equal specificity with each of 57 biochemically confirmed isolates of C. fetus but not with any other Campylobacter species. The C. hyointestinalis probe reacted with 47 of 48 biochemically confirmed field isolates of C. hyointestinalis. In Southern blot hybridization of BglII digests of genomic DNA, the respective probes reacted within three restriction fragments of either C. hyointestinalis (7.2, 8.2, and 10.1 kb) or C. fetus (7.0, 7.7, and 9.0 kb). This suggests multiple copies of genes encoding 16S rRNA. Images PMID:1723076

  10. Intrinsic challenges in ancient microbiome reconstruction using 16S rRNA gene amplification.

    PubMed

    Ziesemer, Kirsten A; Mann, Allison E; Sankaranarayanan, Krithivasan; Schroeder, Hannes; Ozga, Andrew T; Brandt, Bernd W; Zaura, Egija; Waters-Rist, Andrea; Hoogland, Menno; Salazar-García, Domingo C; Aldenderfer, Mark; Speller, Camilla; Hendy, Jessica; Weston, Darlene A; MacDonald, Sandy J; Thomas, Gavin H; Collins, Matthew J; Lewis, Cecil M; Hofman, Corinne; Warinner, Christina

    2015-01-01

    To date, characterization of ancient oral (dental calculus) and gut (coprolite) microbiota has been primarily accomplished through a metataxonomic approach involving targeted amplification of one or more variable regions in the 16S rRNA gene. Specifically, the V3 region (E. coli 341-534) of this gene has been suggested as an excellent candidate for ancient DNA amplification and microbial community reconstruction. However, in practice this metataxonomic approach often produces highly skewed taxonomic frequency data. In this study, we use non-targeted (shotgun metagenomics) sequencing methods to better understand skewed microbial profiles observed in four ancient dental calculus specimens previously analyzed by amplicon sequencing. Through comparisons of microbial taxonomic counts from paired amplicon (V3 U341F/534R) and shotgun sequencing datasets, we demonstrate that extensive length polymorphisms in the V3 region are a consistent and major cause of differential amplification leading to taxonomic bias in ancient microbiome reconstructions based on amplicon sequencing. We conclude that systematic amplification bias confounds attempts to accurately reconstruct microbiome taxonomic profiles from 16S rRNA V3 amplicon data generated using universal primers. Because in silico analysis indicates that alternative 16S rRNA hypervariable regions will present similar challenges, we advocate for the use of a shotgun metagenomics approach in ancient microbiome reconstructions. PMID:26563586

  11. Rare Events of Intragenus and Intraspecies Horizontal Transfer of the 16S rRNA Gene

    PubMed Central

    Tian, Ren-Mao; Cai, Lin; Zhang, Wei-Peng; Cao, Hui-Luo; Qian, Pei-Yuan

    2015-01-01

    Horizontal gene transfer (HGT) of operational genes has been widely reported in prokaryotic organisms. However, informational genes such as those involved in transcription and translation processes are very difficult to be horizontally transferred, as described by Woese’s complexity hypothesis. Here, we analyzed all of the completed prokaryotic genome sequences (2,143 genomes) in the NCBI (National Center for Biotechnology Information) database, scanned for genomes with high intragenomic heterogeneity of 16S rRNA gene copies, and explored potential HGT events of ribosomal RNA genes based on the phylogeny, genomic organization, and secondary structures of the ribosomal RNA genes. Our results revealed 28 genomes with relatively high intragenomic heterogeneity of multiple 16S rRNA gene copies (lowest pairwise identity <98.0%), and further analysis revealed HGT events and potential donors of the heterogeneous copies (such as HGT from Chlamydia suis to Chlamydia trachomatis) and mutation events of some heterogeneous copies (such as Streptococcus suis JS14). Interestingly, HGT of the 16S rRNA gene only occurred at intragenus or intraspecies levels, which is quite different from the HGT of operational genes. Our results improve our understanding regarding the exchange of informational genes. PMID:26220935

  12. Efficient Nucleic Acid Extraction and 16S rRNA Gene Sequencing for Bacterial Community Characterization.

    PubMed

    Anahtar, Melis N; Bowman, Brittany A; Kwon, Douglas S

    2016-01-01

    There is a growing appreciation for the role of microbial communities as critical modulators of human health and disease. High throughput sequencing technologies have allowed for the rapid and efficient characterization of bacterial communities using 16S rRNA gene sequencing from a variety of sources. Although readily available tools for 16S rRNA sequence analysis have standardized computational workflows, sample processing for DNA extraction remains a continued source of variability across studies. Here we describe an efficient, robust, and cost effective method for extracting nucleic acid from swabs. We also delineate downstream methods for 16S rRNA gene sequencing, including generation of sequencing libraries, data quality control, and sequence analysis. The workflow can accommodate multiple samples types, including stool and swabs collected from a variety of anatomical locations and host species. Additionally, recovered DNA and RNA can be separated and used for other applications, including whole genome sequencing or RNA-seq. The method described allows for a common processing approach for multiple sample types and accommodates downstream analysis of genomic, metagenomic and transcriptional information. PMID:27168460

  13. Efficient Nucleic Acid Extraction and 16S rRNA Gene Sequencing for Bacterial Community Characterization

    PubMed Central

    Anahtar, Melis N.; Bowman, Brittany A.; Kwon, Douglas S.

    2016-01-01

    There is a growing appreciation for the role of microbial communities as critical modulators of human health and disease. High throughput sequencing technologies have allowed for the rapid and efficient characterization of bacterial communities using 16S rRNA gene sequencing from a variety of sources. Although readily available tools for 16S rRNA sequence analysis have standardized computational workflows, sample processing for DNA extraction remains a continued source of variability across studies. Here we describe an efficient, robust, and cost effective method for extracting nucleic acid from swabs. We also delineate downstream methods for 16S rRNA gene sequencing, including generation of sequencing libraries, data quality control, and sequence analysis. The workflow can accommodate multiple samples types, including stool and swabs collected from a variety of anatomical locations and host species. Additionally, recovered DNA and RNA can be separated and used for other applications, including whole genome sequencing or RNA-seq. The method described allows for a common processing approach for multiple sample types and accommodates downstream analysis of genomic, metagenomic and transcriptional information. PMID:27168460

  14. Sponge-associated actinobacterial diversity: validation of the methods of actinobacterial DNA extraction and optimization of 16S rRNA gene amplification.

    PubMed

    Yang, Qi; Franco, Christopher M M; Zhang, Wei

    2015-10-01

    Experiments were designed to validate the two common DNA extraction protocols (CTAB-based method and DNeasy Blood & Tissue Kit) used to effectively recover actinobacterial DNA from sponge samples in order to study the sponge-associated actinobacterial diversity. This was done by artificially spiking sponge samples with actinobacteria (spores, mycelia and a combination of the two). Our results demonstrated that both DNA extraction methods were effective in obtaining DNA from the sponge samples as well as the sponge samples spiked with different amounts of actinobacteria. However, it was noted that in the presence of the sponge, the bacterial 16S rRNA gene could not be amplified unless the combined DNA template was diluted. To test the hypothesis that the extracted sponge DNA contained inhibitors, dilutions of the DNA extracts were tested for six sponge species representing five orders. The results suggested that the inhibitors were co-extracted with the sponge DNA, and a high dilution of this DNA was required for the successful PCR amplification for most of the samples. The optimized PCR conditions, including primer selection, PCR reaction system and program optimization, further improved the PCR performance. However, no single PCR condition was found to be suitable for the diverse sponge samples using various primer sets. These results highlight for the first time that the DNA extraction methods used are effective in obtaining actinobacterial DNA and that the presence of inhibitors in the sponge DNA requires high dilution coupled with fine tuning of the PCR conditions to achieve success in the study of sponge-associated actinobacterial diversity. PMID:26245685

  15. Introduction of a novel 18S rDNA gene arrangement along with distinct ITS region in the saline water microalga Dunaliella

    PubMed Central

    2010-01-01

    Comparison of 18S rDNA gene sequences is a very promising method for identification and classification of living organisms. Molecular identification and discrimination of different Dunaliella species were carried out based on the size of 18S rDNA gene and, number and position of introns in the gene. Three types of 18S rDNA structure have already been reported: the gene with a size of ~1770 bp lacking any intron, with a size of ~2170 bp consisting one intron near 5' terminus, and with a size of ~2570 bp harbouring two introns near 5' and 3' termini. Hereby, we report a new 18S rDNA gene arrangement in terms of intron localization and nucleotide sequence in a Dunaliella isolated from Iranian salt lakes (ABRIINW-M1/2). PCR amplification with genus-specific primers resulted in production of a ~2170 bp DNA band, which is similar to that of D. salina 18S rDNA gene containing only one intron near 5' terminus. Whilst, sequence composition of the gene revealed the lack of any intron near 5' terminus in our isolate. Furthermore, another alteration was observed due to the presence of a 440 bp DNA fragment near 3' terminus. Accordingly, 18S rDNA gene of the isolate is clearly different from those of D. salina and any other Dunaliella species reported so far. Moreover, analysis of ITS region sequence showed the diversity of this region compared to the previously reported species. 18S rDNA and ITS sequences of our isolate were submitted with accesion numbers of EU678868 and EU927373 in NCBI database, respectively. The optimum growth rate of this isolate occured at the salinity level of 1 M NaCl. The maximum carotenoid content under stress condition of intense light (400 μmol photon m-2 s-1), high salinity (4 M NaCl) and deficiency of nitrate and phosphate nutritions reached to 240 ng/cell after 15 days. PMID:20377865

  16. Use of 16S rRNA Gene Profiling by Terminal Restriction Fragment Length Polymorphism Analysis To Compare Bacterial Communities in Sputum and Mouthwash Samples from Patients with Cystic Fibrosis†

    PubMed Central

    Rogers, G. B.; Carroll, M. P.; Serisier, D. J.; Hockey, P. M.; Jones, G.; Kehagia, V.; Connett, G. J.; Bruce, K. D.

    2006-01-01

    The bacterial communities present in the oral cavity and the lungs of 19 adult cystic fibrosis (CF) patients were compared by using terminal restriction fragment length polymorphism analysis of 16S rRNA gene PCR products amplified from nucleic acids extracted directly from bacteria in clinical samples. Sputum samples were not found to be subject to profound contamination by oral cavity bacteria. Evidence of colonization of the CF lung by certain oral bacterial species was found. PMID:16825392

  17. Molecular microbial diversity of an anaerobic digestor as determined by small-subunit rDNA sequence analysis.

    PubMed Central

    Godon, J J; Zumstein, E; Dabert, P; Habouzit, F; Moletta, R

    1997-01-01

    The bacterial community structure of a fluidized-bed reactor fed by vinasses (wine distillation waste) was analyzed. After PCR amplification, four small-subunit (SSU) rDNA clone libraries of Bacteria, Archaea, Procarya, and Eucarya populations were established. The community structure was determined by operational taxonomic unit (OTU) phylogenetic analyses of 579 partial rDNA sequences (about 500 bp long). A total of 146 OTUs were found, comprising 133, 6, and 7 from the Bacteria, Archaea, and Eucarya domains, respectively. A total of 117 bacterial OTU were affiliated with major phyla: low-G+C gram-positive bacteria, Cytophaga-Flexibacter-Bacteroides, Proteobacteria, high-G+C gram-positive bacteria, and Spirochaetes, where the clone distribution was 3