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Sample records for lato mediante pcr

  1. Detection of tick blood parasites in Egypt using PCR assay II- Borrelia burgdorferi sensu lato.

    PubMed

    Adham, Fatma K; El-Samie-Abd, Emtithal M; Gabre, Refaat M; El Hussein, Hala

    2010-12-01

    The prevalence of Borrelia burgdorferi sensu lato (s.l.), the etiologic agent of Lyme borrelosis (LB), was determined for the first time in Egypt by using polymerase chain reaction (PCR). Questing 5243 hard and soft ticks were collected from animal farms throughout Giza Governorate. DNA from 500 individual tick species was extracted and PCR was performed. Primers verified from the sequence of German strain Pko of Borrelia afzelii were used. Fragments of 642 bp were generated and sequenced. The prevalence of B. burgdorferi sensu lato (s.l.) was 28% of examined soft and hard ticks. High infection rate (66%) of B. burgdorferi s.l. was observed in both nymph and adult soft ticks Ornithodoros savignyi. Beside, the role of hard ticks as potential vectors of Lyme disease in Egypt, where the infection rate was between 0.0-50.0%. Sequence analysis of PCR product of Borrelia burgdorferi sensu lato shares high degree of similarity in sequence compared to similar species in GenBank.

  2. Accurate and sensitive real-time PCR assays using intergenic spacer 1 region to differentiate Cryptococcus gattii sensu lato and Cryptococcus neoformans sensu lato.

    PubMed

    Tavares, Eliandro Reis; Azevedo, Caroline Souza; Panagio, Luciano Aparecido; Pelisson, Marsileni; Pinge-Filho, Phileno; Venancio, Emerson José; Barros, Tânia Fraga; Yamada-Ogatta, Sueli Fumie; Yamauchi, Lucy Megumi

    2016-01-01

    In this work, two accurate and sensitive real-time polymerase chain reaction (PCR) assays to differentiate pathogenic Cryptococcus gattii sensu lato (s.l.) and C. neoformans sensu lato (s.l.) targeting the intergenic spacer 1 (IGS1) region from rDNA locus were developed. Specific primers were designed based on their IGS1 sequence analyses and the optimal real-time PCR assays showed that the dissociation curves generated two different melting peaks, at 82.8 and 84.2ºC for C. gattii s.l. and C. neoformans s.l., respectively. No amplifications were observed in the negative template control. The minimum limit of detection of both primers was 100 plasmid copies per reaction, and they were highly specific when tested with a range of fungal DNAs. Overall, the results showed that the designed primers completely differentiated C. gattii s.l. and C. neoformans s.l. from clinical and environmental sources with great accuracy when compared to phenotypic identification, with no cross-reactivity to other fungal DNA.

  3. Real-time PCR-based identification of Borrelia burgdorferi sensu lato species in ticks collected from humans in Romania.

    PubMed

    Briciu, Violeta T; Meyer, Fabian; Sebah, Daniela; Tăţulescu, Doina F; Coroiu, Georgiana; Lupşe, Mihaela; Carstina, Dumitru; Mihalca, Andrei D; Hizo-Teufel, Cecilia; Klier, Christiane; Huber, Ingrid; Fingerle, Volker

    2014-09-01

    The aims of our study were to determine (i) which tick species bite humans in Romania and (ii) the prevalence of Borrelia (B.) burgdorferi genospecies in these ticks. All ticks collected from patients who presented to the Clinic of Infectious Diseases Cluj Napoca in spring/summer 2010 were morphologically identified by an entomologist and tested for B. burgdorferi genospecies prevalence by a real-time PCR assay targeting the hbb gene and melting curve analysis. Out of 532 ticks, 518 were Ixodes ricinus, 10 Dermacentor marginatus, and 3 Haemaphysalis spp. ticks, and one unidentified tick due to destruction. Since evaluation of the hbb PCR revealed that it was not possible to differentiate between B. spielmanii/B. valaisiana and B. garinii/B. bavariensis, sequencing of an 800-bp fragment of the ospA gene was performed in these cases. Out of 389 investigated ticks, 43 were positive by hbb PCR for B. burgdorferi sensu lato. The positive samples were 42 Ixodes ricinus (11.1% B. burgdorferi sensu lato prevalence) and the one unidentified tick. Species identification revealed the presence of mainly B. afzelii, but also of B. garinii, B. burgdorferi sensu stricto, B. valaisiana, and B. lusitaniae. In 4 samples, differentiation between B. spielmanii/B. valaisiana was impossible. Our study shows that the most relevant human pathogenic B. burgdorferi genospecies - predominantly B. afzelii - are present in ticks collected from Romanian patients.

  4. Immunohistochemistry and real-time PCR as diagnostic tools for detection of Borrelia burgdorferi sensu lato in ticks collected from humans.

    PubMed

    Briciu, Violeta T; Sebah, Daniela; Coroiu, Georgiana; Lupşe, Mihaela; Cârstina, Dumitru; Ţăţulescu, Doina F; Mihalca, Andrei D; Gherman, Călin M; Leucuţa, Daniel; Meyer, Fabian; Hizo-Teufel, Cecilia; Fingerle, Volker; Huber, Ingrid

    2016-05-01

    The objective of this study was to evaluate different methods used for detection of Borrelia burgdorferi sensu lato (s.l.) in ticks: immunohistochemistry followed by focus floating microscopy (FFM) and real-time polymerase chain reaction (real-time PCR) targeting the ospA and hbb genes. Additionally, an optimized ospA real-time PCR assay was developed with an integrated internal amplification control (IAC) for the detection of inhibition in the PCR assay and was validated as an improved screening tool for B. burgdorferi. One hundred and thirty-six ticks collected from humans in a hospital from Cluj-Napoca, Romania, were investigated regarding genus, stage of development and sex, and then tested by all three assays. A poor quality of agreement was found between FFM and each of the two real-time PCR assays, as assessed by concordance analysis (Cohen's kappa), whereas the agreement between the two real-time PCR assays was moderate. The present study argues for a low sensitivity of FFM and underlines that discordant results of different assays used for detection of B. burgdorferi in ticks are frequent.

  5. First Detection of Borrelia burgdorferi sensu lato DNA in Serum of the Wild Boar (Sus scrofa) in Northern Portugal by Nested-PCR.

    PubMed

    Faria, Ana S; Paiva-Cardoso, Maria das Neves; Nunes, Mónica; Carreira, Teresa; Vale-Gonçalves, Hélia M; Veloso, Octávia; Coelho, Catarina; Cabral, João A; Vieira-Pinto, Madalena; Vieira, Maria L

    2015-03-01

    Lyme borreliosis is the most common tick-borne zoonosis in the northern hemisphere. Several vertebrates are crucial in the epidemiological cycle of Borrelia burgdorferi sensu lato, but the role of wild boar as a reservoir is still unknown. Sera were collected from 90 wild boars shot in the Trás-os-Montes region, Northern Portugal (hunting season 2011/2012). In this study, Borrelia DNA was detected for the first time by nested-PCR in three different sera, suggesting that the wild boar may be a potential reservoir for this spirochete. Sequencing results show 100% similarity with Borrelia afzelii. Further studies are needed to evaluate the public health risks associated with boar hunting.

  6. Establishment of a minor groove binder-probe based quantitative real time PCR to detect Borrelia burgdorferi sensu lato and differentiation of Borrelia spielmanii by ospA-specific conventional PCR

    PubMed Central

    2010-01-01

    Background Borrelia burgdorferi sensu lato (sl), the causative agent of Lyme borreliosis, is transmitted by ticks of the genus Ixodes as vector. For identification of Borrelia infections in ticks a TaqMan™ minor groove binder (MGB) probe-based quantitative real time PCR (qPCR) was established targeting the 5S-23S intergenic spacer. Extension to a duplex qPCR included an Ixodes spp. positive control to verify successful DNA isolation. Besides qPCR, an ospA-specific conventional PCR for species-specific identification of B. spielmanii was established. Afterwards 1000 I. ricinus flagged in the city of Hanover, Germany, were investigated for B. burgdorferi sl infections followed by species identification. Furthermore, I. hexagonus ticks were investigated to proof applicability of the PCRs. Results Quantitative real time PCR (qPCR) identifying B. burgdorferi sl in ticks was able to detect 1-10 copies per reaction. B. spielmanii ospA-specific conventional PCR was also highly specific and showed no cross reactions with the other tested Borrelia species. From 1000 hanoveranian ticks 24.3% were positive compared to only 7.4% positives by dark-field microscopy. Related to tick stage 1.7% larvae, 18.1% nymphs, and 34.6% adults were positive. The most frequent species was B. garinii, followed by B. afzelii, B. spielmanii, B. valaisiana and B. burgdorferi sensu stricto (ss). 70.6% of I. ricinus were mono-infected, whereas 28.0% and 1.4% were infected with two and three Borrelia species, respectively. From 232 I. hexagonus collected from hedgehogs in different sites of Germany, qPCR detected 5.7% to be infected with B. burgdorferi sl, which were identified as B. afzelii, B. garinii and B. spielmanii. Conclusions The evaluated qPCR to detect B. burgdorferi sl in Ixodes spp. is highly specific and sensitive. As a duplex qPCR including detection of Ixodes spp. DNA it is the first DNA based technique incorporating a control for successful DNA isolation from the vector tick

  7. Accurate Identification of Candida parapsilosis (Sensu Lato) by Use of Mitochondrial DNA and Real-Time PCR

    PubMed Central

    Souza, Ana Carolina R.; Ferreira, Renata C.; Gonçalves, Sarah S.; Quindós, Guillermo; Eraso, Elena; Bizerra, Fernando C.; Briones, Marcelo R. S.

    2012-01-01

    Candida parapsilosis is the Candida species isolated the second most frequently from blood cultures in South America and some European countries, such as Spain. Since 2005, this species has been considered a complex of 3 closely related species: C. parapsilosis, Candida metapsilosis, and Candida orthopsilosis. Here, we describe a real-time TaqMan-MGB PCR assay, using mitochondrial DNA (mtDNA) as the target, which readily distinguishes these 3 species. We first used comparative genomics to locate syntenic regions between these 3 mitochondrial genomes and then selected NADH5 as the target for the real-time PCR assay. Probes were designed to include a combination of different single-nucleotide polymorphisms that are able to differentiate each species within the C. parapsilosis complex. This new methodology was first tested using mtDNA and then genomic DNA from 4 reference and 5 clinical strains. For assay validation, a total of 96 clinical isolates and 4 American Type Culture Collection (ATCC) isolates previously identified by internal transcribed spacer (ITS) ribosomal DNA (rDNA) sequencing were tested. Real-time PCR using genomic DNA was able to differentiate the 3 species with 100% accuracy. No amplification was observed when DNA from other species was used as the template. We observed 100% congruence with ITS rDNA sequencing identification, including for 30 strains used in blind testing. This novel method allows a quick and accurate intracomplex identification of C. parapsilosis and saves time compared with sequencing, which so far has been considered the “gold standard” for Candida yeast identification. In addition, this assay provides a useful tool for epidemiological and clinical studies of these emergent species. PMID:22535986

  8. Genetic Variability within Borrelia burgdorferi Sensu Lato Genospecies Established by PCR-Single-Strand Conformation Polymorphism Analysis of the rrfA-rrlB Intergenic Spacer in Ixodes ricinus Ticks from the Czech Republic

    PubMed Central

    Derdáková, Markéta; Beati, Lorenza; Pet'ko, Branislav; Stanko, Michal; Fish, Durland

    2003-01-01

    In Europe the Borrelia burgdorferi sensu lato complex is represented by five distinct genospecies: Borrelia burgdorferi sensu stricto, Borrelia afzelii, Borrelia garinii, Borrelia valaisiana, and Borrelia lusitaniae. These taxonomic entities are known to differ in their specific associations with vertebrate hosts and to provoke distinct clinical manifestations in human patients. However, exceptions to these rules have often been observed, indicating that strains belonging to a single genospecies may be more heterogeneous than expected. It is, therefore, important to develop alternative identification tools which are able to distinguish Borrelia strains not only at the specific level but also at the intraspecific level. DNA from a sample of 370 Ixodes ricinus ticks collected in the Czech Republic was analyzed by PCR for the presence of a ∼230-bp fragment of the rrfA-rrlB intergenic spacer of Borrelia spp. A total of 20.5% of the ticks were found to be positive. The infecting genospecies were identified by analyzing the amplified products by the restriction fragment length polymorphism (RFLP) method with restriction enzyme MseI and by single-strand conformation polymorphism (SSCP) analysis. The two methods were compared, and PCR-SSCP analysis appeared to be a valuable tool for rapid identification of spirochetes at the intraspecific level, particularly when large samples are examined. Furthermore, by using PCR-SSCP analysis we identified a previously unknown Borrelia genotype, genotype I-77, which would have gone unnoticed if RFLP analysis alone had been used. PMID:12514035

  9. Genetic variability within Borrelia burgdorferi sensu lato genospecies established by PCR-single-strand conformation polymorphism analysis of the rrfA-rrlB intergenic spacer in ixodes ricinus ticks from the Czech Republic.

    PubMed

    Derdáková, Markéta; Beati, Lorenza; Pet'ko, Branislav; Stanko, Michal; Fish, Durland

    2003-01-01

    In Europe the Borrelia burgdorferi sensu lato complex is represented by five distinct genospecies: Borrelia burgdorferi sensu stricto, Borrelia afzelii, Borrelia garinii, Borrelia valaisiana, and Borrelia lusitaniae. These taxonomic entities are known to differ in their specific associations with vertebrate hosts and to provoke distinct clinical manifestations in human patients. However, exceptions to these rules have often been observed, indicating that strains belonging to a single genospecies may be more heterogeneous than expected. It is, therefore, important to develop alternative identification tools which are able to distinguish Borrelia strains not only at the specific level but also at the intraspecific level. DNA from a sample of 370 Ixodes ricinus ticks collected in the Czech Republic was analyzed by PCR for the presence of a approximately 230-bp fragment of the rrfA-rrlB intergenic spacer of Borrelia spp. A total of 20.5% of the ticks were found to be positive. The infecting genospecies were identified by analyzing the amplified products by the restriction fragment length polymorphism (RFLP) method with restriction enzyme MseI and by single-strand conformation polymorphism (SSCP) analysis. The two methods were compared, and PCR-SSCP analysis appeared to be a valuable tool for rapid identification of spirochetes at the intraspecific level, particularly when large samples are examined. Furthermore, by using PCR-SSCP analysis we identified a previously unknown Borrelia genotype, genotype I-77, which would have gone unnoticed if RFLP analysis alone had been used.

  10. Molecular Identification and Analysis of Borrelia burgdorferi Sensu Lato in Lizards in the Southeastern United States

    PubMed Central

    Clark, Kerry; Hendricks, Amanda; Burge, David

    2005-01-01

    Lyme borreliosis (LB) group spirochetes, collectively known as Borrelia burgdorferi sensu lato, are distributed worldwide. Wild rodents are acknowledged as the most important reservoir hosts. Ixodes scapularis is the primary vector of B. burgdorferi sensu lato in the eastern United States, and in the southeastern United States, the larvae and nymphs mostly parasitize certain species of lizards. The primary aim of the present study was to determine whether wild lizards in the southeastern United States are naturally infected with Lyme borreliae. Blood samples obtained from lizards in Florida and South Carolina were tested for the presence of LB spirochetes primarily by using B. burgdorferi sensu lato-specific PCR assays that amplify portions of the flagellin (flaB), outer surface protein A (ospA), and 66-kDa protein (p66) genes. Attempts to isolate spirochetes from a small number of PCR-positive lizards failed. However, PCR amplification and sequence analysis of partial flaB, ospA, and p66 gene fragments confirmed numerous strains of B. burgdorferi sensu lato, including Borrelia andersonii, Borrelia bissettii, and B. burgdorferi sensu stricto, in blood from lizards from both states. B. burgdorferi sensu lato DNA was identified in 86 of 160 (54%) lizards representing nine species and six genera. The high infection prevalence and broad distribution of infection among different lizard species at different sites and at different times of the year suggest that LB spirochetes are established in lizards in the southeastern United States. PMID:15870353

  11. Distinct Combinations of Borrelia burgdorferi Sensu Lato Genospecies Found in Individual Questing Ticks from Europe

    PubMed Central

    Kurtenbach, Klaus; De Michelis, Simona; Sewell, Henna-Sisko; Etti, Susanne; Schäfer, Stefanie M.; Hails, Rosie; Collares-Pereira, Margarida; Santos-Reis, Margarida; Haninçová, Klára; Labuda, Milan; Bormane, Antra; Donaghy, Michael

    2001-01-01

    The genetic diversity of Borrelia burgdorferi sensu lato was assessed in individual adult Ixodes ricinus ticks from Europe by direct PCR amplification of spirochetal DNA followed by genospecies-specific hybridization. Analysis of mixed infections in the ticks showed that B. garinii and B. valaisiana segregate from B. afzelii. This and previous findings suggest that host complement interacts with spirochetes in the tick, thereby playing an important role in the ecology of Lyme borreliosis. PMID:11571205

  12. The Phylogeny of Bacillus cereus sensu lato.

    PubMed

    Okinaka, Richard T; Keim, Paul

    2016-02-01

    The three main species of the Bacillus cereus sensu lato, B. cereus, B. thuringiensis, and B. anthracis, were recognized and established by the early 1900 s because they each exhibited distinct phenotypic traits. B. thuringiensis isolates and their parasporal crystal proteins have long been established as a natural pesticide and insect pathogen. B. anthracis, the etiological agent for anthrax, was used by Robert Koch in the 19th century as a model to develop the germ theory of disease, and B. cereus, a common soil organism, is also an occasional opportunistic pathogen of humans. In addition to these three historical species designations, are three less-recognized and -understood species: B. mycoides, B. weihenstephanensis, and B. pseudomycoides. All of these "species" combined comprise the Bacillus cereus sensu lato group. Despite these apparently clear phenotypic definitions, early molecular approaches to separate the first three by various DNA hybridization and 16S/23S ribosomal sequence analyses led to some "confusion" because there were limited differences to differentiate between these species. These and other results have led to frequent suggestions that a taxonomic change was warranted to reclassify this group to a single species. But the pathogenic properties of B. anthracis and the biopesticide applications of B. thuringiensis appear to "have outweighed pure taxonomic considerations" and the separate species categories are still being maintained. B. cereus sensu lato represents a classic example of a now common bacterial species taxonomic quandary.

  13. Echinococcus granulosus sensu lato and Taenia hydatigena in pig in southern Brazil.

    PubMed

    Monteiro, Danieli Urach; Botton, Sônia de Avila; Tonin, Alexandre Alberto; Haag, Karen Luisa; Musskopf, Germano; Azevedo, Maria Isabel; Weiblen, Carla; Ribeiro, Tatiana Correa; de la Rue, Mário Luiz

    2015-01-01

    This study aimed to identify the parasitical etiologic agents of visceral cysts in pigs from the central/northern region of Rio Grande do Sul State, Brazil. Fifty-eight cysts were found in livers during veterinary inspection of swine slaughtered from January 2008 to 2012. Collected samples were submitted to macroscopic and molecular analyzes. Polymerase chain reaction (PCR), DNA sequencing and BLAST alignment of sequences was used to molecular characterization of the samples. By PCR 10.3% (6/58) of tested samples were positive for Echinococcus granulosus sensu lato and 56.9% (33/58) for Cysticercus tenuicollis. In this study, it was verified the occurrence of larval forms of E. granulosus sensu lato and Taenia hydatigena in pig herds from the central/northern region of Rio Grande do Sul State. The presence of both parasites is relevant due to the economic losses for the meat industry. Additionally, E. granulosus sensu lato has zoonotic importance and may be infecting pig herds in southern Brazil.

  14. Molecular Typing of Borrelia burgdorferi Sensu Lato: Taxonomic, Epidemiological, and Clinical Implications

    PubMed Central

    Wang, Guiqing; van Dam, Alje P.; Schwartz, Ira; Dankert, Jacob

    1999-01-01

    Borrelia burgdorferi sensu lato, the spirochete that causes human Lyme borreliosis (LB), is a genetically and phenotypically divergent species. In the past several years, various molecular approaches have been developed and used to determine the phenotypic and genetic heterogeneity within the LB-related spirochetes and their potential association with distinct clinical syndromes. These methods include serotyping, multilocus enzyme electrophoresis, DNA-DNA reassociation analysis, rRNA gene restriction analysis (ribotyping), pulsed-field gel electrophoresis, plasmid fingerprinting, randomly amplified polymorphic DNA fingerprinting analysis, species-specific PCR and PCR-based restriction fragment length polymorphism (RFLP) analysis, and sequence analysis of 16S rRNA and other conserved genes. On the basis of DNA-DNA reassociation analysis, 10 different Borrelia species have been described within the B. burgdorferi sensu lato complex: B. burgdorferi sensu stricto, Borrelia garinii, Borrelia afzelii, Borrelia japonica, Borrelia andersonii, Borrelia valaisiana, Borrelia lusitaniae, Borrelia tanukii, Borrelia turdi, and Borrelia bissettii sp. nov. To date, only B. burgdorferi sensu stricto, B. garinii, and B. afzelii are well known to be responsible for causing human disease. Different Borrelia species have been associated with distinct clinical manifestations of LB. In addition, Borrelia species are differentially distributed worldwide and may be maintained through different transmission cycles in nature. In this paper, the molecular methods used for typing of B. burgdorferi sensu lato are reviewed. The current taxonomic status of B. burgdorferi sensu lato and its epidemiological and clinical implications, especiallly correlation between the variable clinical presentations and the infecting Borrelia species, are discussed in detail. PMID:10515907

  15. Genetic Diversity of Borrelia burgdorferi Sensu Lato in Ticks from Mainland Portugal

    PubMed Central

    De Michelis, Simona; Sewell, Henna-Sisko; Collares-Pereira, Margarida; Santos-Reis, Margarida; Schouls, Leo M.; Benes, Vladimir; Holmes, Edward C.; Kurtenbach, Klaus

    2000-01-01

    To date Borrelia lusitaniae is the only genospecies of Borrelia burgdorferi sensu lato isolated from Ixodes ricinus ticks collected in Portugal and Tunisia. This suggests that the genospecies diversity of B. burgdorferi sensu lato decreases toward the southwestern margin of its Old World subtropical range. In order to further explore the genetic diversity of B. burgdorferi sensu lato from this region, 55 I. ricinus and 27 Hyalomma marginatum questing adults, collected during the spring of 1998 from a sylvatic habitat south of Lisbon, Portugal, were analyzed. Infection prevalences of 75% in I. ricinus ticks and 7% in H. marginatum ticks were detected by a nested PCR that targets the rrf (5S)-rrl (23S) spacer of B. burgdorferi sensu lato. Restriction fragment length polymorphism (RFLP) analysis of the I. ricinus-derived amplicons showed that the sequences in the majority of samples were similar to those of B. lusitaniae type strains (76% for strain PotiB1, 5% for strain PotiB3). Two novel RFLP patterns were obtained from 12% of the samples. The remaining 7% of samples gave mixed RFLP patterns. Phylogenetic analysis of rrf-rrl spacer sequences revealed a diverse population of B. lusitaniae in questing adult I. ricinus ticks (the sequences did not cluster with those of any other genospecies). This population consisted of 10 distinct sequence types, suggesting that multiple strains of B. lusitaniae were present in the local I. ricinus population. We hypothesize that B. lusitaniae has a narrow ecological niche that involves host species restricted to the Mediterranean Basin. PMID:10834965

  16. Molecular Evidence of Coinfection of Ticks with Borrelia burgdorferi Sensu Lato and the Human Granulocytic Ehrlichiosis Agent in Switzerland

    PubMed Central

    Leutenegger, Christian M.; Pusterla, Nicola; Mislin, Caroline N.; Weber, Rainer; Lutz, Hans

    1999-01-01

    Adult Ixodes ricinus ticks were collected in Switzerland and tested for the presence of coinfection with Borrelia burgdorferi sensu lato and the human granulocytic ehrlichiosis (HGE) agent by real-time PCR. Of 100 ticks, 49% were positive for B. burgdorferi and 2% were positive for the HGE agent. The two HGE agent-positive ticks were also found to be positive for B. burgdorferi. PMID:10488215

  17. Assessment of two new molecular methods for identification of Candida parapsilosis sensu lato species.

    PubMed

    Garcia-Effron, Guillermo; Canton, Emilia; Pemán, Javier; Dilger, Amanda; Romá, Eva; Perlin, David S

    2011-09-01

    Candida parapsilosis sensu stricto, C. orthopsilosis, and C. metapsilosis replaced C. parapsilosis groups I, II, and III in 2005. Since then, an increased interest in studying their epidemiology has arisen based on the observed differences in antifungal susceptibilities and virulence the three species. A strict differentiation of these species cannot be achieved by phenotypic methods. We evaluate two new molecular methodologies to differentiate among these species by the use of a collection of 293 bloodstream infection isolates of C. parapsilosis sensu lato. For the first method, the isolates were studied using PCR amplification of a fragment of the C. parapsilosis sensu lato FKS1 gene and a universal primer pair followed by EcoRI enzyme digestion. The other method used the allele discrimination ability of molecular beacons in a multiplex real-time PCR format. Both methods of identification showed 100% concordance with internal transcribed spacer 1 (ITS1)/ITS2 sequencing and proved to be effective for clinical applications, even with mixed-species DNAs.

  18. Geographical and genospecies distribution of Borrelia burgdorferi sensu lato DNA detected in humans in the USA.

    PubMed

    Clark, Kerry L; Leydet, Brian F; Threlkeld, Clifford

    2014-05-01

    The present study investigated the cause of illness in human patients primarily in the southern USA with suspected Lyme disease based on erythema migrans-like skin lesions and/or symptoms consistent with early localized or late disseminated Lyme borreliosis. The study also included some patients from other states throughout the USA. Several PCR assays specific for either members of the genus Borrelia or only for Lyme group Borrelia spp. (Borrelia burgdorferi sensu lato), and DNA sequence analysis, were used to identify Borrelia spp. DNA in blood and skin biopsy samples from human patients. B. burgdorferi sensu lato DNA was found in both blood and skin biopsy samples from patients residing in the southern states and elsewhere in the USA, but no evidence of DNA from other Borrelia spp. was detected. Based on phylogenetic analysis of partial flagellin (flaB) gene sequences, strains that clustered separately with B. burgdorferi sensu stricto, Borrelia americana or Borrelia andersonii were associated with Lyme disease-like signs and symptoms in patients from the southern states, as well as from some other areas of the country. Strains most similar to B. burgdorferi sensu stricto and B. americana were found most commonly and appeared to be widely distributed among patients residing throughout the USA. The study findings suggest that human cases of Lyme disease in the southern USA may be more common than previously recognized and may also be caused by more than one species of B. burgdorferi sensu lato. This study provides further evidence that B. burgdorferi sensu stricto is not the only species associated with signs and/or symptoms consistent with Lyme borreliosis in the USA.

  19. First isolation and cultivation of Borrelia burgdorferi sensu lato from Missouri.

    PubMed

    Oliver, J H; Kollars, T M; Chandler, F W; James, A M; Masters, E J; Lane, R S; Huey, L O

    1998-01-01

    Five Borrelia burgdorferi sensu lato isolates from Missouri are described. This represents the first report and characterization of such isolates from that state. The isolates were obtained from either Ixodes dentatus or Amblyomma americanum ticks that had been feeding on cottontail rabbits (Sylvilagus floridanus) from a farm in Bollinger County, Mo., where a human case of Lyme disease had been reported. All isolates were screened immunologically by indirect immunofluorescence by using monoclonal antibodies to B. burgdorferi-specific outer surface protein A (OspA) (antibodies H3TS and H5332), B. burgdorferi-specific OspB (antibody H6831), Borrelia (genus)-specific antiflagellin (antibody H9724), and Borrelia hermsii-specific antibody (antibody H9826). Analysis of the isolates also involved a comparison of their protein profiles by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Finally, the isolates were analyzed by PCR with six pairs of primers known to amplify selected DNA target sequences specifically found in the reference strain B. burgdorferi B-31. Although some genetic variability was detected among the five isolates as well as between them and the B-31 strain, enough similarities were found to classify them as B. burgdorferi sensu lato.

  20. Prevalence of Borrelia burgdorferi sensu lato-infected ticks on migrating birds.

    PubMed Central

    Olsén, B; Jaenson, T G; Bergström, S

    1995-01-01

    The prevalence of Lyme disease Borrelia-infected ticks on migrating birds was studied in Scandinavia. A total of 22,998 birds were caught at eight different bird observatories and examined for ticks. Five different species of ticks were found infesting the birds. The dominant species, Ixodesricinus, constituted 98.3% of the ticks collected. The presence of spirochetes was determined by an immunofluorescence assay of tick larvae and DNA amplification by PCR on all ticks. To determine which Borrelia burgdorferi sensu lato species were present, a species classification was performed by DNA amplification with species-specific 16S rDNA primers and by DNA sequencing (rDNA is DNA coding for rRNA). Flagellin gene sequences of all species of B. burgdorferi sensu lato previously recorded in Europe were observed. Borrelia garinii was the most prevalent Lyme disease Borrelia species in ticks collected from birds arriving from the South or Southeast in the spring, whereas the distribution was more heterogeneous in ticks from birds migrating from the Southwest. These data support the notion that birds are partly responsible for the heterogeneous distribution of Lyme disease Borrelia spirochetes in Europe. PMID:7487041

  1. Molecular epidemiology and in vitro antifungal susceptibility testing of 108 clinical Cryptococcus neoformans sensu lato and Cryptococcus gattii sensu lato isolates from Denmark.

    PubMed

    Hagen, Ferry; Hare Jensen, Rasmus; Meis, Jacques F; Arendrup, Maiken Cavling

    2016-09-01

    Cryptococcosis is mainly caused by members of the Cryptococcus gattii/Cryptococcus neoformans species complexes. Here, we report the molecular characterisation and in vitro antifungal susceptibility of Danish clinical cryptococcal isolates. Species, genotype, serotype and mating type were determined by amplified fragment length polymorphism (AFLP) fingerprinting and qPCR. EUCAST E.Def 7.2 MICs were determined for amphotericin B, flucytosine, fluconazole, voriconazole and isavuconazole. Most isolates were C. neoformans (serotype A; n = 66) and belonged to genotype AFLP1/VNI (n = 61) or AFLP1B/VNII (n = 5) followed by Cryptococcus deneoformans (serotype D; genotype AFLP2, n = 20), C. neoformans × C. deneoformans hybrids (serotype AD; genotype AFLP3, n = 13) and Cryptococcus curvatus (n = 2). Six isolates were C. gattii sensu lato, and one isolate was a C. deneoformans × C. gattii hybrid (genotype AFLP8). All isolates were amphotericin B susceptible. Flucytosine susceptibility was uniform MIC50 of 4-8 mg l(-1) except for C. curvatus (MICs >32 mg l(-1) ). Cryptococcus gattii sensu lato isolates were somewhat less susceptible to the azoles. MICs of fluconazole (>32 mg l(-1) ), voriconazole (≥0.5 mg l(-1) ) and isavuconazole (0.06 and 0.25 mg l(-1) respectively) were elevated compared to the wild-type population for 1/19 C. deneoformans and 1/2 C. curvatus isolates. Flucytosine MIC was elevated for 1/61 C. neoformans (>32 mg l(-1) ). Antifungal susceptibility revealed species-specific differential susceptibility, but suggested acquired resistance was an infrequent phenomenon.

  2. Borrelia miyamotoi sensu lato seroreactivity and seroprevalence in the northeastern United States.

    PubMed

    Krause, Peter J; Narasimhan, Sukanya; Wormser, Gary P; Barbour, Alan G; Platonov, Alexander E; Brancato, Janna; Lepore, Timothy; Dardick, Kenneth; Mamula, Mark; Rollend, Lindsay; Steeves, Tanner K; Diuk-Wasser, Maria; Usmani-Brown, Sahar; Williamson, Phillip; Sarksyan, Denis S; Fikrig, Erol; Fish, Durland

    2014-07-01

    Borrelia miyamotoi sensu lato, a relapsing fever Borrelia sp., is transmitted by the same ticks that transmit B. burgdorferi (the Lyme disease pathogen) and occurs in all Lyme disease-endemic areas of the United States. To determine the seroprevalence of IgG against B. miyamotoi sensu lato in the northeastern United States and assess whether serum from B. miyamotoi sensu lato-infected persons is reactive to B. burgdorferi antigens, we tested archived serum samples from area residents during 1991-2012. Of 639 samples from healthy persons, 25 were positive for B. miyamotoi sensu lato and 60 for B. burgdorferi. Samples from ≈10% of B. miyamotoi sensu lato-seropositive persons without a recent history of Lyme disease were seropositive for B. burgdorferi. Our results suggest that human B. miyamotoi sensu lato infection may be common in southern New England and that B. burgdorferi antibody testing is not an effective surrogate for detecting B. miyamotoi sensu lato infection.

  3. Prevalence of Borrelia burgdorferi sensu lato in Ixodes ricinus ticks collected from southern Poland.

    PubMed

    Strzelczyk, Joanna K; Gaździcka, Jadwiga; Cuber, Piotr; Asman, Marek; Trapp, Gizela; Gołąbek, Karolina; Zalewska-Ziob, Marzena; Nowak-Chmura, Magdalena; Siuda, Krzysztof; Wiczkowski, Andrzej; Solarz, Krzysztof

    2015-12-01

    In 2008-2011 ticks were collected from southern Poland. Out of 6336 individuals collected and identified as Ixodes ricinus, 768 (2 larvae, 84 nymphs, 417 females, 265 males) were included in molecular study. The aim of this study was to investigate the prevalence and types of genospecies of Borrelia burgdorferi sensu lato in ticks. The polymerase chain reaction (PCR) was applied to detect the presence of pathogens in ticks. Subsequently the amplified DNA was digested with TasI enzyme. The infection rate was 15% (116) of examined ticks. PCR-RFLP analysis allowed distinguishing three genospecies of B. burgdorferi s.l.: B. burgdorferi sensu stricto, B. afzelii, and B. garinii. RFLP analyses of 116 positive samples revealed 96 (83%) monoinfections and 13 (11%) coinfections, whereas unidentified genospecies were present in 7 (6%) of positive samples. In the case of monoinfections, B. burgdorferi s.s. was the predominant species of pathogen in infected ticks - 61.4%. Other genospecies: B. garinii and B. afzelii were detected in 22.9% and 15.6% of the samples, respectively. To sum up, 15 % of ticks were infected by B. burgdorferi s.l which increases the risk of human infections in the recreational areas of southern Poland. Furthermore, there is a need to increase public awareness and implement more preventive measures concerning Lyme disease.

  4. Isolation of Borrelia burgdorferi sensu lato from the skin of the European badger (Meles meles) in Switzerland.

    PubMed

    Gern, Lise; Sell, Katy

    2009-04-01

    No data are available on the role of badgers in the ecology of Lyme borreliosis spirochetes in Europe. In a recent study describing validation of a molecular method allowing host DNA identification and Borrelia burgdorferi sensu lato detection in Ixodes ricinus, the simultaneous presence of B. afzelii DNA and of European badger (Meles meles) DNA was detected in I. ricinus ticks in Switzerland. This suggested that badgers might be reservoir hosts for B. afzelii. Here, we present results obtained in a study on badgers conducted in 1996-1997. Thirty-one tissue samples (ear biopsy: n = 25, aspiration fluid: n = 6) from 8 badgers were placed in BSK medium to isolate B. burgdorferi sensu lato and were then examined by polymerase chain reaction/restriction fragment length polymorphism (PCR/RFLP). Globally, six Borrelia isolates (6/31, 19.4%) were obtained from 3/8 (37.5%) badgers. These isolates were identified as B. afzelii (n = 3) and B. valaisiana (n = 3).

  5. Borrelia burgdorferi sensu lato in Ixodes ricinus ticks collected from migratory birds in Southern Norway

    PubMed Central

    2010-01-01

    Background Borrelia burgdorferi sensu lato (s.l.) are the causative agent for Lyme borreliosis (LB), the most common tick-borne disease in the northern hemisphere. Birds are considered important in the global dispersal of ticks and tick-borne pathogens through their migration. The present study is the first description of B. burgdorferi prevalence and genotypes in Ixodes ricinus ticks feeding on birds during spring and autumn migration in Norway. Methods 6538 migratory birds were captured and examined for ticks at Lista Bird Observatory during the spring and the autumn migration in 2008. 822 immature I. ricinus ticks were collected from 215 infested birds. Ticks were investigated for infection with B. burgdorferi s.l. by real-time PCR amplification of the 16S rRNA gene, and B. burgdorferi s.l. were thereafter genotyped by melting curve analysis after real-time PCR amplification of the hbb gene, or by direct sequencing of the PCR amplicon generated from the rrs (16S)-rrl (23S) intergenetic spacer. Results B. burgdorferi s.l. were detected in 4.4% of the ticks. The most prevalent B. burgdorferi genospecies identified were B. garinii (77.8%), followed by B.valaisiana (11.1%), B. afzelii (8.3%) and B. burgdorferi sensu stricto (2.8%). Conclusion Infection rate in ticks and genospecies composition were similar in spring and autumn migration, however, the prevalence of ticks on birds was higher during spring migration. The study supports the notion that birds are important in the dispersal of ticks, and that they may be partly responsible for the heterogeneous distribution of B. burgdorferi s.l. in Europe. PMID:21054890

  6. Detection of Lyme Disease Bacterium, Borrelia burgdorferi sensu lato, in Blacklegged Ticks Collected in the Grand River Valley, Ontario, Canada

    PubMed Central

    Scott, John D.; Foley, Janet E.; Anderson, John F.; Clark, Kerry L.; Durden, Lance A.

    2017-01-01

    We document the presence of blacklegged ticks, Ixodes scapularis, in the Grand River valley, Centre Wellington, Ontario. Overall, 15 (36%) of 42 I. scapularis adults collected from 41 mammalian hosts (dogs, cats, humans) were positive for the Lyme disease bacterium, Borrelia burgdorferi sensu lato (s.l.). Using real-time PCR testing and DNA sequencing of the flagellin (fla) gene, we determined that Borrelia amplicons extracted from I. scapularis adults belonged to B. burgdorferi sensu stricto (s.s.), which is pathogenic to humans and certain domestic animals. Based on the distribution of I. scapularis adults within the river basin, it appears likely that migratory birds provide an annual influx of I. scapularis immatures during northward spring migration. Health-care providers need to be aware that local residents can present with Lyme disease symptoms anytime during the year. PMID:28260991

  7. Tick vectors of Cercopithifilaria bainae in dogs: Rhipicephalus sanguineus sensu lato versus Ixodes ricinus.

    PubMed

    Ramos, Rafael Antonio Nascimento; Giannelli, Alessio; Brianti, Emanuele; Annoscia, Giada; Cantacessi, Cinzia; Dantas-Torres, Filipe; Otranto, Domenico

    2013-08-01

    Recently, dermal microfilariae of a Cercopithifilaria species (Spirurida, Onchocercidae), namely Cercopithifilaria bainae , were detected in dogs from several geographical areas of the Mediterranean basin. Evidence from both laboratory and field studies support the role of the brown dog tick, Rhipicephalus sanguineus sensu lato, as an intermediate host of this nematode. In the present study, we investigated the competence of Ixodes ricinus nymphs as vectors of C. bainae. On November 2012, fully engorged nymphs of I. ricinus (n = 174) and R. sanguineus s.l. (n = 10) were collected from a dog infected by C. bainae. The presence of C. bainae in I. ricinus was assessed by both microscopic dissection of specimens and detection of nematode DNA (PCR), at days 3, 10, 20 and 30 (T1-T4) post-collection; due to the small number of specimens available, R. sanguineus s.l. were examined using the same methods at T4 only. No developing larva of C. bainae was detected in I. ricinus specimens at different time points (T1-T4), even if four of these specimens were PCR-positive at T1. Seven out of ten R. sanguineus s.l. were positive for C. bainae third-stage larvae (L3) at both microscopical and molecular analysis at T4. This study indicates that C. bainae does not develop in I. ricinus nymphs, which preclude the role of this tick as an intermediate host of this parasite. Data presented herein provide new insights into the biology of this filarioid species and will lead to a better understanding of the role of different tick species as vectors of nematodes.

  8. Borrelia burgdorferi sensu lato in Ixodes cf. neuquenensis and Ixodes sigelos ticks from the Patagonian region of Argentina.

    PubMed

    Sebastian, Patrick S; Bottero, Maria Noelia Saracho; Carvalho, Luis; Mackenstedt, Ute; Lareschi, Marcela; Venzal, José M; Nava, Santiago

    2016-10-01

    This study was conducted to detect Borrelia burgdorferi sensu lato infection in ixodid ticks from the Patagonia region in the south of Argentina. Therefore, ticks were collected on rodents in the provinces of Chubut, Río Negro and Santa Cruz. These ticks were identified as nymphs of Ixodes cf. neuquenensis and Ixodes sigelos. The B. burgdorferi s.l. infection was tested by a battery of PCR methods targeting the gene flagellin (fla) and the rrfA-rrlB intergenic spacer region (IGS). Three pools of I. sigelos nymphs from Chubut and Santa Cruz provinces as well as one pool of I. cf. neuquenensis nymphs from Río Negro province were tested positive in the fla-PCR. The samples of I. sigelos were also positive for the IGS-PCR. Phylogenetically, the haplotypes found in the positive ticks belong to the B. burgdorferi s.l. complex, and they were closely related to Borrelia chilensis, a genospecies isolated from Ixodes stilesi in Chile. The pathogenic relevance of the Borrelia genospecies detected in both I. neuquenensis and I. sigelos is unknown.

  9. Lyme borreliosis caused by diverse genospecies of Borrelia burgdorferi sensu lato in northeastern China.

    PubMed

    Ni, X-B; Jia, N; Jiang, B-G; Sun, T; Zheng, Y-C; Huo, Q-B; Liu, K; Ma, L; Zhao, Q-M; Yang, H; Wang, X; Jiang, J-F; Cao, W-C

    2014-08-01

    The variety of Borrelia burgdorferi sensu lato (B. burgdorferi) genospecies leads to distinction in clinical manifestations of Lyme borreliosis (LB). There are reports of LB clinical characteristics in China, where the B. burgdorferi genospecies in ticks and animal hosts are different from those in Europe and North America. During May to September in 2010 and 2011, all patients who had erythema migrans (EM, more than 5 cm in diameter) after a recent tick-bite, and sought medical care at Mudanjiang Forestry Central Hospital, Heilongjiang Province of northeastern China, were enrolled in the study. Specific PCR was used to determine the B. burgdorferi genospecies in the disseminated patients. Of 265 EM patients, B. burgdorferi DNA was detected in blood specimens from 15 of 55 disseminated patients. Sequence analyses of 5S-23S rRNA, flagellin, ospC, 16S rRNA and ospA genes revealed that 11 patients were infected with Borrelia garinii, three with Borrelia afzelii and one with Borrelia valaisiana-related genospecies. Among 15 patients, 40%, 13.3% and 13.3% manifested pruritus, pain and ulceration, respectively. Systemic symptoms, arthralgia or a swollen joint and lymphadenopathy were observed in 26.7%, 13.3% and 6.7% patients, respectively. In northeastern China, three genospecies of LB patients were detected. The B. burgdorferi genospecies identified in this study was predominantly B. garinii. A case infected with B. valaisiana-related genospecies was reported for the first time.

  10. Automated purification of Borrelia burgdorferi s.l. PCR products with KingFisher™ magnetic particle processor prior to genome sequencing

    NASA Astrophysics Data System (ADS)

    Mäkinen, Johanna; Marttila, Harri; Viljanen, Matti K.

    2001-01-01

    Borrelia burgdorferi sensu lato genospecies were differentiated by PCR-based sequencing of the borrelial flagellin gene. To evaluate the usefulness of KingFisher™ magnetic particle processor in PCR product purification, borrelia PCR products were purified with KingFisher™ magnetic particle processor prior to cycle sequencing and the quality of the sequence data received was analyzed. KingFisher was found to offer a rapid and reliable alternative for borrelial PCR product purification.

  11. Infection of Ixodes ricinus (Acari: Ixodidae) by Borrelia burgdorferi sensu lato in North Africa

    USGS Publications Warehouse

    Zhioua, E.; Bouattour, A.; Hu, C.M.; Gharbi, M.; Aeschliman, A.; Ginsberg, H.S.; Gern, L.

    1999-01-01

    Free-living adult Ixodes ricinus L. were collected in Amdoun, situated in the Kroumiry mountains in northwestern Tunisia (North Africa). Using direct fluorescence antibody assay, the infection rate of field-collected I. ricinus by Borrelia burgdorferi sensu lato was 30.5% (n = 72). No difference in infection rate was observed between male and female ticks. Spirochetes that had been isolated from I. ricinus from Ain Drahim (Kroumiry Mountains) in 1988 were identified as Borrelia lusitaniae (formerly genospecies PotiB2). This is the first identification of a genospecies of Borrelia burgdorferi sensu lato from the continent of Africa.

  12. The Western Progression of Lyme Disease: Infectious and Nonclonal Borrelia burgdorferi Sensu Lato Populations in Grand Forks County, North Dakota

    PubMed Central

    Stone, Brandee L.; Russart, Nathan M.; Gaultney, Robert A.; Floden, Angela M.; Vaughan, Jefferson A.

    2014-01-01

    Scant attention has been paid to Lyme disease, Borrelia burgdorferi, Ixodes scapularis, or reservoirs in eastern North Dakota despite the fact that it borders high-risk counties in Minnesota. Recent reports of B. burgdorferi and I. scapularis in North Dakota, however, prompted a more detailed examination. Spirochetes cultured from the hearts of five rodents trapped in Grand Forks County, ND, were identified as B. burgdorferi sensu lato through sequence analyses of the 16S rRNA gene, the 16S rRNA gene-ileT intergenic spacer region, flaB, ospA, ospC, and p66. OspC typing revealed the presence of groups A, B, E, F, L, and I. Two rodents were concurrently carrying multiple OspC types. Multilocus sequence typing suggested the eastern North Dakota strains are most closely related to those found in neighboring regions of the upper Midwest and Canada. BALB/c mice were infected with B. burgdorferi isolate M3 (OspC group B) by needle inoculation or tick bite. Tibiotarsal joints and ear pinnae were culture positive, and B. burgdorferi M3 was detected by quantitative PCR (qPCR) in the tibiotarsal joints, hearts, and ear pinnae of infected mice. Uninfected larval I. scapularis ticks were able to acquire B. burgdorferi M3 from infected mice; M3 was maintained in I. scapularis during the molt from larva to nymph; and further, M3 was transmitted from infected I. scapularis nymphs to naive mice, as evidenced by cultures and qPCR analyses. These results demonstrate that isolate M3 is capable of disseminated infection by both artificial and natural routes of infection. This study confirms the presence of unique (nonclonal) and infectious B. burgdorferi populations in eastern North Dakota. PMID:25304515

  13. The Western progression of lyme disease: infectious and Nonclonal Borrelia burgdorferi Sensu Lato populations in Grand Forks County, North Dakota.

    PubMed

    Stone, Brandee L; Russart, Nathan M; Gaultney, Robert A; Floden, Angela M; Vaughan, Jefferson A; Brissette, Catherine A

    2015-01-01

    Scant attention has been paid to Lyme disease, Borrelia burgdorferi, Ixodes scapularis, or reservoirs in eastern North Dakota despite the fact that it borders high-risk counties in Minnesota. Recent reports of B. burgdorferi and I. scapularis in North Dakota, however, prompted a more detailed examination. Spirochetes cultured from the hearts of five rodents trapped in Grand Forks County, ND, were identified as B. burgdorferi sensu lato through sequence analyses of the 16S rRNA gene, the 16S rRNA gene-ileT intergenic spacer region, flaB, ospA, ospC, and p66. OspC typing revealed the presence of groups A, B, E, F, L, and I. Two rodents were concurrently carrying multiple OspC types. Multilocus sequence typing suggested the eastern North Dakota strains are most closely related to those found in neighboring regions of the upper Midwest and Canada. BALB/c mice were infected with B. burgdorferi isolate M3 (OspC group B) by needle inoculation or tick bite. Tibiotarsal joints and ear pinnae were culture positive, and B. burgdorferi M3 was detected by quantitative PCR (qPCR) in the tibiotarsal joints, hearts, and ear pinnae of infected mice. Uninfected larval I. scapularis ticks were able to acquire B. burgdorferi M3 from infected mice; M3 was maintained in I. scapularis during the molt from larva to nymph; and further, M3 was transmitted from infected I. scapularis nymphs to naive mice, as evidenced by cultures and qPCR analyses. These results demonstrate that isolate M3 is capable of disseminated infection by both artificial and natural routes of infection. This study confirms the presence of unique (nonclonal) and infectious B. burgdorferi populations in eastern North Dakota.

  14. Borrelia burgdorferi sensu lato infection in larval Ixodes ricinus (Acari: Ixodidae) feeding on blackbirds in northwestern Italy.

    PubMed

    Mannelli, Alessandro; Nebbia, Patrizia; Tramuta, Clara; Grego, Elena; Tomassone, Laura; Ainardi, Romina; Venturini, Lucia; De Meneghi, Daniele; Meneguz, Pier Giuseppe

    2005-03-01

    Birds belonging to 59 species (n = 1,206) were live captured in Piemonte, northwestern Italy, in 2001. Ixodes ricinus (L.) larvae were collected from 59 birds belonging to nine species, and nymphs were recovered on 79 birds belonging to 10 species. Eurasian blackbirds, Turdus merula L., had significantly higher levels of infestation by ticks than other passerine species. Larval I. ricinus of blackbirds peaked in summer, when prevalence was 39% (95% confidence interval 24.2-55.5) and mean number of ticks per host was 3.3 (1.6-7.2), whereas nymphs peaked in spring, when prevalence was 72.2% (54.8-85.8) and mean number of ticks per host was 6.9 (4.4-10.7). Immature I. ricinus were coincidentally aggregated on blackbirds, with 15 blackbirds feeding 67.4% of nymphs and 40.3% of larvae, and coinfestation by both stages was relatively high in summer: Kappa = 0.64 (0.40-0.88). Borrelia burgdorferi sensu lato was identified by polymerase chain reaction (PCR) in 58.3% (35.9-78.5) of larvae with engorgement ratio > or = 3 that were collected from blackbirds. Larvae that were collected from other passerine species gave negative PCR results. Sixteen of 21 PCR-positive samples belonged to B. garinii (76.2%), and five (23.8%) were Borrelia valaisiana. Results of this study suggest that blackbirds play an important role as hosts for immature I. ricinus and as reservoir of Borrelia garinii in northwestern Italy.

  15. Multilocus sequence typing of Pseudomonas syringae sensu lato confirms previously described genomospecies and permits rapid identification.

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Since 2002, severe leaf spotting on parsley (Petroselinum crispum L.) has occurred in Monterey County, California. One of two different pathovars of Pseudomonas syringae sensu lato were isolated from diseased leaves from seven distinct outbreaks and twice from the same outbreak (2002 and 2009). Frag...

  16. A new species of Chaenusa Haliday sensu lato (Hymenoptera: Braconidae) from the Nearctic Region

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Chaenusa psillosae Kula, new species from the Nearctic Region is described. Specimens upon which the new species is described were reared from an undetermined species of Hydrellia Robineau-Desvoidy infesting Sagittaria latifolia Willd. A key to the New World species of Chaenusa sensu lato is amended...

  17. Habitat-Specific Diversity of Borrelia burgdorferi Sensu Lato in Europe, Exemplified by Data from Latvia

    PubMed Central

    Etti, Susanne; Hails, Rosie; Schäfer, Stefanie M.; De Michelis, Simona; Sewell, Henna-Sisko; Bormane, Antra; Donaghy, Michael; Kurtenbach, Klaus

    2003-01-01

    The distribution of Borrelia burgdorferi sensu lato genospecies in questing Ixodes ricinus ticks from ecologically distinct habitats in Latvia was analyzed. A significant variation in the frequency of the genospecies across sites was observed, pointing to the importance of the host community in the ecology of Lyme borreliosis. PMID:12732580

  18. Use of FTA(®) card methodology for sampling and molecular characterization of Echinococcus granulosus sensu lato in Africa.

    PubMed

    Boué, Franck; El Berbri, Ikhlass; Hormaz, Vanessa; Boucher, Jean-Marc; El Mamy, Ahmed Bezeid; Traore, Abdallah; Fihri, Ouafaa Fassi; Petavy, Anne-Françoise; Dakkak, Allal; Umhang, Gérald

    2017-02-01

    Cystic Echinococcosis is a parasitic disease caused by the cestode Echinococcus granulosus widely distributed in Africa. Monitoring of this parasite requires access to cyst samples on intermediate hosts observed at the slaughterhouse. In order to facilitate sampling in the field and analysis, the French National Reference Laboratory for Echinococcus spp. has developed a tissue derived from DNA sampling with FTA(®) card technology. The DNA samples were taken by applying the FTA(®) paper on the germinal layer after opening the cysts. The sampling technique was validated using frozen cysts (n = 76) stored in the laboratory and from field samples (n = 134) taken at the slaughterhouse by veterinarian technicians during meat inspection in Morocco, Mali and Mauritania. DNA was extracted after several weeks of storage at room temperature. PCR assays were performed using primers for generic cestode (cox1) and amplified fragments were sequenced. All samples taken in the lab and 80% of field samples were capable of molecular characterization. Cyst-derived DNA from FTA(®) samples can be useful for easy sampling, storage and rapid, safe and cheap shipment. The use of the FTA methodology will facilitate studies in the field to investigate the presence and genetic characterization of E. granulosus sensu lato in African countries.

  19. Evaluation of Borrelia real time PCR DNA targeting OspA, FlaB and 5S-23S IGS and Borrelia 16S rRNA RT-qPCR.

    PubMed

    de Leeuw, Bertie H C G M; Maraha, Boulos; Hollemans, Leonie; Sprong, Hein; Brandenburg, Afke H; Westenend, Pieter J; Kusters, Johannes G

    2014-12-01

    Borrelia burgdorferi non-sensu lato (s.l.) strains occurred in the Netherlands. A multiplex OspA, FlaB, IGS real time PCR was compared to 16S rRNA/rDNA RT-qPCR with lower average Cycle threshold (Ct) and LOD on strain dilutions. Multiplexing increased sensitivity on CSF samples (n=74), distinguishing B. burgdorferi s.l. from non-s.l. strains.

  20. Environmental Contamination by Echinococcus granulosus sensu lato Eggs in Relation to Slaughterhouses in Urban and Rural Areas in Tunisia

    PubMed Central

    Chaâbane-Banaoues, Raja; Oudni-M’rad, Myriam; M’rad, Selim; Mezhoud, Habib; Babba, Hamouda

    2016-01-01

    Hydatidosis has become a real concern for health care institutions and animal rearers in Tunisia. The Tunisian endemicity is aggravated by the growing number of dogs and the difficulty of getting rid of contaminated viscera because of the lack of equipment in most slaughterhouses. Therefore, microscopic and molecular tools were applied to evaluate the role of slaughterhouses in canine infection and Echinococcus granulosus sensu lato (s. l.) egg dissemination. Exposure risk to E. granulosus s. l. eggs in urban and rural areas was explored in order to implant preventive and adapted control strategies. Microscopic examinations detected taeniid eggs in 152 amongst 553 fecal samples. The copro-PCR demonstrated that 138 of 152 taeniid samples analyzed were positive for E. granulosus s. l. DNA. PCR-RFLP demonstrated that all isolated samples belonged to E. granulosus sensu stricto (s. s.). An important environmental contamination index (25.0%) by E. granulosus s. l. eggs was demonstrated. The average contamination index from the regions around slaughterhouses (23.3%; 95% CI: 17.7-28.9%) was in the same range as detected in areas located far from slaughterhouses (26.0%, 95% CI: 21.3-30.8%). Echinococcosis endemic areas were extended in both rural (29.9%, 95% CI: 24.8-34.9%) and urban locations (18.1%, 95% CI: 13.0-22.9%). The pathogen dissemination is related neither to the presence/absence of slaughterhouses nor to the location in urban or rural areas, but is probably influenced by human activities (home slaughtering) and behavior towards the infected viscera. PMID:26951990

  1. Molecular phylogeny of the megadiverse insect infraorder Bibionomorpha sensu lato (Diptera)

    PubMed Central

    Kaspřák, David; Mantič, Michal; Fitzgerald, Scott; Ševčíková, Tereza; Tóthová, Andrea; Jaschhof, Mathias

    2016-01-01

    The phylogeny of the insect infraorder Bibionomorpha (Diptera) is reconstructed based on the combined analysis of three nuclear (18S, 28S, CAD) and three mitochondrial (12S, 16S, COI) gene markers. All the analyses strongly support the monophyly of Bibionomorpha in both the narrow (sensu stricto) and the broader (sensu lato) concepts. The major lineages of Bibionomorpha sensu lato (Sciaroidea, Bibionoidea, Anisopodoidea, and Scatopsoidea) and most of the included families are supported as monophyletic groups. Axymyiidae was not found to be part of Bibionomorpha nor was it found to be its sister group. Bibionidae was paraphyletic with respect to Hesperinidae and Keroplatidae was paraphyletic with respect to Lygistorrhinidae. The included Sciaroidea incertae sedis (except Ohakunea Edwards) were found to belong to one clade, but the relationships within this group and its position within Sciaroidea require further study. PMID:27781163

  2. Rhipicephalus sanguineus sensu lato (Ixodidae) in synantropic rodents in Rio Grande do Sul, Brazil.

    PubMed

    Winkel, Kathleen Tavares; Ribeiro, Paulo Bretanha; Antunes, Lidiane Oliveira; Cárcamo, Marcial Corrêa; Vianna, Elvia Elena Silveira

    2014-01-01

    Rhipicephalus sanguineus, the brown dog tick, is responsible for maintaining and transmitting various pathogens, both in animals and human beings, and it is of great sanitary importance. This communication reports the first occurrence of Rhipicephalus sanguineus sensu lato parasitizing Rattus norvegicus in the state of Rio Grande do Sul, Brazil, and it is also the first record of this tick species parasitizing Rattus rattus in Brazil. The rodents were captured from the port area, located in the city of Pelotas, Rio Grande do Sul, Brazil. We collected 6 larvae of this tick species from 2 male R. rattus individuals, and 3 larvae from 2 female R. norvegicus individuals; parasitized specimens of both rodent species were captured from different sites within the experimental area. This record broadens the number of Rhipicephalus sanguineus sensu lato hosts in urban areas, indicating the need for continued monitoring on population density for both R. sanguineus and synanthropic rodents.

  3. Whole genome sequence of an unusual Borrelia burgdorferi sensu lato isolate

    SciTech Connect

    Casjens, S.R.; Dunn, J.; Fraser-Liggett, C. M.; Mongodin, E. F.; Qiu, W. G.; Luft, B. J.; Schutzer, S. E.

    2011-03-01

    Human Lyme disease is caused by a number of related Borrelia burgdorferi sensu lato species. We report here the complete genome sequence of Borrelia sp. isolate SV1 from Finland. This isolate is to date the closest known relative of B. burgdorferi sensu stricto, but it is sufficiently genetically distinct from that species that it and its close relatives warrant its candidacy for new-species status. We suggest that this isolate should be named 'Borrelia finlandensis.'

  4. Divergence of the SigB regulon and pathogenesis of the Bacillus cereus sensu lato group

    PubMed Central

    2012-01-01

    Background The Bacillus cereus sensu lato group currently includes seven species (B. cereus, B. anthracis, B. mycoides, B. pseudomycoides, B. thuringiensis, B. weihenstephanensis and B. cytotoxicus) that recent phylogenetic and phylogenomic analyses suggest are likely a single species, despite their varied phenotypes. Although horizontal gene transfer and insertion-deletion events are clearly important for promoting divergence among these genomes, recent studies have demonstrated that a major basis for phenotypic diversity in these organisms may be differential regulation of the highly similar gene content shared by these organisms. To explore this hypothesis, we used an in silico approach to evaluate the relationship of pathogenic potential and the divergence of the SigB-dependent general stress response within the B. cereus sensu lato group, since SigB has been demonstrated to support pathogenesis in Bacillus, Listeria and Staphylococcus species. Results During the divergence of these organisms from a common “SigB-less” ancestor, the placement of SigB promoters at varied locations in the B. cereus sensu lato genomes predict alternative structures for the SigB regulon in different organisms. Predicted promoter changes suggesting differential transcriptional control of a common gene pool predominate over evidence of indels or horizontal gene transfer for explaining SigB regulon divergence. Conclusions Four lineages of the SigB regulon have arisen that encompass different gene contents and suggest different strategies for supporting pathogenesis. This is consistent with the hypothesis that divergence within the B. cereus sensu lato group rests in part on alternative strategies for regulation of a common gene pool. PMID:23088190

  5. Fourier transform infrared spectroscopy of DNA from Borrelia burgdorferi sensu lato and Ixodes ricinus ticks

    NASA Astrophysics Data System (ADS)

    Muntean, Cristina M.; Stefan, Razvan; Bindea, Maria; Cozma, Vasile

    2013-06-01

    In this work we present a method for detection of motile and immotile Borrelia burgdorferi genomic DNA, in relation with infectious and noninfectious spirochetes. An FT-IR study of DNA isolated from B. burgdorferi sensu lato strains and from positive and negative Ixodes ricinus ticks, respectively, is reported. Motile bacterial cells from the species B. burgdorferi sensu stricto, Borrelia garinii and Borrelia afzelii were of interest. Also, FT-IR absorbance spectra of DNA from immotile spirochetes of B. burgdorferi sensu stricto, in the absence and presence of different antibiotics (doxycycline, erythromycin, gentamicin, penicillin V or phenoxymethylpenicillin, tetracycline, respectively) were investigated. FT-IR spectra, providing a high molecular structural information, have been analyzed in the wavenumber range 400-1800 cm-1. FT-IR signatures, spectroscopic band assignments and structural interpretations of these DNAs are reported. Spectral differences between FT-IR absorbances of DNAs from motile bacterial cells and immotile spirochetes, respectively, have been found. Particularly, alterations of the sugar-phosphate B-form chain in the case of DNA from Borrelia immotile cells, as compared with DNA from B. burgdorferi sensu lato motile cells have been observed. Based on this work, specific B. burgdorferi sensu lato and I. ricinus DNA-ligand interactions, respectively, might be further investigated using Fourier transform infrared spectroscopy.

  6. Molecular and Pathogenic Characterization of Borrelia burgdorferi Sensu Lato Isolates from Spain

    PubMed Central

    Escudero, Raquel; Barral, Marta; Pérez, Azucena; Vitutia, M. Mar; García-Pérez, Ana L.; Jiménez, Santos; Sellek, Ricela E.; Anda, Pedro

    2000-01-01

    Fifteen Borrelia burgdorferi sensu lato isolates from questing ticks and skin biopsy specimens from erythema migrans patients in three different areas of Spain were characterized. Four different genospecies were found (nine Borrelia garinii, including the two human isolates, three B. burgdorferi sensu stricto, two B. valaisiana, and one B. lusitaniae), showing a diverse spectrum of B. burgdorferi sensu lato species. B. garinii isolates were highly variable in terms of pulsed-field gel electrophoresis pattern and OspA serotype, with four of the seven serotypes described. One of the human isolates was OspA serotype 5, the same found in four of seven tick isolates. The second human isolate was OspA serotype 3, which was not present in ticks from the same area. Seven B. garinii isolates were able to disseminate through the skin of C3H/HeN mice and to cause severe inflammation of joints. One of the two B. valaisiana isolates also caused disease in mice. Only one B. burgdorferi sensu stricto isolate was recovered from the urinary bladder. One isolate each of B. valaisiana and B. lusitaniae were not able to disseminate through the skin of mice or to infect internal organs. In summary, there is substantial diversity in the species and in the pathogenicity of B. burgdorferi sensu lato in areas in northern Spain where Lyme disease is endemic. PMID:11060064

  7. Incidence, Antibiotic Susceptibility, and Toxin Profiles of Bacillus cereus sensu lato Isolated from Korean Fermented Soybean Products.

    PubMed

    Yim, Jin-Hyeok; Kim, Kwang-Yeop; Chon, Jung-Whan; Kim, Dong-Hyeon; Kim, Hong-Seok; Choi, Da-Som; Choi, In-Soo; Seo, Kun-Ho

    2015-06-01

    Korean fermented soybean products, such as doenjang, kochujang, ssamjang, and cho-kochujang, can harbor foodborne pathogens such as Bacillus cereus sensu lato (B. cereus sensu lato). The aim of this study was to characterize the toxin gene profiles, biochemical characteristics, and antibiotic resistance patterns of B. cereus sensu lato strains isolated from Korean fermented soybean products. Eighty-eight samples of Korean fermented soybean products purchased from retails in Seoul were tested. Thirteen of 26 doenjang samples, 13 of 23 kochujang samples, 16 of 30 ssamjang samples, and 5 of 9 cho-kochujang samples were positive for B. cereus sensu lato strains. The contamination level of all positive samples did not exceed 4 log CFU/g of food (maximum levels of Korea Food Code). Eighty-seven B. cereus sensu lato strains were isolated from 47 positive samples, and all isolates carried at least one enterotoxin gene. The detection rates of hblCDA, nheABC, cytK, and entFM enterotoxin genes among all isolates were 34.5%, 98.9%, 57.5%, and 100%, respectively. Fifteen strains (17.2%) harbored the emetic toxin gene. Most strains tested positive for salicin fermentation (62.1%), starch hydrolysis (66.7%), hemolysis (98.9%), motility test (100%), and lecithinase production (96.6%). The B. cereus sensu lato strains were highly resistant to β-lactam antibiotics such as ampicillin, penicillin, cefepime, imipenem, and oxacillin. Although B. cereus sensu lato levels in Korean fermented soybean products did not exceed the maximum levels permitted in South Korea (<10(4) CFU/g), these results indicate that the bacterial isolates have the potential to cause diarrheal or emetic gastrointestinal diseases.

  8. Expression Profiles of Toll-Like Receptors in the Differentiation of an Infection with Borrelia burgdorferi Sensu Lato Spirochetes.

    PubMed

    Dudek, Slawomir; Ziółko, Ewa; Kimsa-Dudek, Magdalena; Solarz, Krzysztof; Mazurek, Urszula; Wierzgoń, Aleksander; Kokot, Teresa; Muc-Wierzgoń, Małgorzata

    2017-04-01

    The similarity of Lyme borreliosis to other diseases and its complex pathogenesis present diagnostic and therapeutic difficulties. The changes that occur at the cellular and molecular levels after a Borrelia sp. infection still remain poorly understood. Therefore, the present study focused on the expression of TLR and TLR-signaling genes in human dermal fibroblasts in the differentiation of an infection with Borrelia burgdorferi sensu lato spirochetes. Normal human dermal fibroblasts were cultured with the spirochetes of Borrelia burgdorferi sensu stricto, Borrelia afzelii and Borrelia garinii. Total RNA was extracted from the cells using TRIzol reagent. The analysis of the expression profiles of TLRs and TLR-related genes was performed using commercially available oligonucleotide microarrays of HG-U133A. The GeneSpring 12.0 platform and significance analysis of microarrays were used for the statistical analysis of microarray data. The analyses using the oligonucleotide microarray and QRT-PCR techniques permitted to identify the genes encoding TLR4 and TLR6 as specific for infection with B. afzelii and B. burgdorferi sensu stricto. In turn, TLR3 was only characteristic for an infection with B. burgdorferi sensu stricto. There were no changes in the TLR gene expression after infection with B. garinii. Our findings confirm that Borrelia has a major effect on fibroblast gene expression. Further characterization of changes in gene expression may lead to valuable insights into the role of the toll-like receptor in the pathogenesis of Lyme disease and may provide guidelines for the development of diagnostic markers for an infection with a particular Borrelia genospecies. Moreover, this will help to identify better treatment strategies for Lyme disease.

  9. Comparative histology of floral elaiophores in the orchids Rudolfiella picta (Schltr.) Hoehne (Maxillariinae sensu lato) and Oncidium ornithorhynchum H.B.K. (Oncidiinae sensu lato)

    PubMed Central

    Davies, Kevin L.; Stpiczyńska, Malgorzata

    2009-01-01

    Background and Aims Floral elaiophores, although widespread amongst orchids, have not previously been described for Maxillariinae sensu lato. Here, two claims that epithelial, floral elaiophores occur in the genus Rudolfiella Hoehne (Bifrenaria clade) are investigated. Presumed elaiophores were compared with those of Oncidiinae Benth. and the floral, resin-secreting tissues of Rhetinantha M.A. Blanco and Heterotaxis Lindl., both genera formerly assigned to Maxillaria Ruiz & Pav. (Maxillariinae sensu stricto). Methods Putative, floral elaiophore tissue of Rudolfiella picta (Schltr.) Hoehne and floral elaiophores of Oncidium ornithorhynchum H.B.K. were examined by means of light microscopy, histochemistry, scanning electron microscopy and transmission electron microscopy. Key Results and Conclusions Floral, epithelial elaiophores are present in Rudolfiella picta, indicating, for the first time, that oil secretion occurs amongst members of the Bifrenaria clade (Maxillariinae sensu lato). However, whereas the elaiophore of R. picta is borne upon the labellar callus, the elaiophores of O. ornithorhynchum occur on the lateral lobes of the labellum. In both species, the elaiophore comprises a single layer of palisade secretory cells and parenchymatous, subsecretory tissue. Cell wall cavities are absent from both and there is no evidence of cuticular distension in response to oil accumulation between the outer tangential wall and the overlying cuticle in R. picta. Distension of the cuticle, however, occurs in O. ornithorhynchum. Secretory cells of R. picta contain characteristic, spherical or oval plastids with abundant plastoglobuli and these more closely resemble plastids found in labellar, secretory cells of representatives of Rhetinantha (formerly Maxillaria acuminata Lindl. alliance) than elaiophore plastids of Oncidiinae. In Rhetinantha, such plastids are involved in the synthesis of resin-like material or wax. Despite these differences, the elaiophore anatomy of

  10. Revisiting phylogenetic diversity and cryptic species of Cenococcum geophilum sensu lato.

    PubMed

    Obase, Keisuke; Douhan, Greg W; Matsuda, Yosuke; Smith, Matthew E

    2016-08-01

    The fungus Cenococcum geophilum Fr. (Dothideomycetes, Ascomycota) is one of the most common ectomycorrhizal fungi in boreal to temperate regions. A series of molecular studies has demonstrated that C. geophilum is monophyletic but a heterogeneous species or a species complex. Here, we revisit the phylogenetic diversity of C. geophilum sensu lato from a regional to intercontinental scale by using new data from Florida (USA) along with existing data in GenBank from Japan, Europe, and North America. The combination of internal transcribed spacer (ITS) ribosomal DNA and the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene resolved six well-supported lineages (87-100 % bootstrap values) that are closely related to each other and a seventh lineage that is phylogenetically distinct. A multi-locus analysis (small subunit (SSU), large subunit (LSU), translational elongation factor (TEF), and the largest and second-largest subunits of RNA polymerase II (RPB1 and RPB2)) revealed that the divergent lineage is the sister group to all other known Cenococcum isolates. Isolates of the divergent lineage grow fast on nutrient media and do not form ectomycorrhizas on seedlings of several pine and oak species. Our results indicate that C. geophilum sensu lato includes more phylogenetically distinct cryptic species than have previously been reported. Furthermore, the divergent lineage appears to be a non-mycorrhizal sister group. We discuss the phylogenetic diversity of C. geophilum sensu lato and argue in favor of species recognition based on phylogenetic and ecological information in addition to morphological characteristics. A new genus and species (Pseudocenococcum floridanum gen. et sp. nov.) is proposed to accommodate a divergent and putatively non-mycorrhizal lineage.

  11. Prevalence of Borrelia burgdorferi Sensu Lato in Ticks Collected from Migratory Birds in Switzerland

    PubMed Central

    Poupon, Marie-Angèle; Lommano, Elena; Humair, Pierre-François; Douet, Véronique; Rais, Olivier; Schaad, Michael; Jenni, Lukas; Gern, Lise

    2006-01-01

    The prevalence of ticks infected by Borrelia burgdorferi sensu lato on birds during their migrations was studied in Switzerland. A total of 1,270 birds captured at two sites were examined for tick infestation. Ixodes ricinus was the dominant tick species. Prevalences of tick infestation were 6% and 18.2% for birds migrating northward and southward, respectively. Borrelia valaisiana was the species detected most frequently in ticks, followed by Borrelia garinii and Borrelia lusitaniae. Among birds infested by infected ticks, 23% (6/26) were infested by B. lusitaniae-infected larvae. Migratory birds appear to be reservoir hosts for B. lusitaniae. PMID:16391149

  12. First documentation of ivermectin resistance in Rhipicephalus sanguineus sensu lato (Acari: Ixodidae).

    PubMed

    Rodriguez-Vivas, R I; Ojeda-Chi, M M; Trinidad-Martinez, I; Pérez de León, A A

    2017-01-15

    The brown dog tick, Rhipicephalus sanguineus sensu lato (Latreille, 1806), is an ectoparasite and disease vector of significant veterinary and public health importance that is distributed widely around the world. The intensive use of synthetic acaricides for tick control exerts a strong selective pressure for brown dog ticks to become resistant to them. Here, we investigated claims from the field regarding treatment failure associated with the use of veterinary products containing ivermectin (IVM) to control brown dog ticks infesting dogs in Yucatan state, Mexico. Dogs in six state municipalities were inspected to sample 15 R. sanguineus s.l.

  13. 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

  14. Sporothrix schenckii Sensu Lato identification in fragments of skin lesion cultured in NNN medium for differential diagnosis of cutaneous leishmaniasis.

    PubMed

    Antonio, Liliane de Fátima; Pimentel, Maria Inês Fernandes; Lyra, Marcelo Rosandiski; Madeira, Maria de Fátima; Miranda, Luciana de Freitas Campos; Paes, Rodrigo Almeida; Brito-Santos, Fábio; Carvalho, Maria Helena Galdino Figueredo; Schubach, Armando de Oliveira

    2017-02-01

    Eighty-nine patients with clinical suspicion of leishmaniasis were referred for differential diagnosis. Sporothrix schenckii sensu lato was isolated in Novy-MacNeal-Nicolle + Schneider media in 98% of 64 patients with final diagnosis of sporotrichosis. This medium may be suitable for diagnosis of sporotrichosis in areas where cutaneous leishmaniasis is also endemic.

  15. An Invasive Mammal (the Gray Squirrel, Sciurus carolinensis) Commonly Hosts Diverse and Atypical Genotypes of the Zoonotic Pathogen Borrelia burgdorferi Sensu Lato.

    PubMed

    Millins, Caroline; Magierecka, Agnieszka; Gilbert, Lucy; Edoff, Alissa; Brereton, Amelia; Kilbride, Elizabeth; Denwood, Matt; Birtles, Richard; Biek, Roman

    2015-07-01

    Invasive vertebrate species can act as hosts for endemic pathogens and may alter pathogen community composition and dynamics. For the zoonotic pathogen Borrelia burgdorferi sensu lato, the agent of Lyme borreliosis, recent work shows invasive rodent species can be of high epidemiological importance and may support host-specific strains. This study examined the role of gray squirrels (Sciurus carolinensis) (n = 679), an invasive species in the United Kingdom, as B. burgdorferi sensu lato hosts. We found that gray squirrels were frequently infested with Ixodes ricinus, the main vector of B. burgdorferi sensu lato in the United Kingdom, and 11.9% were infected with B. burgdorferi sensu lato. All four genospecies that occur in the United Kingdom were detected in gray squirrels, and unexpectedly, the bird-associated genospecies Borrelia garinii was most common. The second most frequent infection was with Borrelia afzelii. Genotyping of B. garinii and B. afzelii produced no evidence for strains associated with gray squirrels. Generalized linear mixed models (GLMM) identified tick infestation and date of capture as significant factors associated with B. burgdorferi sensu lato infection in gray squirrels, with infection elevated in early summer in squirrels infested with ticks. Invasive gray squirrels appear to become infected with locally circulating strains of B. burgdorferi sensu lato, and further studies are required to determine their role in community disease dynamics. Our findings highlight the fact that the role of introduced host species in B. burgdorferi sensu lato epidemiology can be highly variable and thus difficult to predict.

  16. Comparison of isolation rate of Borrelia burgdorferi sensu lato in two different culture media, MKP and BSK-H.

    PubMed

    Ružić-Sabljić, E; Maraspin, V; Cimperman, J; Strle, F; Lotrič-Furlan, S; Stupica, D; Cerar, T

    2014-07-01

    The aim of the study was to evaluate two culture media for Borrelia burgdorferi sensu lato isolation from a 5 × 2 × 2 mm skin biopsy that was dissected into two pieces and inoculated into modified Kelly-Pettenkofer (MKP) and Barbour-Stoenner-Kelly-H (BSK-H) medium. Samples were incubated at 33°C for up to 9 weeks. Borrelia species was determined by MluI-restriction of whole genome or by MseI-restriction of PCR product. We determined the proportion of isolation rate, 'slow-growers', contaminated specimens and Borrelia species in the two media. In each of the two media 235 skin specimens were cultivated. We found 90/470 (19.1%) contaminated cultures (BSK-H 67/235, 28.5%; MKP 23/235, 9.8%; p <0.0001). Borrelia growth was ascertained in 59/235 (25.1%) BSK-H and 102/235 (43.4%) MKP cultures (p <0.0001); the corresponding values for non-contaminated cultures were 59/168 (35.1%) and 102/212 (48.1%); (p 0.003). Fourteen specimens were positive only in BSK-H, 57 solely in MKP, and 43 in both culture media. Slow growth was present in 8/59 (13.6%) BSK-H and in 4/98 (4.1%) MKP positive cultures (p 0.019). Borrelia afzelii was identified in 44/51 (86.3%) BSK-H and in 88/98 (89.8%) MKP culture-positive samples; the corresponding findings for Boreelia garinii and B. burgdorferi sensu stricto were 6/51 (11.8%) and 9/98 (9.2%), and 1/51 (1.9%) and 1/98 (1.0%), for BSK-H and MKP, respectively. Comparison of MKP and BSK-H medium for Borrelia culturing from skin specimens of European patients with erythema migrans revealed the advantage of MKP over BSK-H.

  17. Infection with Ehrlichia canis and Anaplasma platys (Rickettsiales: Anaplasmataceae) in two lineages of Rhipicephalus sanguineus sensu lato (Acari: Ixodidae) from Argentina.

    PubMed

    Cicuttin, Gabriel L; Tarragona, Evelina L; De Salvo, M Nazarena; Mangold, Atilio J; Nava, Santiago

    2015-09-01

    Natural infection with Ehrlichia canis and Anaplasma platys in ticks belonging to the tropical and temperate lineages of Rhipicephalus sanguineus sensu lato from Argentina was evaluated. Samples were tested for Ehrlichia canis infection by PCR assays using 16S rRNA, dsb and p28 gene, while detection of A. platys was performed with 16S rRNA and groESL gene. The assignment of the ticks to each lineage was corroborated with 16S rDNA sequences. All ticks infected with E. canis and A. platys belonged to the tropical lineage. These results constitute the first record of E. canis infection in R. sanguineus s.l ticks from Argentina. No ticks from the temperate lineage were found to be infected with E. canis, coinciding with previous studies performed in Argentina and Uruguay where E. canis infection was not detected in R. sanguineus s.l from the temperate lineage. Because the presence of the tropical lineage of R. sanguineus s.l has been documented in tropical areas of northern Argentina between 22° and 24° of south latitude, the findings of this work indicate that transmission of E. canis and A. platys to dogs by R. sanguineus s.l probably occurs along this region.

  18. Borrelia chilensis, a new member of the Borrelia burgdorferi sensu lato complex that extends the range of this genospecies in the Southern Hemisphere

    PubMed Central

    Ivanova, Larisa B.; Tomova, Alexandra; González-Acuña, Daniel; Murúa, Roberto; Moreno, Claudia X.; Hernández, Claudio; Cabello, Javier; Cabello, Carlos; Daniels, Thomas J.; Godfrey, Henry P.; Cabello, Felipe C.

    2014-01-01

    Summary Borrelia burgdorferi sensu lato (s.l.), transmitted by Ixodes spp. ticks, is the causative agent of Lyme disease. Although Ixodes spp. ticks are distributed in both Northern and Southern Hemispheres, evidence for the presence of B. burgdorferi s.l. in South America apart from Uruguay is lacking. We now report the presence of culturable spirochetes with flat-wave morphology and borrelial DNA in endemic Ixodes stilesi ticks collected in Chile from environmental vegetation and long-tailed rice rats (Oligoryzomys longicaudatus). Cultured spirochetes and borrelial DNA in ticks were characterized by multilocus sequence typing and by sequencing five other loci (16S and 23S ribosomal genes, 5S-23S intergenic spacer, flaB, ospC). Phylogenetic analysis placed this spirochete as a new genospecies within the Lyme borreliosis group. Its plasmid profile determined by PCR and pulsed-field gel electrophoresis differed from that of B. burgdorferi B31A3. We propose naming this new South American member of the Lyme borreliosis group Borrelia chilensis VA1, in honor of its country of origin. PMID:24148079

  19. Borrelia mayonii sp. nov., a member of the Borrelia burgdorferi sensu lato complex, detected in patients and ticks in the upper midwestern United States.

    PubMed

    Pritt, Bobbi S; Respicio-Kingry, Laurel B; Sloan, Lynne M; Schriefer, Martin E; Replogle, Adam J; Bjork, Jenna; Liu, Gongping; Kingry, Luke C; Mead, Paul S; Neitzel, David F; Schiffman, Elizabeth; Hoang Johnson, Diep K; Davis, Jeffrey P; Paskewitz, Susan M; Boxrud, David; Deedon, Alecia; Lee, Xia; Miller, Tracy K; Feist, Michelle A; Steward, Christopher R; Theel, Elitza S; Patel, Robin; Irish, Cole L; Petersen, Jeannine M

    2016-11-01

    Lyme borreliosis (LB) is a multisystem disease caused by spirochetes in the Borrelia burgdorferisensu lato (Bbsl) genospecies complex. We previously described a novel Bbsl genospecies (type strain MN14-1420T) that causes LB among patients with exposures to ticks in the upper midwestern USA. Patients infected with the novel Bbsl genospecies demonstrated higher levels of spirochetemia and somewhat differing clinical symptoms as compared with those infected with other Bbsl genospecies. The organism was detected from human specimens using PCR, microscopy, serology and culture. The taxonomic status was determined using an eight-housekeeping-gene (uvrA, rplB, recG, pyrG, pepX, clpX, clpA and nifS) multi-locus sequence analysis (MLSA) and comparison of 16S rRNA gene, flaB, rrf-rrl, ospC and oppA2 nucleotide sequences. Using a system threshold of 98.3 % similarity for delineation of Bbsl genospecies by MLSA, we demonstrated that the novel species is a member of the Bbsl genospecies complex, most closely related to B. burgdorferisensu stricto (94.7-94.9 % similarity). This same species was identified in Ixodes scapularis ticks collected in Minnesota and Wisconsin. This novel species, Borrelia mayonii sp. nov, is formally described here. The type strain, MN14-1420, is available through the Deutsche Sammlung von Mikroorganismen und Zelkulturen GmbH (DSM 102811) and the American Type Culture Collection (ATCC BAA-2743).

  20. Emergence of tick-borne pathogens (Borrelia burgdorferi sensu lato, Anaplasma phagocytophilum, Ricketsia raoultii and Babesia microti) in the Kyiv urban parks, Ukraine.

    PubMed

    Didyk, Yuliya M; Blaňárová, Lucia; Pogrebnyak, Svyatoslav; Akimov, Igor; Peťko, Branislav; Víchová, Bronislava

    2017-02-01

    To date, only limited data about the presence of ticks and circulation of tick-borne pathogens in urban parks of Kyiv in northern Ukraine are available. In total, 767 ticks (696 Ixodes ricinus and 69 Dermacentor reticulatus) collected in seven urban parks and one suburban oak wood park in Kyiv were individually analyzed by the PCR assays. Tick-borne pathogens, namely spirochetes from Borrelia burgdorferi sensu lato complex, Anaplasma phagocytophilum, and Babesia microti, were detected in 11.1% of tested I. ricinus ticks. In total, 4% of I. ricinus ticks tested positive for the presence of B. burdorferi s.l. (Borrelia afzelii and Borrelia garinii), 5.2% for A. phagocytophilum, and Ba. microti was confirmed in 1.9% of examined ticks. Mixed infections were recorded in four DNA samples, representing the prevalence of 0.6%. One female and two I. ricinus nymphs were simultaneously infected with B. afzelii and A. phagocytophilum, and one female carried B. afzelii and Ba. microti. In addition, 10.1% of D. reticulatus ticks tested positive for Rickettsia raoultii. Identification of infectious agents and their diversity, assessment of the relative epidemiological importance and determination of the prevalence in questing ticks from central parts of the cities are crucial steps towards the tick-borne diseases surveillance in urban environment.

  1. Human seroprevalence against Borrelia burgdorferi sensu lato in two comparable regions of the eastern Alps is not correlated to vector infection rates.

    PubMed

    Sonnleitner, S T; Margos, G; Wex, F; Simeoni, J; Zelger, R; Schmutzhard, E; Lass-Flörl, C; Walder, G

    2015-04-01

    Seroprevalences were determined by testing sera of 1607 blood donors from North, East, and South Tyrol. In the Tyrols, the continental divide delimitates areas with high seroprevalences of IgG antibodies against Borrelia burgdorferi sensu lato in the North (7.2%) from areas with low seroprevalences in the South (1.5%). To determine Borrelia prevalences in unfed Ixodes ricinus ticks, 755 questing ticks were tested by PCR. Prevalences in nymphal and adult ticks were found to be 19.7% (n=132) and 21.5% (n=205) in North Tyrol and 23% (n=43) and 23.7% (n=376) in South Tyrol, respectively. Sequencing of 46 Borrelia-positive ticks yielded 74% Borrelia (B.) afzelii, 11% B. garinii, 7% B. lusitaniae, 7% B. burgdorferi sensu stricto, and 2% B. valaisiana infections. Distinct genetic clusters could not be delimitated on either side of the continental divide. This study describes occurrence and geographic dispersion of Borrelia spp. in the Tyrols, discusses possible reasons for significant differences in human seroprevalence, and indicates that prevalence of Borrelia in vector ticks is not a direct predictive factor for the local seroprevalence in humans.

  2. Molecular, biological, and morphometric comparisons between different geographical populations of Rhipicephalus sanguineus sensu lato (Acari: Ixodidae).

    PubMed

    Sanches, Gustavo S; Évora, Patrícia M; Mangold, Atílio J; Jittapalapong, Sattaporn; Rodriguez-Mallon, Alina; Guzmán, Pedro E E; Bechara, Gervásio H; Camargo-Mathias, Maria I

    2016-01-15

    In this study, different geographical populations of Rhipicephalus sanguineus sensu lato were compared by molecular, biological, and morphometric methods. Phylogenetic trees were constructed using 12S and 16S rDNA sequences and showed two distinct clades: one composed of ticks from Brazil (Jaboticabal, SP), Cuba (Havana) Thailand (Bangkok) and the so-called "tropical strain" ticks. The second clade was composed of ticks from Spain (Zaragoza), Argentina (Rafaela, Santa Fe) and the so-called "temperate strain" ticks. Morphometric analysis showed good separation between females of the two clades and within the temperate clade. Males also exhibited separation between the two clades, but with some overlap. Multiple biological parameters revealed differences between the two clades, especially the weight of the engorged female. These results confirm the existence of at least two species under the name "R. sanguineus".

  3. Larvae of Ixodiphagus wasps (Hymenoptera: Encyrtidae) in Rhipicephalus sanguineus sensu lato ticks (Acari: Ixodidae) from Brazil.

    PubMed

    Bezerra Santos, Marcos Antônio; de Macedo, Lucia Oliveira; de Souza, Islanne Barbosa; do Nascimento Ramos, Carlos Alberto; Alves, Leucio Câmara; Ramos, Rafael Antonio Nascimento; de Carvalho, Gílcia Aparecida

    2017-03-21

    The biological control of ticks represents an alternative method to the chemical control, given its ecological-friendly approach. Amongst the alternatives, the use of parasitoids of the genus Ixodiphagus (Hymenoptera: Encyrtidae) has been largely investigated. The aim of this study was to document and molecularly characterize Ixodiphagus wasps in ticks from a tropical region of Brazil. From October 2015 to March 2016, Rhipicephalus sanguineus sensu lato ticks (n=1814) were collected from naturally infested dogs and Ixodiphagus larvae were detected by microscopic examination. In addition, adult wasps were obtained in the laboratory. Larvae and adults were molecularly identified as Ixodiphagus hookeri. These findings suggest that this type of parasitism deserves to be studied in local tick populations, in order to elucidate the role of these wasps as a potential alternative to chemical tick control.

  4. Helicobacter heilmannii sensu lato: An overview of the infection in humans

    PubMed Central

    Bento-Miranda, Mario; Figueiredo, Ceu

    2014-01-01

    Helicobacter heilmannii sensu lato (H. heilmannii s.l.) is a group of gastric non-Helicobacter pylori Helicobacter species that are morphologically indistinguishable from each other. H. heilmannii s.l. infect the stomach of several animals and may have zoonotic potential. Although the prevalence of these infections in humans is low, they are associated with gastric pathology, including mucosa-associated lymphoid tissue lymphoma, making them a significant health issue. Here, the taxonomy, epidemiology, microbiology, diagnosis, and treatment of these infections will be reviewed. The gastric pathology associated with H. heilmannii s.l. infections in humans will also be addressed. Finally, the features of the complete bacterial genomes available and studies on species-specific pathogenesis will be reviewed. The understanding of the mechanisms that underlie gastric disease development mediated by the different bacterial species that constitute H. heilmannii s.l. is essential for developing strategies for prevention and treatment of these infections. PMID:25548476

  5. The dynamic history of plastid genomes in the Campanulaceae sensu lato is unique among angiosperms

    PubMed Central

    Knox, Eric B.

    2014-01-01

    Why have some plants lost the organizational stability in plastid genomes (plastomes) that evolved in their algal ancestors? During the endosymbiotic transformation of a cyanobacterium into the eukaryotic plastid, most cyanobacterial genes were transferred to the nucleus or otherwise lost from the plastome, and the resulting plastome architecture in land plants confers organizational stability, as evidenced by the conserved gene order among bryophytes and lycophytes, whereas ferns, gymnosperms, and angiosperms share a single, 30-kb inversion. Although some additional gene losses have occurred, gene additions to angiosperm plastomes were previously unknown. Plastomes in the Campanulaceae sensu lato have incorporated dozens of large ORFs (putative protein-coding genes). These insertions apparently caused many of the 125+ large inversions now known in this small eudicot clade. This phylogenetically restricted phenomenon is not biogeographically localized, which indicates that these ORFs came from the nucleus or (less likely) a cryptic endosymbiont. PMID:25024223

  6. Advances in antitumor polysaccharides from phellinus sensu lato: Production, isolation, structure, antitumor activity, and mechanisms.

    PubMed

    Yan, Jing-Kun; Pei, Juan-Juan; Ma, Hai-Le; Wang, Zhen-Bin; Liu, Yuan-Shuai

    2017-04-13

    Edible and medicinal fungi (mushrooms) are widely applied to functional foods and nutraceutical products because of their proven nutritive and medicinal properties. Phellinus sensu lato is a well-known medicinal mushroom that has long been used in preventing ailments, including gastroenteric dysfunction, diarrhea, hemorrhage, and cancers, in oriental countries, particularly in China, Japan, and Korea. Polysaccharides represent a major class of bioactive molecules in Phellinus s. l., which have notable antitumor, immunomodulatory, and medicinal properties. Polysaccharides that were isolated from fruiting bodies, cultured mycelia, and filtrates of Phellinus s. l. have not only activated different immune responses of the host organism but have also directly suppressed tumor growth and metastasis. Studies suggest that polysaccharides from Phellinus s. l. are promising alternative anticancer agents or synergizers for existing antitumor drugs. This review summarizes the recent development of polysaccharides from Phellinus s. l., including polysaccharide production, extraction and isolation methods, chemical structure, antitumor activities, and mechanisms of action.

  7. A taxonomic review of the Neoserica (sensu lato) septemlamellata group (Coleoptera, Scarabaeidae, Sericini)

    PubMed Central

    Ahrens, Dirk; Liu, Wan-Gang; Fabrizi, Silvia; Bai, Ming; Yang, Xing-Ke

    2014-01-01

    Abstract In the present paper the species belonging to the Neoserica (sensu lato) septemlamellata group, that included so far only four known species, are revised. Here we describe eleven new species originating mainly from Indochina and Southern China: N. daweishanica sp. n., N. gaoligongshanica sp. n., N. guangpingensis sp. n., N. igori sp. n., N. jiulongensis sp. n., N. plurilamellata sp. n., N. weishanica sp. n., N. yanzigouensis sp. n. (China) N. sapaensis sp. n. (China, Vietnam), N. bansongchana sp. n., N. takakuwai sp. n. (Laos). The lectotypes of Neoserica septemlamellata Brenske, 1898 and N. septemfoliata Moser, 1915 are designated. Keys to the species and species groups are given, the genitalia of all species and their habitus are illustrated and distribution maps are included. PMID:24843263

  8. Borrelia burgdorferi sensu lato-infected Ixodes ricinus collected from vegetation near the Arctic Circle.

    PubMed

    Hvidsten, Dag; Stordal, Frode; Lager, Malin; Rognerud, Bjørg; Kristiansen, Bjørn-Erik; Matussek, Andreas; Gray, Jeremy; Stuen, Snorre

    2015-09-01

    This is the first study to determine the density of questing Ixodes ricinus in northern Norway. It was performed at two sites in Brønnøy, which has been known for its tick permissive habitats for decades and is one of the northernmost habitats with an abundant I. ricinus population in the world. From April to November 2011, all stages of host-seeking I. ricinus were collected from the two sites. The overall prevalence of nymphs infected with Borrelia burgdorferi sensu lato was 21% and that of adult ticks 46%. The rates of the genospecies Borrelia afzelii, Borrelia garinii, and Borrelia valaisiana were similar to findings in most other studies in Scandinavia, with B. afzelii by far the most prevalent at 76%. The high Borrelia-infection prevalence in ticks from Brønnøy may explain the high incidence rate of reported Lyme borreliosis in the municipality.

  9. 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.

  10. 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.

  11. Update of phylogenetic and genetic diversity of Sporothrix schenckii sensu lato.

    PubMed

    Rangel-Gamboa, Lucía; Martínez-Hernandez, Fernando; Maravilla, Pablo; Arenas-Guzmán, Roberto; Flisser, Ana

    2016-03-01

    Sporothrix schenckii sensu lato causes subcutaneous mycosis. In this article we analysed its phylogeny and genetic diversity using calmodulin DNA sequences deposited in GenBank database. Population genetics indices were calculated, plus phylogenetic and haplotype network trees were built. Five clades with high values of posterior probability, 47 haplotypes and high diversity in the complex were found. Analysis of partial calmodulin sequences alignment revealed conserved and polymorphic regions that could be used as reference for taxonomic identification. The use of population genetics analysis allowed understanding the phylogenetic proximity of S. schenckii s. str. and S. brasiliensis; scarce genetic flow among them with low migration index and high ancestry coefficient was found. Similarly, S. globosa, S. mexicana and S. pallida sequences showed highly differentiated species with no genetic exchange. The phylogenetic tree suggests that S. mexicana shared a common ancestor with S. pallida; while S. globosa and S. brasiliensis are more related to S. schenckii s. str. and showed less haplotype diversity and restrictions in geographic distribution. In the haplotype network tree S. schenckii s. str. species displayed worldwide distribution without dispersion centres; while S. brasiliensis and S. globosa, exhibited Brazil and Euro-Asia as dispersion centres, respectively. Our data suggest that S. schenckii complex has been submitted to a divergent evolution process, probably due to the pressure of the environment and of the host. In contrast, S. brasiliensis could have been submitted to purifying selection or expansion process.

  12. Current advances in Phellinus sensu lato: medicinal species, functions, metabolites and mechanisms.

    PubMed

    Dai, Yu-Cheng; Zhou, Li-Wei; Cui, Bao-Kai; Chen, Yan-Qiu; Decock, Cony

    2010-08-01

    Twenty-six species of Phellinus sensu lato, reported as medicinal mushrooms, are enumerated in this review. The species' names were checked and revised according to contemporary taxonomy and the latest version of the International Code of Botanical Nomenclature (Vienna Code); two misapplied names of Phellinus baumii Pilát and Phellinus himalayensis Y.C. Dai in previous reports are also discussed. Of the 20 types of medicinal functions, the most shared functions are antitumor and improving immunity, both of which may be viewed as the basal functions of Phellinus s. l. In addition, alleviating septic shock, anti-inflammatory, and antioxidation are also a routine functions mentioned often. The main medicinal metabolites, including several kinds of polysaccharides and polyphenols, are introduced. Different methods and conditions could purify various polysaccharides with difference in activity level even from the same species, while all polyphenols are hispidin and its derivatives in general. Three aspects of mechanism contribute to antitumor activities of polysaccharides: (1) promoting an immune response, (2) inducing cell apoptosis, and (3) inhibiting metastasis. Other general mechanisms of the metabolites in antioxidant activity, and in treating diabetes, as well as complications are summarized. We also elaborate on potential scientific strategies for obtaining the medicinal metabolites from Phellinus s. l., such as artificial cultivation, the discoveries of more species with medicinal functions, the utilization of species growing quickly, and the optimization of culture conditions and media supplements in fermentation.

  13. Identification of Bacillus anthracis spore component antigens conserved across diverse Bacillus cereus sensu lato strains.

    PubMed

    Mukhopadhyay, Sanghamitra; Akmal, Arya; Stewart, Andrew C; Hsia, Ru-Ching; Read, Timothy D

    2009-06-01

    We sought to identify proteins in the Bacillus anthracis spore, conserved in other strains of the closely related Bacillus cereus group, that elicit an immune response in mammals. Two high throughput approaches were used. First, an in silico screening identified 200 conserved putative B. anthracis spore components. A total of 192 of those candidate genes were expressed and purified in vitro, 75 of which reacted with the rabbit immune sera generated against B. anthracis spores. The second approach was to screen for cross-reacting antigens in the spore proteome of 10 diverse B. cereus group strains. Two-dimensional electrophoresis resolved more than 200 protein spots in each spore preparation. About 72% of the protein spots were found in all the strains. 18 of these conserved proteins reacted against anti-B. anthracis spore rabbit immune sera, two of which (alanine racemase, Dal-1 and the methionine transporter, MetN) overlapped the set of proteins identified using the in silico screen. A conserved repeat domain protein (Crd) was the most immunoreactive protein found broadly across B. cereus sensu lato strains. We have established an approach for finding conserved targets across a species using population genomics and proteomics. The results of these screens suggest the possibility of a multiepitope antigen for broad host range diagnostics or therapeutics against Bacillus spore infection.

  14. A molecular phylogeny and classification of Leptochloa (Poaceae: Chloridoideae: Chlorideae) sensu lato and related genera

    PubMed Central

    Peterson, Paul M.; Romaschenko, Konstantin; Snow, Neil; Johnson, Gabriel

    2012-01-01

    Background and Aims Leptochloa (including Diplachne) sensu lato (s.l.) comprises a diverse assemblage of C4 (NAD-ME and PCK) grasses with approx. 32 annual or perennial species. Evolutionary relationships and a modern classification of Leptochloa spp. based on the study of molecular characters have only been superficially investigated in four species. The goals of this study were to reconstruct the evolutionary history of Leptochloa s.l. with molecular data and broad taxon sampling. Methods A phylogenetic analysis was conducted of 130 species (mostly Chloridoideae), of which 22 are placed in Leptochloa, using five plastid (rpL32-trn-L, ndhA intron, rps16 intron, rps16-trnK and ccsA) and the nuclear ITS 1 and 2 (ribosomal internal transcribed spacer regions) to infer evolutionary relationships and revise the classification. Key results Leptochloa s.l. is polyphyletic and strong support was found for five lineages. Embedded within the Leptochloa sensu stricto (s.s.) clade are two Trichloris spp. and embedded in Dinebra are Drake-brockmania and 19 Leptochloa spp. Conclusions The molecular results support the dissolution of Leptochloa s.l. into the following five genera: Dinebra with 23 species, Diplachne with two species, Disakisperma with three species, Leptochloa s.s. with five species and a new genus, Trigonochloa, with two species. PMID:22628365

  15. Structures of xyloglucans in primary cell walls of gymnosperms, monilophytes (ferns sensu lato) and lycophytes.

    PubMed

    Hsieh, Yves S Y; Harris, Philip J

    2012-07-01

    Little is known about the structures of the xyloglucans in the primary cell walls of vascular plants (tracheophytes) other than angiosperms. Xyloglucan structures were examined in 13 species of gymnosperms, 13 species of monilophytes (ferns sensu lato), and two species of lycophytes. Wall preparations were obtained, extracted with 6 M sodium hydroxide, and the extracts treated with a xyloglucan-specific endo-(1→4)-β-glucanase preparation. The oligosaccharides released were analysed by matrix-assisted laser-desorption ionisation time-of-flight mass spectrometry and by high-performance anion-exchange chromatography. The xyloglucan oligosaccharide profiles from the gymnosperm walls were similar to those from the walls of most eudicotyledons and non-commelinid monocotyledons, indicating that the xyloglucans were fucogalactoxyloglucans, containing the fucosylated units XXFG and XLFG. The xyloglucan oligosaccharide profiles for six of the monilophyte species were similar to those of the gymnosperms, indicating they were also fucogalactoxyloglucans. Phylogenetically, these monilophyte species were from both basal and more derived orders. However, the profiles for the other monilophyte species showed various significant differences, including additional oligosaccharides. In three of the species, these additional oligosaccharides contained arabinosyl residues which were most abundant in the profile of Equisetum hyemale. The two species of lycophytes examined, Selaginella kraussiana and Lycopodium cernuum, had quite different xyloglucan oligosaccharide profiles, but neither were fucogalactoxyloglucans. The S. kraussiana profile had abundant oligosaccharides containing arabinosyl residues. The L. cernuum profile indicated the xyloglucan had a very complex structure.

  16. Delineation of a new species of the Borrelia burgdorferi Sensu Lato Complex, Borrelia americana sp. nov.

    PubMed

    Rudenko, Nataliia; Golovchenko, Maryna; Lin, Tao; Gao, Lihui; Grubhoffer, Libor; Oliver, James H

    2009-12-01

    Analysis of borrelia isolates collected from ticks, birds, and rodents from the southeastern United States revealed the presence of well-established populations of Borrelia burgdorferi sensu stricto, Borrelia bissettii, Borrelia carolinensis, and Borrelia sp. nov. Multilocus sequence analysis of five genomic loci from seven samples representing Borrelia sp. nov. isolated from nymphal Ixodes minor collected in South Carolina showed their close relatedness to California strains known as genomospecies 1 and separation from any other known species of the B. burgdorferi sensu lato complex. One nucleotide difference in the size of the 5S-23S intergenic spacer region, one substitution in 16S rRNA gene signature nucleotides, and silent nucleotide substitutions in sequences of the gene encoding flagellin and the gene p66 clearly separate Borrelia sp. nov. isolates from South Carolina into two subgroups. The sequences of isolates of each subgroup share the same restriction fragment length polymorphism patterns of the 5S-23S intergenic spacer region and contain unique signature nucleotides in the 16S rRNA gene. We propose that seven Borrelia sp. nov. isolates from South Carolina and two California isolates designated as genomospecies 1 comprise a single species, which we name Borrelia americana sp. nov. The currently recognized geographic distribution of B. americana is South Carolina and California. All strains are associated with Ixodes pacificus or Ixodes minor and their rodent and bird hosts.

  17. Why are there several species of Borrelia burgdorferi sensu lato detected in dogs and humans?

    PubMed

    Skotarczak, Bogumiła

    2014-04-01

    Borrelia burgdorferi sensu lato is a group of spirochete bacteria species some of which cause borreliosis in humans and dogs. Humans and dogs are susceptible to illness from many of the same tick-borne pathogens, including B. burgdorferi s.l. (Bbsl). Little is known about the pathogenic role of the species of Bbsl in canines. The molecular methods which detect and amplify the DNA of borreliae and allow differentiating borreliae species or strains have not been used in canine diagnostics yet. Until now, it has been believed that in European dogs, like in humans, at least three pathogenic species occur but the most frequently described symptoms may be associated with the infection caused by B. burgdorferi sensu stricto species. A dog as well as a human is a host for many species of Bbsl, because borreliacidal ability of serum of dogs and humans is evident only in certain genospecies of Bbsl. Therefore both a dog and a human harbor more species than in case of some wild animal species which create older phylogenetic Bbsl species-host systems and these animals may act even as a non-competent reservoir host. Apart from many genospecies of Bbsl, a dog harbors other tick-borne agents and dual or triple infections may occur.

  18. Genomic characterization of the Bacillus cereus sensu lato species: backdrop to the evolution of Bacillus anthracis.

    PubMed

    Zwick, Michael E; Joseph, Sandeep J; Didelot, Xavier; Chen, Peter E; Bishop-Lilly, Kimberly A; Stewart, Andrew C; Willner, Kristin; Nolan, Nichole; Lentz, Shannon; Thomason, Maureen K; Sozhamannan, Shanmuga; Mateczun, Alfred J; Du, Lei; Read, Timothy D

    2012-08-01

    The key genes required for Bacillus anthracis to cause anthrax have been acquired recently by horizontal gene transfer. To understand the genetic background for the evolution of B. anthracis virulence, we obtained high-redundancy genome sequences of 45 strains of the Bacillus cereus sensu lato (s.l.) species that were chosen for their genetic diversity within the species based on the existing multilocus sequence typing scheme. From the resulting data, we called more than 324,000 new genes representing more than 12,333 new gene families for this group. The core genome size for the B. cereus s.l. group was ∼1750 genes, with another 2150 genes found in almost every genome constituting the extended core. There was a paucity of genes specific and conserved in any clade. We found no evidence of recent large-scale gene loss in B. anthracis or for unusual accumulation of nonsynonymous DNA substitutions in the chromosome; however, several B. cereus genomes isolated from soil and not previously associated with human disease were degraded to various degrees. Although B. anthracis has undergone an ecological shift within the species, its chromosome does not appear to be exceptional on a macroscopic scale compared with close relatives.

  19. First report of amitraz and cypermethrin resistance in Rhipicephalus sanguineus sensu lato infesting dogs in Mexico.

    PubMed

    Rodriguez-Vivas, R I; Ojeda-Chi, M M; Trinidad-Martinez, I; Bolio-González, M E

    2017-03-01

    Engorged female Rhipicephalus sanguineus sensu lato (Ixodida: Ixodidae) were collected from dogs in the state of Yucatán, Mexico. Fourteen tick populations were collected from dogs at seven veterinary clinics, four residential homes and three cattle farms. The larval immersion test was used in the progeny of collected adult females to test susceptibility to amitraz and cypermethrin. Dose-mortality regressions, 50% lethal concentrations (LC50 ), confidence intervals and slope were estimated by probit analysis. For amitraz, 12 tick populations (85.7%) were classified as resistant and low inter-population variation in the phenotypic level of resistance was evident [resistance ratios (RRs) at LC50 : 1.0-13.0]. For cypermethrin, 12 tick populations (85.7%) were classified as resistant and substantial inter-population variation in the phenotypic level of resistance was evident (RRs at LC50 : 1.0-104.0). Thus, amitraz resistance in R. sanguineus s.l. is common, but generally occurs at low levels; however, alarmingly high levels of cypermethrin resistance are present in R. sanguineus s.l. populations in dogs in Yucatán, Mexico. The intensive use of both acaricides to control ectoparasites on dogs is likely to lead to more serious resistance problems that may cause high levels of control failure in the future.

  20. Established Population of Blacklegged Ticks with High Infection Prevalence for the Lyme Disease Bacterium, Borrelia burgdorferi Sensu Lato, on Corkscrew Island, Kenora District, Ontario

    PubMed Central

    Scott, John D.; Foley, Janet E.; Clark, Kerry L.; Anderson, John F.; Durden, Lance A.; Manord, Jodi M.; Smith, Morgan L.

    2016-01-01

    We document an established population of blacklegged ticks, Ixodes scapularis, on Corkscrew Island, Kenora District, Ontario, Canada. Primers of the outer surface protein A (OspA) gene, the flagellin (fla) gene, and the flagellin B (flaB) gene were used in the PCR assays to detect Borrelia burgdorferi sensu lato (s.l.), the Lyme disease bacterium. In all, 60 (73%) of 82 adult I. scapularis, were infected with B. burgdorferi s.l. As well, 6 (43%) of 14 unfed I. scapularis nymphs were positive for B. burgdorferi s.l. An I. scapularis larva was also collected from a deer mouse, and several unfed larvae were gathered by flagging leaf litter. Based on DNA sequencing of randomly selected Borrelia amplicons from six nymphal and adult I. scapularis ticks, primers for the flagellin (fla) and flagellin B (flaB) genes reveal the presence of B. burgdorferi sensu stricto (s.s.), a genospecies pathogenic to humans and certain domestic animals. We collected all 3 host-feeding life stages of I. scapularis in a single year, and report the northernmost established population of I. scapularis in Ontario. Corkscrew Island is hyperendemic for Lyme disease and has the highest prevalence of B. burgdorferi s.l. for any established population in Canada. Because of this very high infection prevalence, this population of I. scapularis has likely been established for decades. Of epidemiological significance, cottage owners, island visitors, outdoors enthusiasts, and medical professionals must be vigilant that B. burgdorferi s.l.-infected I. scapularis on Corkscrew Island pose a serious public health risk. PMID:27877080

  1. Occurrence of different Borrelia burgdorferi sensu lato genospecies including B. afzelii, B. bavariensis, and B. spielmanii in hedgehogs (Erinaceus spp.) in Europe.

    PubMed

    Skuballa, Jasmin; Petney, Trevor; Pfäffle, Miriam; Oehme, Rainer; Hartelt, Kathrin; Fingerle, Volker; Kimmig, Peter; Taraschewski, Horst

    2012-02-01

    In order to determine whether European hedgehogs (Erinaceus europaeus and E. roumanicus) play a role in the epidemiological cycle of Borrelia burgdorferi sensu lato in Central Europe and Great Britain, tissue samples of hedgehogs from Germany (n=211), Austria (n=4), the Czech Republic (n=22), and the U.K. (n=32) were tested for the presence of these tick-borne pathogens. PCR for amplification of the B. burgdorferi s.l.-specific 5S-23S intergenic spacer region as well as the outer surface protein A (ospA) gene were used. B. burgdorferi s.l. DNA was detected in 35 of the 259 E. europaeus and in 2 of 10 E. roumanicus. B. burgdorferi prevalences in E. europaeus ranged from 0% (U.K.) to 37.5% (Czech Republic), for E. roumanicus from 0% (Czech Republic) to 50.0% (Austria). Sequencing revealed the occurrence of 3 different B. burgdorferi genospecies in E. europaeus: B. afzelii was the dominant genospecies, followed by B. bavariensis (previously B. garinii OspA serotype 4) and B. spielmanii, the latter was detected for the first time in Hamburg (Germany). B. afzelii and B. bavariensis were also found in E. roumanicus. Our results suggest that hedgehogs modulate the epidemiology of certain species of the B. burgdorferi s.l. complex, potentially affecting the distribution and abundance of individual B. burgdorferi s.l. genospecies in various habitats. We hypothesise that juvenile or individuals with low immune competence in particular, have a high reservoir potential for the 3 genospecies identified here.

  2. Established Population of Blacklegged Ticks with High Infection Prevalence for the Lyme Disease Bacterium, Borrelia burgdorferi Sensu Lato, on Corkscrew Island, Kenora District, Ontario.

    PubMed

    Scott, John D; Foley, Janet E; Clark, Kerry L; Anderson, John F; Durden, Lance A; Manord, Jodi M; Smith, Morgan L

    2016-01-01

    We document an established population of blacklegged ticks, Ixodes scapularis, on Corkscrew Island, Kenora District, Ontario, Canada. Primers of the outer surface protein A (OspA) gene, the flagellin (fla) gene, and the flagellin B (flaB) gene were used in the PCR assays to detect Borrelia burgdorferi sensu lato (s.l.), the Lyme disease bacterium. In all, 60 (73%) of 82 adult I. scapularis, were infected with B. burgdorferi s.l. As well, 6 (43%) of 14 unfed I. scapularis nymphs were positive for B. burgdorferi s.l. An I. scapularis larva was also collected from a deer mouse, and several unfed larvae were gathered by flagging leaf litter. Based on DNA sequencing of randomly selected Borrelia amplicons from six nymphal and adult I. scapularis ticks, primers for the flagellin (fla) and flagellin B (flaB) genes reveal the presence of B. burgdorferi sensu stricto (s.s.), a genospecies pathogenic to humans and certain domestic animals. We collected all 3 host-feeding life stages of I. scapularis in a single year, and report the northernmost established population of I. scapularis in Ontario. Corkscrew Island is hyperendemic for Lyme disease and has the highest prevalence of B. burgdorferi s.l. for any established population in Canada. Because of this very high infection prevalence, this population of I. scapularis has likely been established for decades. Of epidemiological significance, cottage owners, island visitors, outdoors enthusiasts, and medical professionals must be vigilant that B. burgdorferi s.l.-infected I. scapularis on Corkscrew Island pose a serious public health risk.

  3. Detection of Borrelia burgdorferi sensu lato in lizards and their ticks from Hungary.

    PubMed

    Földvári, Gábor; Rigó, Krisztina; Majláthová, Viktória; Majláth, Igor; Farkas, Róbert; Pet'ko, Branislav

    2009-06-01

    To investigate the involvement of lizard species in the natural cycle of Borrelia burgdorferi sensu lato (s.l.) in Hungary, a total of 186 reptiles belonging to three species--126 green lizards (Lacerta viridis), 40 Balkan wall lizards (Podarcis taurica), and 20 sand lizards (Lacerta agilis)--were captured in 2007 and 2008. All ticks removed from the lizards were Ixodes ricinus, either larvae (324/472; 68.6%) or nymphs (148/472; 31.4%). More than half (66/126; 52.4%) of L. viridis individuals were infested, and the prevalence of tick infestation on both the other two species was 35% each. All 472 I. ricinus ticks and tissue samples collected from 134 collar scales and 62 toe clips of lizards were further analyzed for the presence of B. burgdorferi s.l. with polymerase chain reaction. The amplification of B. burgdorferi s.l. DNA was successful in 8% (n = 92) of L. viridis, 9% (n = 32) of P. taurica, and 10% (n = 10) of L. agilis tissue samples. Restriction fragment length polymorphism genotyping identified the species Borrelia lusitaniae in all tested lizard samples. Prevalence of B. burgdorferi s.l. in ticks collected from L. viridis, P. taurica, and L. agilis was 8%, 2%, and 0%, respectively. Most of the infected ticks carried B. lusitaniae (74% of genotyped positives); however, Borrelia afzelii (5%) and B. burgdorferi sensu stricto (21%) were detected in ticks removed from green lizards and Balkan wall lizards, respectively. We conclude that lizards, particularly L. viridis, can be important hosts for I. ricinus larvae and nymphs; thus, they can be regarded as reservoirs of these important pathogen vectors. The role of green lizards has been confirmed, and the implication of Balkan wall lizards is suggested in the natural cycle of B. lusitaniae at our study site.

  4. Trematode diversity in the freshwater snail Bithynia siamensis goniomphalos sensu lato from Thailand and Lao PDR.

    PubMed

    Kiatsopit, N; Sithithaworn, P; Kopolrat, K; Namsanor, J; Andrews, R H; Petney, T N

    2016-05-01

    In order to obtain a comprehensive understanding of trematode diversity in Bithynia siamensis goniomphalos sensu lato, the first intermediate host of the liver fluke Opisthorchis viverrini s.l., the prevalence of larval trematode species was investigated in different localities in Thailand and Lao People's Democratic Republic (Lao PDR). In Thailand, snail samples were collected from 29 localities in the nine provinces: Buri Ram, Surin, Chaiya Phum, Maha Sarakham, Khon Kaen, Kalasin, Mukdahan, Sakon Nakhon and Nakhon Phanom. In Lao PDR, snail samples were collected from 21 localities in Vientiane Province and six localities in Savannakhet Province. Snails were identified by standard morphological criteria and then examined for trematode infection using the cercarial shedding method. Twenty different types of cercariae were detected and identified, based on morphological criteria. Virgulate type 1 emerged as the most common cercaria, with an average prevalence of 10.90% (range 0.26-54.22%) in Thailand and 6.58% (range 1.15-89.77%) in Lao PDR. Opisthorchis viverrini s.l. cercariae were the fourth most common in Thailand, with an average prevalence of 1.59% (0.15-6.93), while in Lao PDR their prevalence was 0.96% (0.08-8.37). The high diversity of trematode cercariae observed in this study indicates that B. s. goniomphalos s.l. is highly susceptible to infection with a variety of trematode species. However, the role of non-opisthorchiid trematodes as fish-borne parasites in human health is not fully known and further molecular identification is required.

  5. Implementation of new tools in molecular epidemiology studies of Echinococcus granulosus sensu lato in South America.

    PubMed

    Avila, Héctor G; Santos, Guilherme B; Cucher, Marcela A; Macchiaroli, Natalia; Pérez, Matías G; Baldi, Germán; Jensen, Oscar; Pérez, Verónica; López, Raúl; Negro, Perla; Scialfa, Exequiel; Zaha, Arnaldo; Ferreira, Henrique B; Rosenzvit, Mara; Kamenetzky, Laura

    2017-06-01

    The aim of this work was to determine Echinococcus granulosus sensu lato species and genotypes in intermediate and definitive hosts and in human isolates from endemic regions of Argentina and Brazil including those where no molecular data is available by a combination of classical and alternative molecular tools. A total of 227 samples were isolated from humans, natural intermediate and definitive hosts. Amplification of cytochrome c oxidase subunit I gene fragment was performed and a combination of AluI digestion assay, High Resolution Melting analysis (HRM) assay and DNA sequencing was implemented for Echinococcus species/genotype determination. E. granulosus sensu stricto (G1) was found in sheep (n=35), cattle (n=67) and dogs (n=5); E. ortleppi (G5) in humans (n=3) and cattle (n=108); E. canadensis (G6) in humans (n=2) and E. canadensis (G7) in pigs (n=7). We reported for the first time the presence of E. ortleppi (G5) and E. canadensis (G6) in humans from San Juan and Catamarca Argentinean provinces and E. canadensis (G7) in pigs from Cordoba Argentinean province. In this work, we widened molecular epidemiology studies of E. granulosus s. l. in South America by analyzing several isolates from definitive and intermediate hosts, including humans from endemic regions were such information was scarce or unavailable. The presence of different species/genotypes in the same region and host species reinforce the need of rapid and specific techniques for accurate determination of Echinococcus species such as the ones proposed in this work.

  6. Borrelia burgdorferi Sensu Lato in Siberian chipmunks (Tamias sibiricus) introduced in suburban forests in France.

    PubMed

    Vourc'h, Gwenaël; Marmet, Julie; Chassagne, Michelle; Bord, Séverine; Chapuis, Jean-Louis

    2007-01-01

    Numerous vertebrate reservoirs have been described for Borrelia burgdorferi sensu lato (sl), which includes the etiological agents of Lyme Borreliosis (LB). The Siberian chipmunk (Tamias sibiricus) is a rodent originating from Asia, where it is suspected to be a B. burgdorferi reservoir. It has been intentionally released into the wild in Europe since the 1970s, but has not yet been subject to any study regarding its association with the LB agent. In this paper we studied Siberian chipmunk infestation with the LB vector (Ixodes ricinus) and infection prevalence by LB spirochetes in a suburban introduced population. We compared these findings with known competent reservoir hosts, the bank vole (Myodes [clethrionomys] glareolus) and wood mouse (Apodemus sylvaticus). All Siberian chipmunks were infested with larvae and larval abundance was higher in this species (mean number of larvae [95% Confidence Interval]: 73.5 [46.0, 117.2]) than in the two other rodent species (bank voles: 4.4 [3.0, 6.3] and wood mice: 10.2 [4.9, 21.2]). Significant factors affecting abundance of larvae were host species and sampling season. Nymphs were most prevalent on chipmunks (86.2%, mean: 5.1 [3.3, 8.0]), one vole carried only two nymphs, and none of the mice had any nymphs. Nymph abundance in chipmunks was affected by sampling season and sex. Furthermore, the infection prevalence of B. burgdorferi sl in the Siberian chipmunk was the highest (33.3%) and predominantly of B. afzelii. The infection prevalence was 14.1% in bank voles, but no wood mouse was found to be infected. Our results suggest that the Siberian chipmunk may be an important reservoir host for LB.

  7. Morphological and genetic diversity of Rhipicephalus sanguineus sensu lato from the New and Old Worlds

    PubMed Central

    2013-01-01

    Background The taxonomic status of the brown dog tick (Rhipicephalus sanguineus sensu stricto), which has long been regarded as the most widespread tick worldwide and a vector of many pathogens to dogs and humans, is currently under dispute. Methods We conducted a comprehensive morphological and genetic study of 278 representative specimens, which belonged to different species (i.e., Rhipicephalus bursa, R. guilhoni, R. microplus, R. muhsamae, R. pusillus, R. sanguineus sensu lato, and R. turanicus) collected from Europe, Asia, Americas, and Oceania. After detailed morphological examination, ticks were molecularly processed for the analysis of partial mitochondrial (16S rDNA, 12S rDNA, and cox1) gene sequences. Results In addition to R. sanguineus s.l. and R. turanicus, three different operational taxonomic units (namely, R. sp. I, R. sp. II, and R. sp. III) were found on dogs. These operational taxonomical units were morphologically and genetically different from R. sanguineus s.l. and R. turanicus. Ticks identified as R. sanguineus s.l., which corresponds to the so-called “tropical species” (=northern lineage), were found in all continents and genetically it represents a sister group of R. guilhoni. R. turanicus was found on a wide range of hosts in Italy and also on dogs in Greece. Conclusions The tropical species and the temperate species (=southern lineage) are paraphyletic groups. The occurrence of R. turanicus in the Mediterranean region is confirmed. A consensual re-description of R. sanguineus s.s. and R. turanicus will be necessary to solve the taxonomic problems within the so-called R. sanguineus group. PMID:23880226

  8. Retreat and extinction of the Late Pleistocene cave bear ( Ursus spelaeus sensu lato)

    NASA Astrophysics Data System (ADS)

    Baca, Mateusz; Popović, Danijela; Stefaniak, Krzysztof; Marciszak, Adrian; Urbanowski, Mikołaj; Nadachowski, Adam; Mackiewicz, Paweł

    2016-12-01

    The cave bear ( Ursus spelaeus sensu lato) is a typical representative of Pleistocene megafauna which became extinct at the end of the Last Glacial. Detailed knowledge of cave bear extinction could explain this spectacular ecological transformation. The paper provides a report on the youngest remains of the cave bear dated to 20,930 ± 140 14C years before present (BP). Ancient DNA analyses proved its affiliation to the Ursus ingressus haplotype. Using this record and 205 other dates, we determined, following eight approaches, the extinction time of this mammal at 26,100-24,300 cal. years BP. The time is only slightly earlier, i.e. 27,000-26,100 cal. years BP, when young dates without associated collagen data are excluded. The demise of cave bear falls within the coldest phase of the last glacial period, Greenland Stadial 3. This finding and the significant decrease in the cave bear records with cooling indicate that the drastic climatic changes were responsible for its extinction. Climate deterioration lowered vegetation productivity, on which the cave bear strongly depended as a strict herbivore. The distribution of the last cave bear records in Europe suggests that this animal was vanishing by fragmentation into subpopulations occupying small habitats. One of them was the Kraków-Częstochowa Upland in Poland, where we discovered the latest record of the cave bear and also two other, younger than 25,000 14C years BP. The relatively long survival of this bear in karst regions may result from suitable microclimate and continuous access to water provided by deep aquifers, indicating a refugial role of such regions in the Pleistocene for many species.

  9. Retreat and extinction of the Late Pleistocene cave bear (Ursus spelaeus sensu lato).

    PubMed

    Baca, Mateusz; Popović, Danijela; Stefaniak, Krzysztof; Marciszak, Adrian; Urbanowski, Mikołaj; Nadachowski, Adam; Mackiewicz, Paweł

    2016-12-01

    The cave bear (Ursus spelaeus sensu lato) is a typical representative of Pleistocene megafauna which became extinct at the end of the Last Glacial. Detailed knowledge of cave bear extinction could explain this spectacular ecological transformation. The paper provides a report on the youngest remains of the cave bear dated to 20,930 ± 140 (14)C years before present (BP). Ancient DNA analyses proved its affiliation to the Ursus ingressus haplotype. Using this record and 205 other dates, we determined, following eight approaches, the extinction time of this mammal at 26,100-24,300 cal. years BP. The time is only slightly earlier, i.e. 27,000-26,100 cal. years BP, when young dates without associated collagen data are excluded. The demise of cave bear falls within the coldest phase of the last glacial period, Greenland Stadial 3. This finding and the significant decrease in the cave bear records with cooling indicate that the drastic climatic changes were responsible for its extinction. Climate deterioration lowered vegetation productivity, on which the cave bear strongly depended as a strict herbivore. The distribution of the last cave bear records in Europe suggests that this animal was vanishing by fragmentation into subpopulations occupying small habitats. One of them was the Kraków-Częstochowa Upland in Poland, where we discovered the latest record of the cave bear and also two other, younger than 25,000 (14)C years BP. The relatively long survival of this bear in karst regions may result from suitable microclimate and continuous access to water provided by deep aquifers, indicating a refugial role of such regions in the Pleistocene for many species.

  10. Epidemiology and echinocandin susceptibility of Candida parapsilosis sensu lato species isolated from bloodstream infections at a Spanish university hospital

    PubMed Central

    Garcia-Effron, Guillermo; Canton, Emilia; Pemán, Javier; Dilger, Amanda; Romá, Eva; Perlin, David S.

    2012-01-01

    Objectives The aims of this work were to study the epidemiological profiles, differences in echinocandin susceptibilities and clinical relevance of the Candida parapsilosis sensu lato species isolated from proven fungaemia cases at La Fe University Hospital of Valencia (Spain) from 1995 to 2007. Results The prevalence of these species was: C. parapsilosis sensu stricto, 74.4%; Candida orthopsilosis, 23.54%; and Candida metapsilosis, 2.05%. The incidence of the species complex as agents of fungaemia remained stationary until 2005 and doubled in 2006. The incidence of C. orthopsilosis showed an increasing trend during the study period, while C. parapsilosis sensu stricto incidence diminished. Also, an important epidemiological change was observed starting in 2004, when 86.5% of the C. parapsilosis sensu lato strains were found in adult patients, while before that year only 13.5% of the isolates were found in this population. Conclusions Echinocandin drug susceptibility testing using the CLSI M27-A3 document showed a wide range of MIC values (0.015–4 mg/L), with micafungin being the most potent in vitro inhibitor followed by anidulafungin and caspofungin (MIC geometric mean of 0.68, 0.74 and 0.87 mg/L, respectively). C. metapsilosis was the most susceptible species of the complex to anidulafungin and micafungin in vitro (MIC50 for anidulafungin and micafungin: 0.06 mg/L), while there were no differences between C. parapsilosis sensu lato species when caspofungin MIC50s were compared (MIC50 1.00 mg/L). Differences in caspofungin in vitro susceptibility were observed between the different clinical service departments of La Fe Hospital. PMID:22868644

  11. Sex Determination Using PCR

    ERIC Educational Resources Information Center

    Kima, Peter E.; Rasche, Madeline E.

    2004-01-01

    PCR has revolutionized many aspects of biochemistry and molecular biology research. In the following exercise, students learn PCR by isolating their own DNA, amplifying specific segments of the X and Y chromosomes, and estimating the sizes of the PCR products using agarose gel electrophoresis. Based on the pattern of PCR products, students can…

  12. Isolation of live Borrelia burgdorferi sensu lato spirochaetes from patients with undefined disorders and symptoms not typical for Lyme borreliosis.

    PubMed

    Rudenko, N; Golovchenko, M; Vancova, M; Clark, K; Grubhoffer, L; Oliver, J H

    2016-03-01

    Lyme borreliosis is a multisystem disorder with a diverse spectrum of clinical manifestations, caused by spirochaetes of the Borrelia burgdorferi sensu lato complex. It is an infectious disease that can be successfully cured by antibiotic therapy in the early stages; however, the possibility of the appearance of persistent signs and symptoms of disease following antibiotic treatment is recognized. It is known that Lyme borreliosis mimics multiple diseases that were never proven to have a spirochaete aetiology. Using complete modified Kelly-Pettenkofer medium we succeeded in cultivating live B. burgdorferi sensu lato spirochaetes from samples taken from people who suffered from undefined disorders, had symptoms not typical for Lyme borreliosis, but who had undergone antibiotic treatment due to a suspicion of having Lyme disease even though they were seronegative. We report the first recovery of live B. burgdorferi sensu stricto from residents of southeastern USA and the first successful cultivation of live Borrelia bissettii-like strain from residents of North America. Our results support the fact that B. bissettii is responsible for human Lyme borreliosis worldwide along with B. burgdorferi s.s. The involvement of new spirochaete species in Lyme borreliosis changes the understanding and recognition of clinical manifestations of this disease.

  13. The propensity of voles and mice to transmit Borrelia burgdorferi sensu lato infection to feeding ticks.

    PubMed

    Radzijevskaja, Jana; Paulauskas, Algimantas; Rosef, Olav; Petkevičius, Saulius; Mažeika, Vytautas; Rekašius, Tomas

    2013-10-18

    Lyme borreliosis (LB) caused by the spirochete Borrelia burgdorferi sensu lato is the most common tick-borne zoonosis in the Northern Hemisphere. B. burgdorferi s.l. can infect humans and wild and domestic animals. Ixodes ricinus is the main vector, and small rodents are the most important mammalian reservoirs hosts of B. burgdorferi s.l. in Europe. The prevalence of B. burgdorferi s.l. in I. ricinus ticks from captured rodents, calculated specific infectivities, and transmission coefficients were estimated in order to investigate the role of voles and mice in transmission of the LB causative agent. A total of 12.3% (53 out of 431) of immature I. ricinus ticks from rodents in Lithuania and 3.25% (21 out of 646) in Norway were infected with B. burgdorferi s.l. In Lithuania a total of 40% infested Microtus arvalis, 29% of Myodes glareolus and 4.8% of Apodemus flavicollis carried infected larvae and 67% of M. glareolus, 36% of M. arvalis but none of A. flavicollis carried infected nymphs. In Norway, 2.4% of larvae and 12.1% of nymphs feeding on A. flavicollis were infected. A total of 9% of infested A. flavicollis carried infected larvae and 13% - infected nymphs. Borrelia afzelii was the single genospecies identified in ticks feeding on rodents in Lithuania, and was predominant in ticks collected from rodents in Norway. According to calculated indices of specific infectivity and tick-to host transmission coefficient, M. arvalis and M. glareolus voles were found to be more efficient in transmitting B. burgdorferi s.l. to ticks than A. flavicollis mice. GLMM analysis showed that rodent species significantly influenced the probability of a larva being infected with B. burgdorferi s.l. The larvae feeding on M. arvalis and M. glareolus were more likely to be infected with B. burgdorferi s.l. than those feeding on A. flavicollis. This is the first study to report the quantitative roles of voles and mice in the transmission of B. burgdorferi s.l. to larval ticks in

  14. Habitat discrimination by gravid Anopheles gambiae sensu lato – a push-pull system

    PubMed Central

    2014-01-01

    Background The non-random distribution of anopheline larvae in natural habitats suggests that gravid females discriminate between habitats of different quality. Whilst physical and chemical cues used by Culex and Aedes vector mosquitoes for selecting an oviposition site have been extensively studied, those for Anopheles remain poorly explored. Here the habitat selection by Anopheles gambiae sensu lato (s.l.), the principal African malaria vector, was investigated when presented with a choice of two infusions made from rabbit food pellets, or soil. Methods Natural colonization and larval survival was evaluated in artificial ponds filled randomly with either infusion. Dual-choice, egg-count bioassays evaluated the responses of caged gravid females to (1) two- to six-day old infusions versus lake water; (2) autoclaved versus non-autoclaved soil infusions; and assessed (3) the olfactory memory of gravid females conditioned in pellet infusion as larvae. Results Wild Anopheles exclusively colonized ponds with soil infusion and avoided those with pellet infusion. When the individual infusions were tested in comparison with lake water, caged An. gambiae sensu stricto (s.s.) showed a dose response: females increasingly avoided the pellet infusion with increasing infusion age (six-day versus lake water: odds ratio (OR) 0.22; 95% confidence interval (CI) 0.1-0.5) and showed increasing preference to lay eggs as soil infusion age increased (six-day versus lake water: OR 2.1; 95% CI 1.4-3.3). Larvae survived in soil infusions equally well as in lake water but died in pellet infusions. Anopheles gambiae s.s. preferred to lay eggs in the non-autoclaved soil (OR 2.6; 95% CI 1.8-3.7) compared with autoclaved soil. There was no change in the avoidance of pellet infusion by individuals reared in the infusion compared with those reared in lake water. Conclusion Wild and caged An. gambiae s.l. females discriminate between potential aquatic habitats for oviposition. These choices benefit

  15. Brown dog tick, Rhipicephalus sanguineus sensu lato, infestation ofsusceptible dog hosts is reduced by slow release of semiochemicalsfrom a less susceptible host

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Domestic dog breeds are hosts for the tick Rhipicephalus sanguineus sensu lato, but infestation levels vary among breeds. Beagles are less susceptible to tick infestations than English cocker spaniels due to enhanced production of 2-hexanone and benzaldehyde that act as tick repellents. We report th...

  16. Comparative Genomics of Listeria Sensu Lato: Genus-Wide Differences in Evolutionary Dynamics and the Progressive Gain of Complex, Potentially Pathogenicity-Related Traits through Lateral Gene Transfer.

    PubMed

    Chiara, Matteo; Caruso, Marta; D'Erchia, Anna Maria; Manzari, Caterina; Fraccalvieri, Rosa; Goffredo, Elisa; Latorre, Laura; Miccolupo, Angela; Padalino, Iolanda; Santagada, Gianfranco; Chiocco, Doriano; Pesole, Graziano; Horner, David S; Parisi, Antonio

    2015-07-15

    Historically, genome-wide and molecular characterization of the genus Listeria has concentrated on the important human pathogen Listeria monocytogenes and a small number of closely related species, together termed Listeria sensu strictu. More recently, a number of genome sequences for more basal, and nonpathogenic, members of the Listeria genus have become available, facilitating a wider perspective on the evolution of pathogenicity and genome level evolutionary dynamics within the entire genus (termed Listeria sensu lato). Here, we have sequenced the genomes of additional Listeria fleischmannii and Listeria newyorkensis isolates and explored the dynamics of genome evolution in Listeria sensu lato. Our analyses suggest that acquisition of genetic material through gene duplication and divergence as well as through lateral gene transfer (mostly from outside Listeria) is widespread throughout the genus. Novel genetic material is apparently subject to rapid turnover. Multiple lines of evidence point to significant differences in evolutionary dynamics between the most basal Listeria subclade and all other congeners, including both sensu strictu and other sensu lato isolates. Strikingly, these differences are likely attributable to stochastic, population-level processes and contribute to observed variation in genome size across the genus. Notably, our analyses indicate that the common ancestor of Listeria sensu lato lacked flagella, which were acquired by lateral gene transfer by a common ancestor of Listeria grayi and Listeria sensu strictu, whereas a recently functionally characterized pathogenicity island, responsible for the capacity to produce cobalamin and utilize ethanolamine/propane-2-diol, was acquired in an ancestor of Listeria sensu strictu.

  17. Identification and molecular survey of Borrelia burgdorferi sensu lato in sika deer (Cervus nippon) from Jilin Province, north-eastern China.

    PubMed

    Zhai, Bintao; Niu, Qingli; Yang, Jifei; Liu, Zhijie; Liu, Junlong; Yin, Hong; Zeng, Qiaoying

    2017-02-01

    Lyme disease caused by Borrelia burgdorferi sensu lato (s.l.) is a common disease of domestic animals and wildlife worldwide. Sika deer is first-grade state-protected wildlife animals in China and have economic consequences for humans. It is reported that sika deer may serve as an important reservoir host for several species of B. burgdorferi s.l. and may transmit these species to humans and animals. However, little is known about the presence of Borrelia pathogens in sika deer in China. In this study, the existence and prevalence of Borrelia sp. in sika deer from four regions of Jilin Province in China was assessed. Seventy-one blood samples of sika deer were collected and tested by nested-PCRs based on 16S ribosomal RNA (16S rRNA), outer surface protein A (OspA), flagenllin (fla), and 5S-23S rRNA intergenic spacer (5S-23S rRNA) genes of B. burgdorferi s.l. Six (8.45%) samples were positive for Borrelia sp. based on sequences of 4 genes. The positive samples were detected 18 for 16S rRNA, 10 for OspA, 16 for fla and 6 for 5S-23S, with the positive rates 25.35% (95% CI=3.8-35.6), 14.08% (95% CI=3.0-21.6), 22.54% (95% CI=4.3-36.9) and 8.45% (95% CI=1.7-22.9), respectively. Sequence analysis of the positive PCR products revealed that the partial 4 genes sequences in this study were all most similar to the sequences of B. garinii and B. burgdorferi sensu stricto (s.s.), no other Borrelia genospecies were found. This is the first report of Borrelia pathogens in sika deer in China. The findings in this study indicated that sika deer as potential natural host and may spread Lyme disease pathogen to animals, ticks, and even humans.

  18. Optimization of the Esperanza window trap for the collection of the African onchocerciasis vector Simulium damnosum sensu lato.

    PubMed

    Toé, Laurent D; Koala, Lassane; Burkett-Cadena, Nathan D; Traoré, Bizini M; Sanfo, Moussa; Kambiré, Sié Roger; Cupp, Eddie W; Traoré, Soungalo; Yameogo, Laurent; Boakye, Daniel; Rodríguez-Pérez, Mario A; Unnasch, Thomas R

    2014-09-01

    A simple inexpensive trap (Esperanza window trap) was shown recently to collect significant numbers of Simulium ochraceum sensu lato, a major vector of Onchocerca volvulus in Mesoamerica. Here, we report studies optimizing this trap for the collection of Simulium damnosum s.l., the major vector of O. volvulus in Africa. A shortened, blue and black striped version of the Esperanza window trap, when baited with a combination of CO2 and worn trousers, rivalled human landing collections in the number of S. damnosum s.l. females collected. Traps baited with a commercially available human skin lure and CO2 resulted in collections that were not significantly different than those obtained from traps baited with worn trousers and CO2. This suggests that the Esperanza window trap may offer a replacement for human landing collections for monitoring onchocerciasis transmission in Africa.

  19. Congruence and indifference between two molecular markers for understanding oral evolution in the Marynidae sensu lato (Ciliophora, Colpodea)

    PubMed Central

    Dunthorn, Micah; Katz, Laura A.; Stoeck, Thorsten; Foissner, Wilhelm

    2012-01-01

    Our understanding of the evolution of oral structures within the Colpodida is confounded by the low number of morphological characters that can be used in constructing hypotheses, and by the low taxon and character sampling in molecular phylogenetic analyses designed to assess these hypotheses. Here we increase character sampling by sequencing the mitochondrial SSU-rDNA locus for three isolates of the Marynidae sensu lato. We show that the inferred mitochondrial and nuclear SSU-rDNA trees, as well as concatenated and constrained analyses, are congruent in not recovering a monophyletic Marynidae. However, due to low node support, the trees are indifferent to whether the morphological characters used to unite the Marynidae are the result of retention of ancestral states or convergence. In light of this indifference and an increased amount of nuclear and mitochondrial SSU-rDNA data, alternative hypotheses of oral evolution in the Colpodida are presented. PMID:22356924

  20. The Identification of Intrinsic Chloramphenicol and Tetracycline Resistance Genes in Members of the Bacillus cereus Group (sensu lato).

    PubMed

    Glenwright, Helen; Pohl, Susanne; Navarro, Ferran; Miro, Elisenda; Jiménez, Guillermo; Blanch, Anicet R; Harwood, Colin R

    2016-01-01

    Bacillus toyonensis strain BCT-7112(T) (NCIMB 14858(T)) has been widely used as an additive in animal nutrition for more than 30 years without reports of adverse toxigenic effects. However, this strain is resistant to chloramphenicol and tetracycline and it is generally considered inadvisable to introduce into the food chain resistance determinants capable of being transferred to other bacterial strains, thereby adding to the pool of such determinants in the gastro-enteric systems of livestock species. We therefore characterized the resistance phenotypes of this strain and its close relatives to determine whether they were of recent origin, and therefore likely to be transmissible. To this end we identified the genes responsible for chloramphenicol (catQ) and tetracycline (tetM) resistance and confirmed the presence of homologs in other members of the B. toyonensis taxonomic unit. Unexpectedly, closely related strains encoding these genes did not exhibit chloramphenicol and tetracycline resistance phenotypes. To understand the differences in the behaviors, we cloned and expressed the genes, together with their upstream regulatory regions, into Bacillus subtilis. The data showed that the genes encoded functional proteins, but were expressed inefficiently from their native promoters. B. toyonensis is a taxonomic unit member of the Bacillus cereus group (sensu lato). We therefore extended the analysis to determine the extent to which homologous chloramphenicol and tetracycline resistance genes were present in other species within this group. This analysis revealed that homologous genes were present in nearly all representative species within the B. cereus group (sensu lato). The absence of known transposition elements and the observations that they are found at the same genomic locations, indicates that these chloramphenicol and tetracycline resistance genes are of ancient origin and intrinsic to this taxonomic group, rather than recent acquisitions. In this context

  1. The Identification of Intrinsic Chloramphenicol and Tetracycline Resistance Genes in Members of the Bacillus cereus Group (sensu lato)

    PubMed Central

    Glenwright, Helen; Pohl, Susanne; Navarro, Ferran; Miro, Elisenda; Jiménez, Guillermo; Blanch, Anicet R.; Harwood, Colin R.

    2017-01-01

    Bacillus toyonensis strain BCT-7112T (NCIMB 14858T) has been widely used as an additive in animal nutrition for more than 30 years without reports of adverse toxigenic effects. However, this strain is resistant to chloramphenicol and tetracycline and it is generally considered inadvisable to introduce into the food chain resistance determinants capable of being transferred to other bacterial strains, thereby adding to the pool of such determinants in the gastro-enteric systems of livestock species. We therefore characterized the resistance phenotypes of this strain and its close relatives to determine whether they were of recent origin, and therefore likely to be transmissible. To this end we identified the genes responsible for chloramphenicol (catQ) and tetracycline (tetM) resistance and confirmed the presence of homologs in other members of the B. toyonensis taxonomic unit. Unexpectedly, closely related strains encoding these genes did not exhibit chloramphenicol and tetracycline resistance phenotypes. To understand the differences in the behaviors, we cloned and expressed the genes, together with their upstream regulatory regions, into Bacillus subtilis. The data showed that the genes encoded functional proteins, but were expressed inefficiently from their native promoters. B. toyonensis is a taxonomic unit member of the Bacillus cereus group (sensu lato). We therefore extended the analysis to determine the extent to which homologous chloramphenicol and tetracycline resistance genes were present in other species within this group. This analysis revealed that homologous genes were present in nearly all representative species within the B. cereus group (sensu lato). The absence of known transposition elements and the observations that they are found at the same genomic locations, indicates that these chloramphenicol and tetracycline resistance genes are of ancient origin and intrinsic to this taxonomic group, rather than recent acquisitions. In this context we

  2. Borrelia burgdorferi sensu lato prevalence and diversity in ticks and small mammals in a Lyme borreliosis endemic Nature Reserve in North-Western Spain. Incidence in surrounding human populations.

    PubMed

    Espí, Alberto; Del Cerro, Ana; Somoano, Aitor; García, Verónica; M Prieto, José; Barandika, José F; García-Pérez, Ana L

    2016-07-18

    To determine the prevalence and diversity of Borrelia burgdorferi sensu lato (s.l.) in an endemic Nature Reserve (Sierra del Sueve) in North-Western Spain, and the risk of human exposure to infected ticks in Asturias, 1013 questing ticks and 70 small mammals were collected between 2012 and 2014. A retrospective descriptive analysis was also carried out on human Lyme borreliosis (LB) cases reported to the local hospital (Cabueñes). Samples were screened for B. burgdorferi s.l. presence by a nested PCR assay, and genospecies were confirmed by sequencing. B. burgdorferi s.l. was detected in 1.4% (12/845) of I. ricinus questing nymphs, 9.1% (2/33) of questing adults, and 12.9% (9/70) of small mammals, as well as in the other tick species. PCR positive samples of 17 questing tick and 6 small mammals were sequenced. Four genospecies were identified: B. afzelii, B. garinii, B. lusitaniae, and B. valaisiana. Phylogenetic analyses based on the flaB gene showed the heterogeneity of B. afzelii in this area. The detection of B. burgdorferi s.l. among questing ticks and small mammals in the study area, as well as the abundance of ticks and of large wild and domestic mammals, indicate a high risk of infection by B. burgdorferi s.l. in the area. Reporting of LB cases to the local hospital support this, and shows the need of thorough monitoring of B. burgdorferi infection in ticks and hosts in the area. More investigations are needed to assess the role of different wildlife species and the risk of transmission to humans.

  3. PCR und Real-Time PCR

    NASA Astrophysics Data System (ADS)

    Konrad, Regina; Busch, Ulrich

    Die vielfältigen Anwendungsmöglichkeiten der Polymerasekettenreaktion (polymerase chain reaction, PCR) machen sie zu einer der wichtigsten und am häufigsten eingesetzten Methoden in der molekularbiologischen Forschung und Diagnostik. Für diese Technologie wurde der Erfinder der Methode, Kary Mullis, 1993 mit dem Nobelpreis ausgezeichnet. Die PCR erlaubt einen hochsensitiven und spezifischen in-vitro-Nachweis von Desoxyribonukleinsäuren (DNA), da im Zuge der Reaktion Sequenzabschnitte gezielt vermehrt werden. Innerhalb weniger Stunden können aus einem einzigen Zielmolekül 1012 identische Moleküle entstehen [1].

  4. Candida parapsilosis (sensu lato) isolated from hospitals located in the Southeast of Brazil: Species distribution, antifungal susceptibility and virulence attributes.

    PubMed

    Ziccardi, Mariangela; Souza, Lucieri O P; Gandra, Rafael M; Galdino, Anna Clara M; Baptista, Andréa R S; Nunes, Ana Paula F; Ribeiro, Mariceli A; Branquinha, Marta H; Santos, André L S

    2015-12-01

    Candida parapsilosis (sensu lato), which represents a fungal complex composed of three genetically related species - Candida parapsilosis sensu stricto, Candida orthopsilosis and Candida metapsilosis, has emerged as an important yeast causing fungemia worldwide. The goal of the present work was to assess the prevalence, antifungal susceptibility and production of virulence traits in 53 clinical isolates previously identified as C. parapsilosis (sensu lato) obtained from hospitals located in the Southeast of Brazil. Species forming this fungal complex are physiologically/morphologically indistinguishable; however, polymerase chain reaction followed by restriction fragment length polymorphism of FKS1 gene has solved the identification inaccuracy, revealing that 43 (81.1%) isolates were identified as C. parapsilosis sensu stricto and 10 (18.9%) as C. orthopsilosis. No C. metapsilosis was found. The geographic distribution of these Candida species was uniform among the studied Brazilian States (São Paulo, Rio de Janeiro and Espírito Santo). All C. orthopsilosis and almost all C. parapsilosis sensu stricto (95.3%) isolates were susceptible to amphotericin B, fluconazole, itraconazole, voriconazole and caspofungin. Nevertheless, one C. parapsilosis sensu stricto isolate was resistant to fluconazole and another one was resistant to caspofungin. C. parapsilosis sensu stricto isolates exhibited higher MIC mean values to amphotericin B, fluconazole and caspofungin than those of C. orthopsilosis, while C. orthopsilosis isolates displayed higher MIC mean to itraconazole compared to C. parapsilosis sensu stricto. Identical MIC mean values to voriconazole were measured for these Candida species. All the isolates of both species were able to form biofilm on polystyrene surface. Impressively, biofilm-growing cells of C. parapsilosis sensu stricto and C. orthopsilosis exhibited a considerable resistance to all antifungal agents tested. Pseudohyphae were observed in 67.4% and 80

  5. Seroprevalence of antibodies to Borrelia burgdorferi sensu lato in healthy adults from western Norway: risk factors and methodological aspects.

    PubMed

    Hjetland, Reidar; Nilsen, Roy M; Grude, Nils; Ulvestad, Elling

    2014-11-01

    The aim of this study was to assess the seroprevalence of antibodies to Borrelia burgdorferi sensu lato in a healthy adult population from Sogn and Fjordane county in western Norway by different assays. Sera from 1213 blood donors at four different blood banks were analysed in Enzygnost Lyme link VlsE/IgG (IgG), Enzygnost Borreliosis IgM (IgM), and Immunetics C6 Lyme ELISA kit (C6). Sera showing positive or grey-zone reactivities were further examined with Borrelia-EUROLine-RN-AT IgG blot and Borrelia-EUROLine-RN-AT IgM blot. The seroprevalences were 9.6%, 8.2%, 8.4%, 6.4% and 5.7%, respectively. The seroprevalence for IgG was lower in the eastern part of the county and in owners of pet animals. It was higher in men, and increased with age and number of tick bites. C6 and IgG gave comparable results. IgM only was found in 4.5%, more often in women, did not increase with age, and showed no relationship with geography, and 56.4% were positive in IgM blot. In conclusion, antibodies to B. burgdorferi s.l. are common in blood donors in western Norway. The results may be used for evaluation of predictive values of test results in patients, as well as a basis for test algorithms in the laboratory.

  6. Pathogenicity and characterization of a novel Bacillus cereus sensu lato isolate toxic to the Mediterranean fruit fly Ceratitis capitata Wied.

    PubMed

    Ruiu, Luca; Falchi, Giovanni; Floris, Ignazio; Marche, Maria Giovanna; Mura, Maria Elena; Satta, Alberto

    2015-03-01

    The lethal and sub-lethal effects of sporulated cultures of a novel Bacillus cereus sensu lato strain lacking detectable cry genes and identified through morphological and genetic analyses, have been studied on the Mediterranean fruit fly Ceratitis capitata. The lethal effects on young larvae were concentration dependent, with a median lethal concentration (LC50) of 4.48 × 10(8)spores/g of diet. Sporulated cultures of this strain significantly extended development time and reduced immature survival, and the size of emerging fly adults. Besides spores, the toxicity has been associated to the insoluble extra-spore fraction characterized through a proteomic approach. The profile of the extra-spore protein fraction (ES) showed major protein bands within the 35-65 kDa range. The results of mass spectrometry analysis highlighted the presence of putative virulence factors, including members of protein families previously associated to the insecticidal action of other microbial entomopathogens. These proteins include metalloproteases, peptidases and other enzymes.

  7. Seasonal transmission of Opisthorchis viverrini sensu lato and a lecithodendriid trematode species in Bithynia siamensis goniomphalos snails in northeast Thailand.

    PubMed

    Namsanor, Jutamas; Sithithaworn, Paiboon; Kopolrat, Kulthida; Kiatsopit, Nadda; Pitaksakulrat, Opal; Tesana, Smarn; Andrews, Ross H; Petney, Trevor N

    2015-07-01

    Seasonal changes play roles in the transmission success of fish-borne zoonotic trematodes (FZT). This study examined the seasonal transmission patterns of Opisthorchis viverrini sensu lato (s.l.) and a virgulate cercaria (family Lecithodendriidae) in the snail intermediate host, Bithynia siamensis goniomphalos in northeast Thailand. Snail samples were collected monthly during the rainy, cool, and hot seasons during 2012-2013 to determine the prevalence and intensity of larval trematode infections. The prevalence of O. viverrini s.l. varied significantly with season, being 0.31%, 1.05%, and 0.37% in the rainy, cool, and hot seasons, respectively (P < 0.05). Similarly, the prevalence of virgulate cercariae was 3.11%, 6.80%, and 1.64% in the rainy, cool, and hot seasons, respectively (P < 0.05). The intensity of larval trematode infections also varied between seasons and peaked in the hot season (P < 0.05) in both species. The snails infected with O. viverrini s.l. were significantly smaller (P < 0.05) and those infected with virgulate cercariae were significantly larger (P < 0.05) than uninfected snails. Seasonal variation and the different sizes of B. s. goniomphalos parasitized by O. viverrini s.l. and virgulate trematodes indicate complex host-parasite interactions with important implications for the epidemiology of O. viverrini s.l.

  8. Changing patterns of prevalence in Opisthorchis viverrini sensu lato infection in children and adolescents in northeast Thailand.

    PubMed

    Khuntikeo, Narong; Sithithaworn, Paiboon; Loilom, Watcharin; Namwat, Nisana; Yongvanit, Puangrat; Thinkhamrop, Bandit; Kiatsopit, Nadda; Andrews, Ross H; Petney, Trevor N

    2016-12-01

    Infection with the liver fluke Opisthorchis viverrini sensu lato (s.l.), a group 1 carcinogen, is the most important risk factor for developing cholangiocarcinoma (CCA) in Southeast Asia. Cholangiocarcinoma is a fatal disease with the world's highest incidence being found in northeast Thailand. Liver fluke infection occurs through eating raw or partially cooked cyprinid fish containing metacercariae and, therefore, the control of O. viverrini s.l. infection should lead to a reduction in CCA incidence. In this report, we review and analyze the age-prevalence profile data of O. viverrini to reveal temporal changes in patterns of prevalence pre- and post-control programs in Thailand. The profiles of O. viverrini prevalence have transformed from high prevalence in school children prior to 1983 to low prevalences after 1994. This pattern strongly suggests the influence of the health education program on the likelihood of school children becoming infected. In conjunction with current developments in health and socioeconomic conditions, we predict that the incidence of CCA will be reduced with time as the population cohorts that experienced the education programs reach the age at which CCA is most likely to develop, i.e. >50 years. The lessons learned in Thailand may be applicable to other areas endemic for human liver flukes.

  9. Seasonal Transmission of Opisthorchis viverrini sensu lato and a Lecithodendriid Trematode Species in Bithynia siamensis goniomphalos Snails in Northeast Thailand

    PubMed Central

    Namsanor, Jutamas; Sithithaworn, Paiboon; Kopolrat, Kulthida; Kiatsopit, Nadda; Pitaksakulrat, Opal; Tesana, Smarn; Andrews, Ross H.; Petney, Trevor N.

    2015-01-01

    Seasonal changes play roles in the transmission success of fish-borne zoonotic trematodes (FZT). This study examined the seasonal transmission patterns of Opisthorchis viverrini sensu lato (s.l.) and a virgulate cercaria (family Lecithodendriidae) in the snail intermediate host, Bithynia siamensis goniomphalos in northeast Thailand. Snail samples were collected monthly during the rainy, cool, and hot seasons during 2012–2013 to determine the prevalence and intensity of larval trematode infections. The prevalence of O. viverrini s.l. varied significantly with season, being 0.31%, 1.05%, and 0.37% in the rainy, cool, and hot seasons, respectively (P < 0.05). Similarly, the prevalence of virgulate cercariae was 3.11%, 6.80%, and 1.64% in the rainy, cool, and hot seasons, respectively (P < 0.05). The intensity of larval trematode infections also varied between seasons and peaked in the hot season (P < 0.05) in both species. The snails infected with O. viverrini s.l. were significantly smaller (P < 0.05) and those infected with virgulate cercariae were significantly larger (P < 0.05) than uninfected snails. Seasonal variation and the different sizes of B. s. goniomphalos parasitized by O. viverrini s.l. and virgulate trematodes indicate complex host–parasite interactions with important implications for the epidemiology of O. viverrini s.l. PMID:25918210

  10. The systematics and population genetics of Opisthorchis viverrini sensu lato: implications in parasite epidemiology and bile duct cancer.

    PubMed

    Sithithaworn, Paiboon; Andrews, Ross H; Petney, Trevor N; Saijuntha, Weerachai; Laoprom, Nonglak

    2012-03-01

    Together with host and environmental factors, the systematics and population genetic variation of Opisthorchis viverrini may contribute to recorded local and regional differences in epidemiology and host morbidity in opisthorchiasis and cholangiocarcinoma (CCA). In this review, we address recent findings that O. viverrini comprises a species complex with varying degrees of population genetic variation which are associated with specific river wetland systems within Thailand as well as the Lao PDR. Having an accurate understanding of systematics is a prerequisite for a meaningful assessment of the population structure of each species within the O. viverrini complex in nature, as well as a better understanding of the magnitude of genetic variation that occurs within different species of hosts in its life cycle. Whether specific genotypes are related to habitat type(s) and/or specific intermediate host species are discussed based on current available data. Most importantly, we focus on whether there is a correlation between incidence of CCA and genotype(s) of O. viverrini. This will provide a solid basis for further comprehensive investigations of the role of genetic variation within each species of O. viverrini sensu lato in human epidemiology and genotype related morbidity as well as co-evolution of parasites with primary and secondary intermediate species of host.

  11. Deep molecular divergence and exceptional morphological stasis in dwarf cannibal snails Nata sensu lato Watson, 1934 (Rhytididae) of southern Africa.

    PubMed

    Moussalli, Adnan; Herbert, David G

    2016-02-01

    The genus Nata Watson, 1934 is a southern African endemic belonging to the Gondwanan family of carnivorous snails, Rhytididae. We present a molecular phylogeny of the genus based on two mitochondrial (16S and COI) and two nuclear genes (ITS2 and 28S RNA), and complement this with an appraisal of morphological characters relating to both the shell and soft parts. We identify four reciprocally monophyletic lineages for which valid names are already available, plus two undescribed species restricted to the Albany Thicket Biome. We show that Nata sensu lato may not be monophyletic. Rather there exist two deep lineages within Nata s.l., one lineage potentially sister to a clade dominated by the Australian and New Zealand radiation, and the other occupying a basal position within Rhytididae. Accordingly we recommend a revision recognising two genera, namely Nata s.s. and Natella respectively. Despite deep molecular divergences within Nata s.s., phenotypic evolution has been remarkably conserved, and contrasts greatly with that exhibited across other major lineages within the Rhytididae.

  12. Borrelia burgdorferi sensu lato and co-infections with Anaplasma phagocytophilum and Rickettsia spp. in Ixodes ricinus in Hamburg, Germany.

    PubMed

    May, K; Jordan, D; Fingerle, V; Strube, C

    2015-12-01

    To obtain initial data on Borrelia burgdorferi sensu lato (Spirochaetales: Spirochaetaceae) in Ixodes ricinus (Ixodida: Ixodidae) ticks in Hamburg, Germany, 1400 questing ticks were collected by flagging at 10 different public recreation areas in 2011 and analysed using probe-based quantitative real-time polymerase chain reaction. The overall rate of infection with B. burgdorferi s.l. was 34.1%; 30.0% of adults were infected (36.7% of females and 26.0% of males), as were 34.5% of nymphs. Significant differences in tick infection rates were observed between the spring and summer/autumn months, as well as among sampling locations. Borrelia genospecies identification by reverse line blotting was successful in 43.6% of positive tick samples. The most frequent genospecies was Borrelia garinii/Borrelia bavariensis, followed by Borrelia afzelii, Borrelia valaisiana, B. burgdorferi sensu stricto, Borrelia spielmanii, Borrelia bissettii and Borrelia lusitaniae. Based on previously published data, co-infection of Borrelia and Rickettsiales spp. was determined in 25.8% of ticks. Overall, 22.9% of ticks were co-infected with Rickettsia spp. (Rickettsiales: Rickettsiaceae), 1.7% with Anaplasma phagocytophilum (Rickettsiales: Anaplasmataceae), and 1.2% with both pathogens. Study results show a high prevalence of Borrelia-positive ticks in recreation areas in the northern German city of Hamburg and the potential health risk to humans in these areas should not be underestimated.

  13. Borrelia burgdorferi sensu lato detected in skin of Norwegian mountain hares (Lepus timidus) without signs of dissemination.

    PubMed

    Kjelland, Vivian; Ytrehus, Bjørnar; Vikørren, Turid; Stuen, Snorre; Skarpaas, Tone; Slettan, Audun

    2011-04-01

    The mountain hare (Lepus timidus) population in southern Norway appears to be in decline. Necropsy and laboratory examinations of 36 hares found dead or diseased during 2007-2009 in Vest- and Aust-Agder counties showed that disease and deaths were attributed to multiple causes, with no specific etiology emerging as a cause for population decline. To investigate whether Borrelia burgdorferi sensu lato (s.l.) infection is associated with mortality in mountain hares, tissues and ticks collected from hares were investigated for infection with the spirochete. Borrelia burgdorferi s.l. DNA was not detected in samples from internal organs, whereas Borrelia afzelii, B. burgdorferi sensu stricto (s.s.), and the not-yet-defined Borrelia sp. SV1 were found in skin samples from hares and in adult and nymphal Ixodes ricinus feeding on hares. Only B. burgdorferi s.s. and Borrelia sp. SV1 were detected in larvae feeding on hares. Our results indicate that disseminated Borrelia infection in hares rarely occurs and, presumably, does not play a central role in the suspected population decline. The results also suggest that the mountain hare to some degree functions as a transmission host for B. burgdorferi s.s. and Borrelia sp. SV1.

  14. Morphological and ontogenetic stratification of abyssal and hadal Eurythenes gryllus sensu lato (Amphipoda: Lysianassoidea) from the Peru-Chile Trench

    NASA Astrophysics Data System (ADS)

    Eustace, Ryan M.; Ritchie, Heather; Kilgallen, Niamh M.; Piertney, Stuart B.; Jamieson, Alan J.

    2016-03-01

    The globally ubiquitous lysianassoid amphipod, Eurythenes gryllus, has been shown to consist of multiple genetically distinct cryptic taxa, with depth considered a major driver of speciation and morphological divergence. Here we examine morphological variation of E. gryllus sensu lato through a continuous depth distribution that spans from abyssal (3000-6000 m) into hadal depths (>6000 m) in the Peru-Chile Trench (SE Pacific Ocean). Three distinct morphospecies were identified: one was confirmed as being E. magellanicus (4602-5329 m) based on DNA sequence and morphological similarity. The other two morphologically distinct species were named based upon depth of occurrence; Abyssal (4602-6173 m) and Hadal (6173-8074 m). The three Eurythenes morphospecies showed vertical ontogenetic stratification across their bathymetric range, where juveniles were found shallower in their depth range and mature females deeper. Potential ecological and evolutionary drivers that explain the observed patterns of intra and inter-specific structure, such as hydrostatic pressure and topographical isolation, are discussed.

  15. Evaluation of a Community-Based Trapping Program to Collect Simulium ochraceum sensu lato for Verification of Onchocerciasis Elimination

    PubMed Central

    Rodríguez-Pérez, Mario A.; Adeleke, Monsuru A.; Rodríguez-Luna, Isabel C.; Cupp, Eddie W.; Unnasch, Thomas R.

    2014-01-01

    Background Collection of the black fly vectors of onchocerciasis worldwide relies upon human landing collections. Recent studies have suggested that the Esperanza Window Trap baited with a human scent lure and CO2 had the potential to replace human hosts for the collection of Simulium ochraceum sensu lato in Southern Chiapas focus, Mexico. The feasibility of utilizing these traps in a community-based approach for the collection of S. ochraceum s.l. was evaluated. Methodology/Principal findings Local residents of a formerly endemic extra-sentinel community for onchocerciasis were trained to carry out collections using the traps. The residents operated the traps over a 60-day period and conducted parallel landing collections, resulting in a total of 28,397 vector black flies collected. None of the flies collected were found to contain parasite DNA when tested by a polymerase chain reaction assay targeting a parasite specific sequence, resulting in a point estimate of infection in the vectors of zero, with an upper bound of the 95% confidence interval 0.13 per 2,000. This meets the accepted criterion for demonstrating an interruption of parasite transmission. Conclusions/Significance These data demonstrate that Esperanza Window Traps may be effectively operated by minimally trained residents of formerly endemic communities, resulting in the collection of sufficient numbers of flies to verify transmission interruption of onchocerciasis. The traps represent a viable alternative to using humans as hosts for the collection of vector flies as part of the verification of onchocerciasis elimination. PMID:25340517

  16. Large Scale Spatial Risk and Comparative Prevalence of Borrelia miyamotoi and Borrelia burgdorferi Sensu Lato in Ixodes pacificus

    PubMed Central

    Padgett, Kerry; Bonilla, Denise; Kjemtrup, Anne; Vilcins, Inger-Marie; Yoshimizu, Melissa Hardstone; Hui, Lucia; Sola, Milagros; Quintana, Miguel; Kramer, Vicki

    2014-01-01

    Borrelia miyamotoi is a newly described emerging pathogen transmitted to people by Ixodes species ticks and found in temperate regions of North America, Europe, and Asia. There is limited understanding of large scale entomological risk patterns of B. miyamotoi and of Borreila burgdorferi sensu stricto (ss), the agent of Lyme disease, in western North America. In this study, B. miyamotoi, a relapsing fever spirochete, was detected in adult (n = 70) and nymphal (n = 36) Ixodes pacificus ticks collected from 24 of 48 California counties that were surveyed over a 13 year period. Statewide prevalence of B. burgdorferi sensu lato (sl), which includes B. burgdorferi ss, and B. miyamotoi were similar in adult I. pacificus (0.6% and 0.8%, respectively). In contrast, the prevalence of B. burgdorferi sl was almost 2.5 times higher than B. miyamotoi in nymphal I. pacificus (3.2% versus 1.4%). These results suggest similar risk of exposure to B. burgdorferi sl and B. miyamotoi from adult I. pacificus tick bites in California, but a higher risk of contracting B. burgdorferi sl than B. miyamotoi from nymphal tick bites. While regional risk of exposure to these two spirochetes varies, the highest risk for both species is found in north and central coastal California and the Sierra Nevada foothill region, and the lowest risk is in southern California; nevertheless, tick-bite avoidance measures should be implemented in all regions of California. This is the first study to comprehensively evaluate entomologic risk for B. miyamotoi and B. burgdorferi for both adult and nymphal I. pacificus, an important human biting tick in western North America. PMID:25333277

  17. Borrelia burgdorferi Sensu Lato Spirochetes in Wild Birds in Northwestern California: Associations with Ecological Factors, Bird Behavior and Tick Infestation

    PubMed Central

    Newman, Erica A.; Eisen, Lars; Eisen, Rebecca J.; Fedorova, Natalia; Hasty, Jeomhee M.; Vaughn, Charles; Lane, Robert S.

    2015-01-01

    Although Borrelia burgdorferi sensu lato (s.l.) are found in a great diversity of vertebrates, most studies in North America have focused on the role of mammals as spirochete reservoir hosts. We investigated the roles of birds as hosts for subadult Ixodes pacificus ticks and potential reservoirs of the Lyme disease spirochete B. burgdorferi sensu stricto (s.s.) in northwestern California. Overall, 623 birds representing 53 species yielded 284 I. pacificus larvae and nymphs. We used generalized linear models and zero-inflated negative binomial models to determine associations of bird behaviors, taxonomic relationships and infestation by I. pacificus with borrelial infection in the birds. Infection status in birds was best explained by taxonomic order, number of infesting nymphs, sampling year, and log-transformed average body weight. Presence and counts of larvae and nymphs could be predicted by ground- or bark-foraging behavior and contact with dense oak woodland. Molecular analysis yielded the first reported detection of Borrelia bissettii in birds. Moreover, our data suggest that the Golden-crowned Sparrow (Zonotrichia atricapilla), a non-resident species, could be an important reservoir for B. burgdorferi s.s. Of 12 individual birds (9 species) that carried B. burgdorferi s.l.-infected larvae, no birds carried the same genospecies of B. burgdorferi s.l. in their blood as were present in the infected larvae removed from them. Possible reasons for this discrepancy are discussed. Our study is the first to explicitly incorporate both taxonomic relationships and behaviors as predictor variables to identify putative avian reservoirs of B. burgdorferi s.l. Our findings underscore the importance of bird behavior to explain local tick infestation and Borrelia infection in these animals, and suggest the potential for bird-mediated geographic spread of vector ticks and spirochetes in the far-western United States. PMID:25714376

  18. Ferritin 1 silencing effect in Rhipicephalus sanguineus sensu lato (Acari: Ixodidae) during experimental infection with Ehrlichia canis.

    PubMed

    Ferrolho, Joana; Antunes, Sandra; Sanches, Gustavo S; Couto, Joana; Évora, Patrícia M; Rosa, Catarina; André, Marcos R; Machado, Rosângela Z; Bechara, Gervásio H; Domingos, Ana

    2017-01-01

    Rhipicephalus sanguineus sensu lato (s.l.) is a very common ectoparasite of domestic dogs able to transmit several pathogens of human and veterinary importance. Tick infestations and tick-borne diseases (TBDs) remain a serious and persistent problem, due to the lack of efficient control measures. It is therefore vital that novel approaches to control are pursued. Whilst vaccination is recognised as a potential control method to reduce tick infestation, no anti-R. sanguineus vaccine is available. Ticks depend on their blood meals to obtain nutrients and to achieve sexual maturity, which exposes them to vast amounts of iron. Although an essential molecule for several biological processes, its excess can lead to oxidative stress. Iron homeostasis is achieved with the help of iron-binding proteins called ferritins, among others, present in several tick tissues and developmental stages. These evolutionarily conserved proteins regulate iron homeostasis by storing and releasing iron in a controlled manner. In this study the R. sanguineus ferritin 1 gene was silenced through RNA interference (RNAi) in adult females exposed to an experimental infection with Ehrlichia canis. The purpose of this study was to assess the role of this protein in tick feeding, ovary development, oogenesis, and pathogen acquisition. Our data has shown that silencing ferritin 1 alters tick competence to normally engorge and causes morphologic and histochemical changes in the ovaries (OV) and oocytes. Furthermore, our data revealed that no E. canis DNA was found in either experimental group. Determining the function of molecules that act in key biological processes, such as blood digestion or reproduction, and that could be considered potential tick antigens will contribute towards the improvement of current control measures against these ectoparasites and the pathogens they vector.

  19. A novel multilocus phylogenetic estimation reveals unrecognized diversity in Asian horned toads, genus Megophrys sensu lato (Anura: Megophryidae).

    PubMed

    Chen, Jin-Min; Zhou, Wei-Wei; Poyarkov, Nikolay A; Stuart, Bryan L; Brown, Rafe M; Lathrop, Amy; Wang, Ying-Yong; Yuan, Zhi-Yong; Jiang, Ke; Hou, Mian; Chen, Hong-Man; Suwannapoom, Chatmongkon; Nguyen, Sang Ngoc; Duong, Tang Van; Papenfuss, Theodore J; Murphy, Robert W; Zhang, Ya-Ping; Che, Jing

    2017-01-01

    The horned toad assemblage, genus Megophrys sensu lato, currently includes three groups previously recognized as the genera Atympanophrys, Xenophrys and Megophrys sensu stricto. The taxonomic status and species composition of the three groups remain controversial due to conflicting phenotypic analyses and insufficient phylogenetic reconstruction; likewise, the position of the monotypic Borneophrys remains uncertain with respect to the horned toads. Further, the diversity of the horned toads remains poorly understood, especially for widespread species. Herein, we evaluate species-level diversity based on 45 of the 57 described species from throughout southern China, Southeast Asia and the Himalayas using Bayesian inference trees and the Generalized Mixed Yule Coalescent (GMYC) approach. We estimate the phylogeny using both mitochondrial and nuclear DNA data. Analyses reveal statistically significant mito-nuclear discordance. All analyses resolve paraphyly for horned toads involving multiple strongly supported clades. These clades correspond with geography. We resurrect the genera Atympanophrys and Xenophrys from the synonymy of Megophrys to eliminate paraphyly of Megophrys s.l. and to account for the morphological, molecular and biogeographic differences among these groups, but we also provide an alternative option. Our study suggests that Borneophrys is junior synonym of Megophrys sensu stricto. We provide an estimation of timeframe for the horned toads. The mitochondrial and nuclear trees indicate the presence of many putative undescribed species. Widespread species, such as Xenophrys major and X. minor, likely have dramatically underestimated diversity. The integration of morphological and molecular evidence can validate this discovery. Montane forest dynamics appear to play a significant role in driving diversification of horned toads.

  20. Borrelia burgdorferi sensu lato spirochetes in wild birds in northwestern California: associations with ecological factors, bird behavior and tick infestation.

    PubMed

    Newman, Erica A; Eisen, Lars; Eisen, Rebecca J; Fedorova, Natalia; Hasty, Jeomhee M; Vaughn, Charles; Lane, Robert S

    2015-01-01

    Although Borrelia burgdorferi sensu lato (s.l.) are found in a great diversity of vertebrates, most studies in North America have focused on the role of mammals as spirochete reservoir hosts. We investigated the roles of birds as hosts for subadult Ixodes pacificus ticks and potential reservoirs of the Lyme disease spirochete B. burgdorferi sensu stricto (s.s.) in northwestern California. Overall, 623 birds representing 53 species yielded 284 I. pacificus larvae and nymphs. We used generalized linear models and zero-inflated negative binomial models to determine associations of bird behaviors, taxonomic relationships and infestation by I. pacificus with borrelial infection in the birds. Infection status in birds was best explained by taxonomic order, number of infesting nymphs, sampling year, and log-transformed average body weight. Presence and counts of larvae and nymphs could be predicted by ground- or bark-foraging behavior and contact with dense oak woodland. Molecular analysis yielded the first reported detection of Borrelia bissettii in birds. Moreover, our data suggest that the Golden-crowned Sparrow (Zonotrichia atricapilla), a non-resident species, could be an important reservoir for B. burgdorferi s.s. Of 12 individual birds (9 species) that carried B. burgdorferi s.l.-infected larvae, no birds carried the same genospecies of B. burgdorferi s.l. in their blood as were present in the infected larvae removed from them. Possible reasons for this discrepancy are discussed. Our study is the first to explicitly incorporate both taxonomic relationships and behaviors as predictor variables to identify putative avian reservoirs of B. burgdorferi s.l. Our findings underscore the importance of bird behavior to explain local tick infestation and Borrelia infection in these animals, and suggest the potential for bird-mediated geographic spread of vector ticks and spirochetes in the far-western United States.

  1. Occurrence of Hepatozoon canis and Cercopithifilaria bainae in an off-host population of Rhipicephalus sanguineus sensu lato ticks.

    PubMed

    Ramos, Rafael Antonio Nascimento; Giannelli, Alessio; Carbone, Domenico; Baneth, Gad; Dantas-Torres, Filipe; Otranto, Domenico

    2014-04-01

    Hepatozoon canis (Eucoccidiorida, Hepatozoidae) and the filarioid Cercopithifilaria bainae (Spirurida, Onchocercidae) are tick-transmitted infectious agents of dogs, highly prevalent in the Mediterranean basin in association with Rhipicephalus sanguineus sensu lato. Ticks were collected from the environment every 25±2 days in a confined location in southern Italy where a community of dogs lives, from August 2012 to July 2013. In order to study the occurrence of H. canis and C. bainae, 1091 tick specimens (770 adults; 271 nymphs, and 50 larvae) were dissected, and oocysts of H. canis and larvae of C. bainae were morphologically identified. Out of 1091 dissected ticks, 13.47% (n=147) were positive for H. canis, with the highest prevalence recorded in unfed adults (16.4%; 126/770), followed by nymphs collected as larvae and allowed to moult (14%; 7/50), unfed nymphs dissected immediately after collection (3%; 8/271), and adults collected as nymphs and allowed to moult (2%; 6/271). The highest number of H. canis-positive ticks (35.5%; 43/121; P<0.05) was recorded during the summer months (i.e., June-July). In addition, 6% of adult ticks (n=66) were positive for third-stage larvae of C. bainae, with the highest number in June (17%; 14/84; P<0.05). Based on the results reported herein, H. canis and C. bainae infections in the study area seem to be dependent on the seasonality of vector tick populations. Hence, dogs living in these areas are more exposed to both pathogens during the warmer months. These findings provide new insights into the ecology of both H. canis and C. bainae.

  2. Large scale spatial risk and comparative prevalence of Borrelia miyamotoi and Borrelia burgdorferi sensu lato in Ixodes pacificus.

    PubMed

    Padgett, Kerry; Bonilla, Denise; Kjemtrup, Anne; Vilcins, Inger-Marie; Yoshimizu, Melissa Hardstone; Hui, Lucia; Sola, Milagros; Quintana, Miguel; Kramer, Vicki

    2014-01-01

    Borrelia miyamotoi is a newly described emerging pathogen transmitted to people by Ixodes species ticks and found in temperate regions of North America, Europe, and Asia. There is limited understanding of large scale entomological risk patterns of B. miyamotoi and of Borreila burgdorferi sensu stricto (ss), the agent of Lyme disease, in western North America. In this study, B. miyamotoi, a relapsing fever spirochete, was detected in adult (n=70) and nymphal (n=36) Ixodes pacificus ticks collected from 24 of 48 California counties that were surveyed over a 13 year period. Statewide prevalence of B. burgdorferi sensu lato (sl), which includes B. burgdorferi ss, and B. miyamotoi were similar in adult I. pacificus (0.6% and 0.8%, respectively). In contrast, the prevalence of B. burgdorferi sl was almost 2.5 times higher than B. miyamotoi in nymphal I. pacificus (3.2% versus 1.4%). These results suggest similar risk of exposure to B. burgdorferi sl and B. miyamotoi from adult I. pacificus tick bites in California, but a higher risk of contracting B. burgdorferi sl than B. miyamotoi from nymphal tick bites. While regional risk of exposure to these two spirochetes varies, the highest risk for both species is found in north and central coastal California and the Sierra Nevada foothill region, and the lowest risk is in southern California; nevertheless, tick-bite avoidance measures should be implemented in all regions of California. This is the first study to comprehensively evaluate entomologic risk for B. miyamotoi and B. burgdorferi for both adult and nymphal I. pacificus, an important human biting tick in western North America.

  3. Comparison of MKP and BSK-H media for the cultivation and isolation of Borrelia burgdorferi sensu lato

    PubMed Central

    Ružić-Sabljić, Eva; Maraspin, Vera; Stupica, Daša; Rojko, Tereza; Bogovič, Petra; Strle, Franc; Cerar, Tjaša

    2017-01-01

    The isolation of B. burgdorferi sensu lato requires the use of complex cultivation media. The aim of the study was to compare the usefulness of BSK-H (a commercial medium produced by HiMedia, India) and MKP medium. MKP and BSK-H media were prepared in accordance with the relevant protocols. Borrelia strains and skin culture biopsies were simultaneously inoculated into both media, incubated and checked for growth. Borrelial growth characteristics, isolation rates and characteristics of the isolated borreliae were analysed and compared. Initially, numbers of spirochaetes were higher in BSK-H than in MKP; however, in comparison with MKP, the strains subcultured in BSK-H medium were more frequently irregular, thin and non-motile, and rapidly died. In addition, the borrelial isolation rate from erythema migrans skin samples was higher in MKP than in BSK-H medium (108/171, 63.2% versus 70/171, 40.9%; p<0.0001). The far most frequently isolated species was Borrelia afzelii (92.9% and 97.2% strains isolated from BSK-H and MKP, respectively). Comparison of strains cultured from individual patients in both media showed differences in plasmid contents in 9/46 (19.6%) strain pairs, and protein profiles differed in 30/43 (69.8%) strain pairs, most often in the expression of OspC (in 27/28 patients OspC was expressed only in strains growing in MKP). BSK-H medium supports the growth of borrelial strains but MKP is superior with regard to the isolation rate, morphology and motility of strains. BSK-H medium supports fast initial growth of borreliae but this is followed by rapid deformation and death of the spirochaetes. PMID:28170447

  4. QUALITY ASSURANCE FOR PCR

    EPA Science Inventory

    The U.S. Environmental Protection Agency (EPA) held a workshop in January 2003 on the detection of viruses in water using polymerase chain reaction (PCR)-based methods. Speakers were asked to address a series of specific questions, including whether a single standard method coul...

  5. QUALITY CONTROLS FOR PCR

    EPA Science Inventory

    The purpose of this presentation is to present an overview of the quality control (QC) sections of a draft EPA document entitled, "Quality Assurance/Quality Control Guidance for Laboratories Performing PCR Analyses on Environmental Samples." This document has been prepared by th...

  6. Pcr by Thermal Convection

    NASA Astrophysics Data System (ADS)

    Braun, Dieter

    The Polymerase Chain Reaction (PCR) allows for highly sensitive and specific amplification of DNA. It is the backbone of many genetic experiments and tests. Recently, three labs independently uncovered a novel and simple way to perform a PCR reaction. Instead of repetitive heating and cooling, a temperature gradient across the reaction vessel drives thermal convection. By convection, the reaction liquid circulates between hot and cold regions of the chamber. The convection triggers DNA amplification as the DNA melts into two single strands in the hot region and replicates into twice the amount in the cold region. The amplification progresses exponentially as the convection moves on. We review the characteristics of the different approaches and show the benefits and prospects of the method.

  7. PCR in forensic genetics.

    PubMed

    Morling, Niels

    2009-04-01

    Since the introduction in the mid-1980s of analyses of minisatellites for DNA analyses, a revolution has taken place in forensic genetics. The subsequent invention of the PCR made it possible to develop forensic genetics tools that allow both very informative routine investigations and still more and more advanced, special investigations in cases concerning crime, paternity, relationship, disaster victim identification etc. The present review gives an update on the use of DNA investigations in forensic genetics.

  8. The discovery after 94 years of the elusive female of a myrmecolacid (Strepsiptera), and the cryptic species of Caenocholax fenyesi Pierce sensu lato.

    PubMed Central

    Kathirithamby, Jeyaraney; Johnston, J Spencer

    2004-01-01

    Due to its extreme sexual dimorphism and disparate hosts, no female myrmecolacid has been matched to its conspecific male to date. Here, for the first time to our knowledge, a morphological description is given of the matched female and male myrmecolacid, Caenocholax fenyesi waloffi ssp. nov. from Veracruz, Mexico: the female parasitic in a cricket and the male parasitic in an ant. For examined segments of DNA, the male and female are identical. Male C. fenyesi Pierce sensu lato was described 94 years ago from Veracruz. The male from Texas USA, which, for the same DNA segments, shows 15% divergence from the morphologically identical male from Veracruz, is given subspecies status, and is named Caenocholax fenyesi texensis ssp. nov. The discovery of the female finally enables many interesting studies to be pursued, such as speciation in morphologically cryptic taxa, the sexes of which parasitize disparate hosts. Caenocholax fenyesi sensu lato may also be evaluated for biocontrol of the red imported fire ant, Solenopsis invicta Buren, which is a pest in the USA and Australia. PMID:15101403

  9. Comparative biology of the tropical and temperate species of Rhipicephalus sanguineus sensu lato (Acari: Ixodidae) under different laboratory conditions.

    PubMed

    Labruna, Marcelo B; Gerardi, Monize; Krawczak, Felipe S; Moraes-Filho, Jonas

    2017-01-01

    Recent studies have shown that the taxon Rhipicephalus sanguineus sensu lato (s.l.) is represented in Latin America by two distinct species, designated as 'tropical species' (distributed from Mexico to Brazil) and 'temperate species' (restricted to the southern cone of South America). Since both tropical and temperate species of R. sanguineus s.l. are parasites primarily of domestic dogs, the reasons for their distinct geographical distribution in South America could be related to particular requirements of abiotic conditions for off-host development. With the purpose to test this hypothesis, this study evaluated the off-host developmental stages (eggs, engorged larvae, nymphs and females) of both tick species simultaneously inside incubators with temperature and photoperiod regimens that simulated the summer and winter conditions of tropical Brazil (where the 'tropical species' occurs) and temperate Brazil (where the 'temperate species' occurs). Results showed that the temperate species had significantly higher survival rates than the tropical species, when engorged ticks (larvae, nymphs and females) and eggs were incubated at lower temperatures simulating winter seasons of many parts of the southern cone of South America, where the temperate species is known to occur. These results suggest that the absence of established populations of the tropical species in temperate areas of South America is related to the low overwinter capacity of the tropical species in those areas. Regarding the temperate species, unfed adults that molted from nymphs under summer conditions of either tropical or temperate Brazil remained dormant, at the state of behavioral diapause for at least 20 weeks. Contrastingly, when engorged nymphs of the temperate species were held at winter conditions for at least 3 months, and then transferred to summer conditions to complete molting, no diapause was observed in adult ticks. These results were corroborated by infestation trials, which showed

  10. Rodent species as natural reservoirs of Borrelia burgdorferi sensu lato in different habitats of Ixodes ricinus in The Netherlands.

    PubMed

    Gassner, Fedor; Takken, Willem; Plas, Carin Lombaers-van der; Kastelein, Pieter; Hoetmer, Arno J; Holdinga, Maarten; van Overbeek, Leonard S

    2013-09-01

    Rodents are natural reservoirs for human pathogenic spirochaetes of the Borrelia burgdorferi complex [B. burgdorferi sensu lato (s.l.)], and the pathogens are transmitted by Ixodes ricinus ticks to humans in The Netherlands. B. burgdorferi s.l. infection prevalence in questing ticks, rodents, and ticks feeding on these rodents, all sampled within the same short time span of five days in three different areas in The Netherlands, were compared in order to establish the relationship between ticks, reservoir hosts, and B. burgdorferi s.l. Questing nymphs were found in all 3 areas and numbers differed per area and even per site within areas. Infection prevalence in questing nymphs ranged between 0 and 20%. Apodemus sylvaticus and Myodes glareolus were the dominant rodents captured, and their numbers differed per area. Infection prevalence, determined by ear biopsies, ranged between 0 and 33.3% for both rodent species. Larvae were most frequently found feeding on these rodents, and their Borrelia infection prevalence ranged between 0 and 6.3% (A. sylvaticus) and between 0 and 29.4% (M. glareolus). The burden of nymphs feeding on rodents was low and varied per area with only 2 of 42 nymphs infected. Comparisons made on the basis of infection prevalence indicated that there was no clear relationship between rodents and questing nymphs when sampled within the same short time span. However, a possible relationship was present when questing ticks were sampled over longer periods in time (months) within or near the same areas (range of infection prevalence between 3.7 and 39.4). Confounding factors thus play a role in the interaction between rodents, ticks, and B. burgdorferi s.l., and it is very likely that other reservoir host species are responsible for the observed fluctuations. It is concluded that the local variations in rodent-Borrelia-tick interactions only partially explain the Lyme borreliosis risk in the sites studied and that other ecological determinants, notably

  11. MAMMALIAN DNA IN PCR REAGENTS

    EPA Science Inventory

    Ancient DNA analysis is becoming widespread. These studies use polymerase chain reaction (PCR) to amplify minute quantities of heavily damaged template. Unusual steps are taken to achieve the sensitivity necessary to detect ancient DNA, including high- cycle PCR amplification t...

  12. Borrelia spirochetes in Russia: Genospecies differentiation by real-time PCR.

    PubMed

    Mukhacheva, T A; Kovalev, S Y

    2014-10-01

    Spirochetes of the Borrelia burgdorferi sensu lato complex are the causative agent of Lyme borreliosis which is widespread in Russia. Nowadays, three clinically important B. burgdorferi s.l. genospecies, B. afzelii, B. garinii, B. bavariensis sp. nov., can be found in Russia, as well as B. miyamotoi, which belongs to the tick-borne relapsing fever group of spirochetes. Several techniques have been developed to differentiate Borrelia genospecies. However, most of them do not allow detection of all of these genospecies simultaneously. Also, no method based on the RT-PCR TaqMan approach has been proposed to differentiate the genetically closely related species B. bavariensis and B. garinii. In the present paper, we investigated two species of ticks, I. persulcatus and I. pavlovskyi (1343 and 92 adults, respectively). Two sets of primers and probes for RT-PCR, with uvrA, glpQ and nifS genes as targets, were designed to detect four Borrelia genospecies in positive samples. The average prevalence of Borrelia sp. was about 40%, with B. afzelii as the most prevalent genospecies. Mixed infections of B. bavariensis and B. garinii were found to be extremely rare. While B. bavariensis was predominant in I. persulcatus, I. pavlovskyi ticks were infected exclusively by B. garinii. The proposed technique proved to be efficient in selection of individual Borrelia species for further genetic analysis, in particular, for multilocus sequence typing. Also, it could be applied for the differentiation of Borrelia genospecies in clinical material.

  13. Copro-PCR prevalence of Echinococcus granulosus infection in dogs in Kerman, south-eastern Iran.

    PubMed

    Mirbadie, S R; Kamyabi, H; Mohammadi, M A; Shamsaddini, S; Harandi, M F

    2017-01-30

    The main objective of this study was to determine the prevalence of taeniid parasites and the specific detection of Echinococcus granulosus using copro-DNA polymerase chain reaction (PCR) analysis in the stray dogs of Kerman, south-eastern Iran. From September 2013 to May 2014, faecal samples of stray dogs were collected from different parts of the city of Kerman and its suburbs. Faecal samples from dogs were collected randomly within 24 h of defecation. All samples were transferred to the research lab and coprological examinations were conducted by the formalin-ether concentration method. In the microscopically positive samples, mitochondrial cytochrome c oxidase subunit 1 (cox1) specific primers were used to determine the taeniid identity of the infection. In addition, another set of primers was used for the specific diagnosis of E. granulosus sensu lato. In total, 307 faecal samples from stray dogs were examined for the presence of the parasites. Taeniidae eggs were detected in 34 dogs (11.07%). All 34 taeniid-positive specimens were PCR positive for cox1 (444 bp). Of all taeniid-positive specimens, 21 samples (6.8% of all dog specimens) were positive according to primers specific for E. granulosus. The findings of the present study revealed that canine echinococcosis is prevalent in the stray dogs in Kerman. The findings of the present study have important implications for hydatid control programmes in the area.

  14. Speciation history and widespread introgression in the European short-call tree frogs (Hyla arborea sensu lato, H. intermedia and H. sarda).

    PubMed

    Gvoždík, Václav; Canestrelli, Daniele; García-París, Mario; Moravec, Jiří; Nascetti, Giuseppe; Recuero, Ernesto; Teixeira, José; Kotlík, Petr

    2015-02-01

    European tree frogs (Hyla) characterized by short temporal parameters of the advertisement call form six genetically differentiated but morphologically cryptic taxa, H. arborea sensu stricto, H. orientalis and H. molleri from across Europe to western Asia (together referred to as H. arborea sensu lato), two putative taxa within H. intermedia (Northern and Southern) from the Italian Peninsula and Sicily, and H. sarda from Sardinia and Corsica. Here, we assess species limits and phylogenetic relationships within these 'short-call tree frogs' based on mitochondrial DNA and nuclear protein-coding markers. The mitochondrial and nuclear genes show partly incongruent phylogeographic patterns, which point to a complex history of gene flow across taxa, particularly in the Balkans. To test the species limits in the short-call tree frogs and to infer the species tree, we used coalescent-based approaches. The monophyly of H. arborea sensu lato is supported by the mtDNA as well as by the all-gene species tree. The Northern and Southern lineages of H. intermedia have been connected by nuclear gene flow (despite their deep mtDNA divergence) and should be treated as conspecific. On the contrary, the parapatric taxa within H. arborea sensu lato should be considered distinct species (H. arborea, H. orientalis, H. molleri) based on the coalescent analysis, although signs of hybridization were detected between them (H. arborea×H. orientalis; H. arborea×H. molleri). A mitochondrial capture upon secondary contact appears to explain the close mtDNA relationship between the geographically remote Iberian H. molleri and H. orientalis from around the Black Sea. Introgressive hybridization occurred also between the Balkan H. arborea and northern Italian H. intermedia, and between the Minor Asiatic H. orientalis and Arabian H. felix arabica (the latter belonging to a different acoustic group/clade). Our results shed light on the species limits in the European short-call tree frogs and show

  15. Seasonal Variation in Biting Rates of Simulium damnosum sensu lato, Vector of Onchocerca volvulus, in Two Sudanese Foci

    PubMed Central

    Zarroug, Isam M. A.; Hashim, Kamal; Elaagip, Arwa H.; Samy, Abdallah M.; Frah, Ehab A.; ElMubarak, Wigdan A.; Mohamed, Hanan A.; Deran, Tong Chor M.; Aziz, Nabil; Higazi, Tarig B.

    2016-01-01

    Background The abundance of onchocerciasis vectors affects the epidemiology of disease in Sudan, therefore, studies of vector dynamics are crucial for onchocerciasis control/elimination programs. This study aims to compare the relative abundance, monthly biting-rates (MBR) and hourly-based distribution of onchocerciasis vectors in Abu-Hamed and Galabat foci. These seasonally-based factors can be used to structure vector control efforts to reduce fly-biting rates as a component of onchocerciasis elimination programs. Methods A cross-sectional study was conducted in four endemic villages in Abu-Hamed and Galabat foci during two non-consecutive years (2007–2008 and 2009–2010). Both adults and aquatic stages of the potential onchocerciasis vector Simulium damnosum sensu lato were collected following standard procedures during wet and dry seasons. Adult flies were collected using human landing capture for 5 days/month. The data was recorded on handheld data collection sheets to calculate the relative abundance, MBR, and hourly-based distribution associated with climatic factors. The data analysis was carried out using ANOVA and Spearman rank correlation tests. Results Data on vector surveillance revealed higher relative abundance of S. damnosum s.l. in Abu- Hamed (39,934 flies) than Galabat (8,202 flies). In Abu-Hamed, vector populations increased in January-April then declined in June-July until they disappeared in August-October. Highest black fly density and MBR were found in March 2007 (N = 9,444, MBR = 58,552.8 bites/person/month), and March 2010 (N = 2,603, MBR = 16,138.6 bites/person/month) while none of flies were collected in August-October (MBR = 0 bites/person/month). In Galabat, vectors increased in September-December, then decreased in February-June. The highest vector density and MBR were recorded in September 2007 (N = 1,138, MBR = 6,828 bites/person/month) and September 2010 (N = 1,163, MBR = 6,978 bites/person/month), whereas, none appeared in

  16. Molecular phylogenetic relationships among members of the family Phytolaccaceae sensu lato inferred from internal transcribed spacer sequences of nuclear ribosomal DNA.

    PubMed

    Lee, J; Kim, S Y; Park, S H; Ali, M A

    2013-02-28

    The phylogeny of a phylogenetically poorly known family, Phytolaccaceae sensu lato (s.l.), was constructed for resolving conflicts concerning taxonomic delimitations. Cladistic analyses were made based on 44 sequences of the internal transcribed spacer of nuclear ribosomal DNA from 11 families (Aizoaceae, Basellaceae, Didiereaceae, Molluginaceae, Nyctaginaceae, Phytolaccaceae s.l., Polygonaceae, Portulacaceae, Sarcobataceae, Tamaricaceae, and Nepenthaceae) of the order Caryophyllales. The maximum parsimony tree from the analysis resolved a monophyletic group of the order Caryophyllales; however, the members, Agdestis, Anisomeria, Gallesia, Gisekia, Hilleria, Ledenbergia, Microtea, Monococcus, Petiveria, Phytolacca, Rivinia, Schindleria, Seguieria, Stegnosperma, and Trichostigma, which belong to the family Phytolaccaceae s.l., did not cluster under a single clade, demonstrating that Phytolaccaceae is polyphyletic.

  17. The optimum cut-off value to differentiate Echinococcus granulosus sensu stricto from other species of E. granulosus sensu lato using larval rostellar hook morphometry.

    PubMed

    Soriano, S V; Pierangeli, N B; Pianciola, L A; Mazzeo, M; Lazzarini, L E; Debiaggi, M F; Bergagna, H F J; Basualdo, J A

    2015-01-01

    Cystic echinococcosis caused by Echinococcus granulosus sensu lato is one of the most important helminth zoonoses in the world; it affects both humans and livestock. The disease is endemic in Argentina and highly endemic in the province of Neuquén. Considerable genetic and phenotypic variation has been demonstrated in E. granulosus, and ten different genotypes (G1-G10) have been identified using molecular tools. Echinococcus granulosus sensu lato may be considered a species complex, comprised of E. granulosus sensu stricto (G1-G3), E. equinus (G4), E. ortleppi (G5) and E. canadensis (G6-G10). In endemic areas, the characterization of cystic echinococcosis molecular epidemiology is important in order to apply adequate control strategies. A cut-off value for larval large hook total length to distinguish E. granulosus sensu stricto isolates from those produced by other species of the complex was defined for the first time. Overall, 1780 larval hooks of 36 isolates obtained from sheep (n= 11, G1), goats (n= 10, G6), cattle (n= 5, G6) and pigs (n= 10, G7) were analysed. Validation against molecular genotyping as gold standard was carried out using the receiver operating characteristic (ROC) curve analysis. The optimum cut-off value was defined as 26.5 μm. The proposed method showed high sensitivity (97.8%) and specificity (91.1%). Since in most endemic regions the molecular epidemiology of echinococcosis includes the coexistence of the widely distributed E. granulosus sensu stricto G1 strain and other species of the complex, this technique could be useful as a quick and economical tool for epidemiological and surveillance field studies, when fertile cysts are present.

  18. Correlation of Borrelia burgdorferi Sensu Lato Prevalence in Questing Ixodes ricinus Ticks with Specific Abiotic Traits in the Western Palearctic▿†

    PubMed Central

    Estrada-Peña, Agustín; Ortega, Carmelo; Sánchez, Nely; DeSimone, Lorenzo; Sudre, Bertrand; Suk, Jonathan E.; Semenza, Jan C.

    2011-01-01

    This meta-analysis of reports examining ticks throughout the Western Palearctic region indicates a distinct geographic pattern for Borrelia burgdorferi sensu lato prevalence in questing nymphal Ixodes ricinus ticks. The greatest prevalence was reported between the 5°E and 25°E longitudes based on an analysis of 123 collection points with 37,940 nymphal tick specimens (87.43% of total nymphs; 56.35% of total ticks in the set of reports over the target area). Climatic traits, such as temperature and vegetation stress, and their seasonality correlated with Borrelia prevalence in questing ticks. The greatest prevalence was associated with mild winter, high summer, and low seasonal amplitude of temperatures within the range of the tick vector, higher vegetation indices in the May-June period, and well-connected vegetation patches below a threshold at which rates suddenly drop. Classification of the target territory using a qualitative risk index derived from the abiotic variables produced an indicator of the probability of finding infected ticks in the Western Palearctic region. No specific temporal trends were detected in the reported prevalence. The ranges of the different B. burgdorferi sensu lato genospecies showed a pattern of high biodiversity between 4°W and 20°E, partially overlapping the area of highest prevalence in ticks. Borrelia afzelii and Borrelia garinii are the dominant species in central Europe (east of ∼25°E), but B. garinii may appear alone at southern latitudes and Borrelia lusitaniae is the main indicator species for meridional territories. PMID:21498767

  19. Taxa and names in Cynoglossum sensu lato (Boraginaceae, Cynoglosseae): an annotated, synonymic inventory, with links to the protologues and mention of original material

    PubMed Central

    Stier, Victoria

    2015-01-01

    Abstract Background An inventory is presented of all names so far validly published in Cynoglossum sensu lato and its segregate genera: Adelocaryum, Afrotysonia, Kuschakewiczia, Lindelofia, Mattiastrum, Paracaryum, Rindera, Solenanthus, Trachelanthus, and their synonyms. Names and designations that were not validly published in the cited place, and later isonyms, are accounted for when they have been included in the International Plant Name Index (IPNI). Problems with IPNI entries, including errors and omissions, are discussed, and the hope is expressed that the present inventory may be of use for fixing them. New information The inventory, generated from a list of structured data, is presented in two Supplements, as a searchable HTML document comprising a sequence of entries with internal cross-links and links to external sources, in particular to protologues accessible online or, copyright restrictions permitting, made available as scanned documents via DOIs, and as machine-readible file. With minor exceptions, all names have been verified in their original place of publication, and all were nomenclaturally assessed. Colour coding is used to distinguish between names (in green) pertaining to Cynoglossum sensu lato, for which complete synonymies are provided; and names (in orange) pertaining to other genera but published under Cynoglossum or its segregates. They are listed together with their basionym and the corresponding correct name (if it exists), but without complete synonymy. Acceptable, potentially correct names appear in bold-face type, both under a broadly defined Cynoglossum (for which purpose validation of 81 new combinations and the name of 1 new species was necessary) and under one or more of its segregates. When a name was published for a new taxon, original material is indicated, usually by direct quotation from the protologue. New type designations are exceptional (two cases), whereas former type designations are cited whenever known. Furthermore

  20. [Rapid PCR authentication Lonicera japanica].

    PubMed

    Jiang, Chao; Hou, Jing-Yi; Huang, Lu-Qi; Yuan, Yuan; Chen, Min; Jin, Yan

    2014-10-01

    To simply and rapid authenticate Lonicera japanica. Rapid allele-specific PCR primer was designed base on trnL-trnF 625 G/T Single nucleotide polymorphism and the PCR reaction systems including annealing temperature was optimized; optimized results were performed to authenticate L. japanica and its 9 adulterants. When 100 x SYBR Green I was added in the PCR product of 87 degrees C initial denatured 1 min; 87 degrees C denatured 5 s, 68 degrees C annealing 5 s, 30 cycle; L. japanica visualize strong green fluorescence under 365 nm UV lamp whereas adulterants without. The results indicate rapid allele-specific PCR could authenticate L. japanica and its adulterants rapidly and simply.

  1. Homogeneous Inflammatory Gene Profiles Induced in Human Dermal Fibroblasts in Response to the Three Main Species of Borrelia burgdorferi sensu lato

    PubMed Central

    Meddeb, Mariam; Carpentier, Wassila; Cagnard, Nicolas; Nadaud, Sophie; Grillon, Antoine; Barthel, Cathy; De Martino, Sylvie Josiane; Jaulhac, Benoît; Boulanger, Nathalie

    2016-01-01

    In Lyme borreliosis, the skin is the key site for bacterial inoculation by the infected tick and for cutaneous manifestations. We previously showed that different strains of Borrelia burgdorferi sensu stricto isolated from tick and from different clinical stages of the Lyme borreliosis (erythema migrans, and acrodermatitis chronica atrophicans) elicited a very similar transcriptional response in normal human dermal fibroblasts. In this study, using whole transcriptome microarray chips, we aimed to compare the transcriptional response of normal human dermal fibroblasts stimulated by 3 Borrelia burgdorferi sensu lato strains belonging to 3 main pathogenic species (B. afzelii, B. garinii and B. burgdorferi sensu stricto) in order to determine whether “species-related” inflammatory pathways could be identified. The three Borrelia strains tested exhibited similar transcriptional profiles, and no species-specific fingerprint of transcriptional changes in fibroblasts was observed. Conversely, a common core of chemokines/cytokines (CCL2, CXCL1, CXCL2, CXCL6, CXCL10, IL-6, IL-8) and interferon-related genes was stimulated by all the 3 strains. Dermal fibroblasts appear to play a key role in the cutaneous infection with Borrelia, inducing a homogeneous inflammatory response, whichever Borrelia species was involved. PMID:27706261

  2. Northern white-breasted hedgehogs Erinaceus roumanicus as hosts for ticks infected with Borrelia burgdorferi sensu lato and Anaplasma phagocytophilum in Romania.

    PubMed

    Dumitrache, Mirabela Oana; Paştiu, Anamaria Ioana; Kalmár, Zsuzsa; Mircean, Viorica; Sándor, Attila David; Gherman, Călin Mircea; Peştean, Cosmin; Mihalca, Andrei Daniel; Cozma, Vasile

    2013-04-01

    Borrelia burgdorferi sensu lato and Anaplasma phagocytophilum are two tick-borne pathogens of medical and/or veterinary importance which are distributed worldwide. Erinaceus roumanicus, the Northern white-breasted hedgehog, is a common synanthropic species that is known to carry not only the hedgehog tick, Ixodes hexagonus, but also I. ricinus, the most common European tick species. I. ricinus is the main vector of both mentioned pathogens. Within this framework and because only limited information is available on the role of E. roumanicus in the ecology of B. burgdorferi s.l. and Anaplasma phagocytophilum in Europe, we carried out an epidemiological surveillance on this species in Romania. From the 57 examined hedgehogs collected in 12 different counties, 24 presented tick infestation. Most ticks (n=959) were morphologically identified as larvae, nymphs, or adults of I. ricinus. The prevalence of B. burgdorferi s.l. was 0.4%, and that of A. phagocytophilum 12%. In all positive cases for B. burgdorferi s.l., restriction fragment length polymorphism revealed the genospecies B. afzelii. In Romania, only limited information is available on the epidemiology of B. burgdorferi s.l. and A. phagocytophilum. As hedgehogs commonly share the same environment with humans and other potential reservoir hosts for tick-borne pathogens, our study provides new epidemiological data of public health importance.

  3. A phylogenetic re-appraisal of the family Liagoraceae sensu lato (Nemaliales, Rhodophyta) based on sequence analyses of two plastid genes and postfertilization development.

    PubMed

    Lin, Showe-Mei; Rodríguez-Prieto, Conxi; Huisman, John M; Guiry, Michael D; Payri, Claude; Nelson, Wendy A; Liu, Shao-Lun

    2015-06-01

    The marine red algal family Liagoraceae sensu lato is shown to be polyphyletic based on analyses of a combined rbcL and psaA data set and the pattern of carposporophyte development. Fifteen of eighteen genera analyzed formed a monophyletic lineage that included the genus Liagora. Nemalion did not cluster with Liagoraceae sensu stricto, and Nemaliaceae is reinstated, characterized morphologically by the formation of the primary gonimolobes by longitudinal divisions of the gonimoblast initial. Yamadaella and Liagoropsis, previously placed in the Dermonemataceae, are shown to be independent lineages and are recognized as two new families Yamadaellaceae and Liagoropsidaceae. Yamadaellaceae is characterized by two gonimoblast initials cut off bilaterally from the fertilized carpogonium and diffusely spreading gonimoblast filaments. Liagoropsidaceae is characterized by at least three gonimoblast initials cut off by longitudinal septa from the fertilized carpogonium. In contrast, Liagoraceae sensu stricto is characterized by a single gonimoblast initial cut off transversely or diagonally from the fertilized carpogonium. Reproductive features, such as diffuse gonimoblasts and unfused carpogonial branches following postfertilization, appear to have evolved on more than one occasion in the Nemaliales and are therefore not taxonomically diagnostic at the family level, although they may be useful in recognizing genera.

  4. Applications of Digital PCR for Clinical Microbiology.

    PubMed

    Kuypers, Jane; Jerome, Keith R

    2017-03-15

    Digital PCR (dPCR) is an important new tool for use in the clinical microbiology laboratory. Its advantages over quantitative PCR (qPCR), including absolute quantification without a standard curve, improved precision, improved accuracy in the presence of inhibitors, and more accurate quantitation when amplification efficiency is low, make dPCR the assay of choice for several specimen testing applications. This mini-review will discuss the advantages and disadvantages of dPCR compared to qPCR, its applications in clinical microbiology and the considerations for implementation of the method in a clinical laboratory.

  5. Serological detection of Anaplasma phagocytophilum, Borrelia burgdorferi sensu lato and Ehrlichia canis antibodies and Dirofilaria immitis antigen in a countrywide survey in dogs in Poland.

    PubMed

    Krämer, Friederike; Schaper, Roland; Schunack, Bettina; Połozowski, Andrzej; Piekarska, Jolanta; Szwedko, Aleksandra; Jodies, Robert; Kowalska, Dagmara; Schüpbach, Dörte; Pantchev, Nikola

    2014-09-01

    Canine vector-borne diseases (CVBDs) have increasingly become a focus of attention in the past few years. Nevertheless, in many parts of Europe information on their occurrence is still scarce. In a large study in Poland 3,094 serum samples taken from dogs throughout all 16 Polish provinces were tested using a commercial kit for the detection of circulating antibodies against Anaplasma phagocytophilum, Borrelia burgdorferi sensu lato and Ehrlichia canis and of Dirofilaria immitis antigen. A total of 12.31% (381/3,094; 95% confidence interval [CI]: 11.18-13.52%) and 3.75% (116/3,094; 95% CI: 3.11-4.48%) of the dogs were positive for A. phagocytophilum and B. burgdorferi s.l. antibodies, respectively. Furthermore, 0.26% (8/3,094; 95% CI: 0.11-0.51%) were positive for E. canis antibodies and 0.16% (5/3,094; 95% CI: 0.05-0.38%) for D. immitis antigen. The highest percentages of A. phagocytophilum-positive dogs were noted in Lesser Poland, Silesia and Łódź Provinces. For B. burgdorferi s.l., the highest prevalence was recorded in Łódź Province. Co-infections with A. phagocytophilum and B. burgdorferi s.l. were recorded in 1.71% of all examined dogs (53/3,094; 95% CI: 1.29-2.23%). One dog even had a triple infection, testing positive for E. canis too. Both A. phagocytophilum and B. burgdorferi s.l. have previously been reported in Poland and were confirmed in the present study by positive samples from all 16 provinces. Concerning E. canis and D. immitis travel history or importation cannot be excluded as factors which may have determined the occurrence of these pathogens in the relevant animals. Practitioners in Poland should be aware of the above mentioned CVBDs and of prophylactic measures to protect dogs and their owners.

  6. Development of a powder formulation based on Bacillus cereus sensu lato strain B25 spores for biological control of Fusarium verticillioides in maize plants.

    PubMed

    Martínez-Álvarez, Juan C; Castro-Martínez, Claudia; Sánchez-Peña, Pedro; Gutiérrez-Dorado, Roberto; Maldonado-Mendoza, Ignacio E

    2016-05-01

    Maize is an economically important crop in northern Mexico. Different fungi cause ear and root rot in maize, including Fusarium verticillioides (Sacc.) Nirenberg. Crop management of this pathogen with chemical fungicides has been difficult. By contrast, the recent use of novel biocontrol strategies, such as seed bacterization with Bacillus cereus sensu lato strain B25, has been effective in field trials. These approaches are not without their problems, since insufficient formulation technology, between other factors, can limit success of biocontrol agents. In response to these drawbacks, we have developed a powder formulation based on Bacillus B25 spores and evaluated some of its characteristics, including shelf life and efficacy against F. verticillioides, in vitro and in maize plants. A talc-based powder formulation containing 1 × 10(9) c.f.u. g(-1) was obtained and evaluated for seed adherence ability, seed germination effect, shelf life and antagonism against F. verticillioides in in vitro and in planta assays. Seed adherence of viable bacterial spores ranged from 1.0 to 1.41 × 10(7) c.f.u. g(-1). Bacteria did not display negative effects on seed germination. Spore viability for the powder formulation slowly decreased over time, and was 53 % after 360 days of storage at room temperature. This formulation was capable of controlling F. verticillioides in greenhouse assays, as well as eight other maize phytopathogenic fungi in vitro. The results suggest that a talc-based powder formulation of Bacillus B25 spores may be sufficient to produce inoculum for biocontrol of maize ear and root rots caused by F. verticillioides.

  7. Brown dog tick, Rhipicephalus sanguineus sensu lato, infestation of susceptible dog hosts is reduced by slow release of semiochemicals from a less susceptible host.

    PubMed

    de Oliveira Filho, Jaires Gomes; Ferreira, Lorena Lopes; Sarria, André Lucio Franceschini; Pickett, John A; Birkett, Michael A; Mascarin, Gabriel Moura; de León, Adalberto A Pérez; Borges, Lígia Miranda Ferreira

    2017-01-01

    Domestic dog breeds are hosts for the brown dog tick, Rhipicephalus sanguineus sensu lato, but infestation levels vary among breeds. Beagles are less susceptible to tick infestations than English cocker spaniels due to enhanced production of 2-hexanone and benzaldehyde that act as volatile tick repellents. We report the use of prototype slow-release formulations of these compounds to reduce the burden of R. sanguineus s. l. on English cocker spaniel dogs. Twelve dogs were randomly assigned to two groups with six dogs each. The treated group received collars with slow-release formulations of the compounds attached, while the control group received collars with clean formulations attached. Five environmental infestations were performed, with the number of ticks (at all stages) on the dogs being counted twice a day for 45days. The counts on the number of tick stages found per dog were individually fitted to linear mixed effects models with repeated measures and normal distribution for errors. The mean tick infestation in the treated group was significantly lower than in the control group. For larvae and nymphs, a decrease in tick infestation was observed at the fifth count, and for adults, lower average counts were observed in all counts. The compounds did not interfere with the distribution of the ticks on the body of the dogs, as a similar percentage of ticks was found on the anterior half of the dogs (54.5% for the control group and 56.2% for the treated group). The biological and reproductive parameters of the ticks were not affected by the repellents. This study highlights for the first time the potential use of a novel allomone (repellent)-based formulation for reduction of tick infestation on susceptible dogs.

  8. ITS1, 5.8S and ITS2 secondary structure modelling for intra-specific differentiation among species of the Colletotrichum gloeosporioides sensu lato species complex.

    PubMed

    Rampersad, Sephra N

    2014-01-01

    The Colletotrichum gloeosporioides species complex is among the most destructive fungal plant pathogens in the world, however, identification of member species which are of quarantine importance is impacted by a number of factors that negatively affect species identification. Structural information of the rRNA marker may be considered to be a conserved marker which can be used as supplementary information for possible species identification. The difficulty in using ITS rDNA sequences for identification lies in the low level of sequence variation at the intra-specific level and the generation of artificially-induced sequence variation due to errors in polymerization of the ITS array during DNA replication. Type and query ITS sequences were subjected to sequence analyses prior to generation of predicted consensus secondary structures, including the pattern of nucleotide polymorphisms and number of indel haplotypes, GC content, and detection of artificially-induced sequence variation. Data pertaining to structure stability, the presence of conserved motifs in secondary structures and mapping of all sequences onto the consensus C. gloeosporioides sensu stricto secondary structure for ITS1, 5.8S and ITS2 markers was then carried out. Motifs that are evolutionarily conserved among eukaryotes were found for all ITS1, 5.8S and ITS2 sequences. The sequences exhibited conserved features typical of functional rRNAs. Generally, polymorphisms occurred within less conserved regions and were seen as bulges, internal and terminal loops or non-canonical G-U base-pairs within regions of the double stranded helices. Importantly, there were also taxonomic motifs and base changes that were unique to specific taxa and which may be used to support intra-specific identification of members of the C. gloeosporioides sensu lato species complex.

  9. Combining a Climatic Niche Model of an Invasive Fungus with Its Host Species Distributions to Identify Risks to Natural Assets: Puccinia psidii Sensu Lato in Australia

    PubMed Central

    Kriticos, Darren J.; Morin, Louise; Leriche, Agathe; Anderson, Robert C.; Caley, Peter

    2013-01-01

    Puccinia psidii sensu lato (s.l.) is an invasive rust fungus threatening a wide range of plant species in the family Myrtaceae. Originating from Central and South America, it has invaded mainland USA and Hawai'i, parts of Asia and Australia. We used CLIMEX to develop a semi-mechanistic global climatic niche model based on new data on the distribution and biology of P. psidii s.l. The model was validated using independent distribution data from recently invaded areas in Australia, China and Japan. We combined this model with distribution data of its potential Myrtaceae host plant species present in Australia to identify areas and ecosystems most at risk. Myrtaceaeous species richness, threatened Myrtaceae and eucalypt plantations within the climatically suitable envelope for P. psidii s.l in Australia were mapped. Globally the model identifies climatically suitable areas for P. psidii s.l. throughout the wet tropics and sub-tropics where moist conditions with moderate temperatures prevail, and also into some cool regions with a mild Mediterranean climate. In Australia, the map of species richness of Myrtaceae within the P. psidii s.l. climatic envelope shows areas where epidemics are hypothetically more likely to be frequent and severe. These hotspots for epidemics are along the eastern coast of New South Wales, including the Sydney Basin, in the Brisbane and Cairns areas in Queensland, and in the coastal region from the south of Bunbury to Esperance in Western Australia. This new climatic niche model for P. psidii s.l. indicates a higher degree of cold tolerance; and hence a potential range that extends into higher altitudes and latitudes than has been indicated previously. The methods demonstrated here provide some insight into the impacts an invasive species might have within its climatically suited range, and can help inform biosecurity policies regarding the management of its spread and protection of valued threatened assets. PMID:23704988

  10. Comparative efficacy of two oral treatments for dogs containing either afoxolaner or fluralaner against Rhipicephalus sanguineus sensu lato and Dermacentor reticulatus.

    PubMed

    Beugnet, Frederic; Liebenberg, Julian; Halos, Lenaïg

    2015-04-15

    The present study compares the efficacy of two recent oral ectoparasiticides containing isoxazolines (NexGard(®), containing afoxolaner and administered at a monthly regimen, and Bravecto™ containing fluralaner and administered at a tri-monthly regimen) against Rhipicephalus sanguineus sensu lato and Dermacentor reticulatus ticks on dogs. 24 dogs were randomly allocated to untreated control, NexGard(®) treated, and Bravecto™ treated groups. The treatments were administered on Days 0, 28 and 56 for afoxolaner and on Day 0 for fluralaner. Tick infestations were performed weekly with 50 unfed adult ticks per each species on each dog from Days 30 to 84 (with the exception of R. sanguineus on Day 63). Ticks were counted at 24h post-infestation. The dogs from both treated groups had statistically significantly (p<0.05) less R. sanguineus and D. reticulatus ticks compared to the untreated dogs on all assessment days. Percent efficacy against R. sanguineus ranged from 86.4% to 99.5% at 24h post-infestation for NexGard(®) and from 65.7% to 100% for Bravecto™. Statistically significantly (p<0.05) less R. sanguineus ticks were recorded for NexGard(®) treated dogs compared to Bravecto™ treated dogs on Day 78. Percent efficacy against D. reticulatus ranged from 85.2% to 99.6% at 24h post-infestation for NexGard(®) and from 63.4% to 99.1% for Bravecto™. Statistically significantly (p<0.05) less D. reticulatus ticks were recorded for NexGard(®) treated dogs compared to Bravecto™ treated dogs on Days 71, 78 and 85.

  11. Co-occurrence and distribution of East (L1014S) and West (L1014F) African knock-down resistance in Anopheles gambiae sensu lato population of Tanzania

    PubMed Central

    Kabula, Bilali; Kisinza, William; Tungu, Patrick; Ndege, Chacha; Batengana, Benard; Kollo, Douglas; Malima, Robert; Kafuko, Jessica; Mohamed, Mahdi; Magesa, Stephen

    2014-01-01

    desarrollo de resistencias en las poblaciones de los vectores Anopheles. Evaluar dichos marcadores es crucial para los programas de monitorización de resistencias. Hemos investigado la presencia y la distribución de las mutaciones de resistencia knockdown (kdr) en Anopheles gambiae s.l. en Tanzania. Métodos Se recolectaron mosquitos Anopheles intradomiciliarios de 10 lugares diferentes y se evaluaron en busca de resistencia a insecticidas utilizando el protocolo estándar de la OMS. Mediante un diagnóstico molecular basado en la PCR se genotiparon los mosquitos y se detectaron los genotipos kdr. Resultados Los An. gambiae evaluados eran resistentes a lambdacialotrina en Muheza, Arumeru y Muleba. De 350 An. gambiae s.l. genotipados, 35% eran An. gambiae s.s. y 65% eran An. arabiensis. Se detectaron mutaciones L1014S y L1014F tanto en An. gambiae s.s. como en An. arabiensis. La mutación puntual L1014S se encontró con una frecuencia alélica de 4-33%, mientras que L1014F tenía una frecuencia alélica de 6-14%. La mutación L1014S estaba ampliamente asociada a An. gambiae s.s. (Chi-Cuadrado = 23.41; P < 0.0001) y la L1014F estaba asociada con An. arabiensis (Chi-Square = 11.21; P = 0.0008). El alelo L1014S estaba significativamente asociado con mosquitos resistentes a la lambdacialotrina (P < 0.001). Conclusión La simultaneidad de mutaciones de L1014S y L1014F junto con informes de resistencia a los insecticidas sugiere que la resistencia a piretroides se está convirtiendo en un fenómeno común entre las poblaciones del vector de la malaria en Tanzania. La presencia de la mutación L1014F en estas poblaciones del Este de África indican la diseminación del gen a lo largo del continente africano. Determinar las implicaciones potenciales a nivel operativo de estos hallazgos sobre el control de la malaria requiere de más estudios. PMID:24386946

  12. Anaplasmataceae and Borrelia burgdorferi sensu lato in the sand lizard Lacerta agilis and co-infection of these bacteria in hosted Ixodes ricinus ticks

    PubMed Central

    2011-01-01

    Background Anaplasmataceae and Borrelia burgdorferi s.l. are important tick-borne bacteria maintained in nature by transmission between ticks and vertebrate hosts. However, the potential role of lizards as hosts has not been sufficiently studied. Results The current study showed that 23 of 171 examined sand lizards Lacerta agilis were PCR positive for Anaplasmataceae. The nucleotide sequences of the several selected PCR products showed 100% homology with Anaplasma spp. found in Ixodes ricinus collected in Tunisia and Morocco (AY672415 - AY672420). 1.2% of lizard collar scale samples were PCR positive for B. lusitaniae. In addition, 12 of 290 examined I. ricinus were PCR positive for B. burgdorferi s.l. and 82 were PCR positive for Anaplasmatacea. The number of ticks per lizard and the number of ticks PCR positive for both microorganisms per lizard were strongly correlated. Moreover, we found a significant correlation between numbers of ticks infected with Anaplasmataceae and with B. burgdorferi s.l. living on the same lizard. However, there was no significant correlation between detection of both bacteria in the same tick. Conclusions To the best of our knowledge, this is the first report of Anaplasmataceae DNA and additionally the second report of B. burgdorferi s.l DNA detection in the sand lizard. PMID:21933412

  13. Multilocus sequence typing of Pseudomonas syringae sensu lato confirms previously described genomospecies and permits rapid identification of P. syringae pv. coriandricola and P. syringae pv. apii causing bacterial leaf spot on parsley.

    PubMed

    Bull, Carolee T; Clarke, Christopher R; Cai, Rongman; Vinatzer, Boris A; Jardini, Teresa M; Koike, Steven T

    2011-07-01

    Since 2002, severe leaf spotting on parsley (Petroselinum crispum) has occurred in Monterey County, CA. Either of two different pathovars of Pseudomonas syringae sensu lato were isolated from diseased leaves from eight distinct outbreaks and once from the same outbreak. Fragment analysis of DNA amplified between repetitive sequence polymerase chain reaction; 16S rDNA sequence analysis; and biochemical, physiological, and host range tests identified the pathogens as Pseudomonas syringae pv. apii and P. syringae pv. coriandricola. Koch's postulates were completed for the isolates from parsley, and host range tests with parsley isolates and pathotype strains demonstrated that P. syringae pv. apii and P. syringae pv. coriandricola cause leaf spot diseases on parsley, celery, and coriander or cilantro. In a multilocus sequence typing (MLST) approach, four housekeeping gene fragments were sequenced from 10 strains isolated from parsley and 56 pathotype strains of P. syringae. Allele sequences were uploaded to the Plant-Associated Microbes Database and a phylogenetic tree was built based on concatenated sequences. Tree topology directly corresponded to P. syringae genomospecies and P. syringae pv. apii was allocated appropriately to genomospecies 3. This is the first demonstration that MLST can accurately allocate new pathogens directly to P. syringae sensu lato genomospecies. According to MLST, P. syringae pv. coriandricola is a member of genomospecies 9, P. cannabina. In a blind test, both P. syringae pv. coriandricola and P. syringae pv. apii isolates from parsley were correctly identified to pathovar. In both cases, MLST described diversity within each pathovar that was previously unknown.

  14. PCR hot-start using duplex primers.

    PubMed

    Kong, Deming; Shen, Hanxi; Huang, Yanping; Mi, Huaifeng

    2004-02-01

    A new technique of PCR hot-start using duplex primers has been developed which can decrease the undesirable products arising throughout PCR amplification thereby giving better results than a manual hot-start method.

  15. Life stage-related differences in density of questing ticks and infection with Borrelia burgdorferi sensu lato within a single cohort of Ixodes pacificus (Acari: Ixodidae).

    PubMed

    Eisen, Rebecca J; Mun, Jeomhee; Eisen, Lars; Lane, Robert S

    2004-07-01

    The primary aims of this study were to quantify the density of Ixodes pacificus Cooley and Kohls nymphs and adults of the same generational cohort collected within a single year in six oak or madrone leaf litter habitats and to compare the prevalence of infection with Borrelia burgdorferi sensu lato (s.l.) in adults originating from nymphal cohorts with a low (<1%) versus high (>10%) infection prevalence. Because adult densities were very low both in and adjacent to several sites, direct comparisons of infection prevalence between nymphs and adults were possible only for two sites. Mean density in these sites decreased from 11.95/100 m2 for nymphs to 0/100 m2 for adults in leaf litter, and infection prevalence with B. burgdorferi s.l. was four-fold higher in nymphs (7.4%) versus adults (1.6%) of the same generational cohort collected in ecotones bordering the leaf litter areas. Assuming a density of adults in leaf litter of 0.04/100 m2 (mean for all six examined sites) and an infection prevalence similar to that found in adults collected from litter ecotones, the risk of encountering infected ticks in leaf litter decreased >1,000-fold from the nymphal to adult stage. Regardless of site-specific infection prevalence in the nymphal stage (n = 2 sites; 0.7 versus 14%), the infection prevalence for the adults of the same generational cohort was similarly low (1.5-1.6%). Peak densities of adult I. pacificus were 0-0.1/100 m2 in leaf litter, 0-6.5/100 m2 in ecotonal grasslands, and 2.0-39.0/100 m2 in ecotonal chaparral. Despite more intensive sampling efforts in leaf litter, the vast majority of the 282 adults collected came from grass or chaparral ecotones (98.9%, n = 279) rather than leaf litter (1.1%, n = 3). The study yielded eight B. burgdorferi s.l.-infected adults; four of these carried B. burgdorferi sensu stricto Johnson, Schmidt, Hyde, Steigerwalt, and Brenner, and the remaining four were infected with currently undescribed B. burgdorferi s.l. spirochetes. This

  16. Divergence of Borrelia burgdorferi sensu lato spirochetes could be driven by the host: diversity of Borrelia strains isolated from ticks feeding on a single bird

    PubMed Central

    2014-01-01

    Background The controversy surrounding the potential impact of birds in spirochete transmission dynamics and their capacity to serve as a reservoir has existed for a long time. The majority of analyzed bird species are able to infect larval ticks with Borrelia. Dispersal of infected ticks due to bird migration is a key to the establishment of new foci of Lyme borreliosis. The dynamics of infection in birds supports the mixing of different species, the horizontal exchange of genetic information, and appearance of recombinant genotypes. Methods Four Borrelia burgdorferi sensu lato strains were cultured from Ixodes minor larvae and four strains were isolated from Ixodes minor nymphs collected from a single Carolina Wren (Thryothorus ludovicianus). A multilocus sequence analysis that included 16S rRNA, a 5S-23S intergenic spacer region, a 16S-23S internal transcribed spacer, flagellin, p66, and ospC separated 8 strains into 3 distinct groups. Additional multilocus sequence typing of 8 housekeeping genes, clpA, clpX, nifS, pepX, pyrG, recG, rplB, and uvrA was used to resolve the taxonomic status of bird-associated strains. Results Results of analysis of 14 genes confirmed that the level of divergence among strains is significantly higher than what would be expected for strains within a single species. The presence of cross-species recombination was revealed: Borrelia burgdorferi sensu stricto housekeeping gene nifS was incorporated into homologous locus of strain, previously assigned to B. americana. Conclusions Genetically diverse Borrelia strains are often found within the same tick or same vertebrate host, presenting a wide opportunity for genetic exchange. We report the cross-species recombination that led to incorporation of a housekeeping gene from the B. burgdorferi sensu stricto strain into a homologous locus of another bird-associated strain. Our results support the hypothesis that recombination maintains a majority of sequence polymorphism within Borrelia

  17. Serological detection of antibodies to Anaplasma spp., Borrelia burgdorferi sensu lato and Ehrlichia canis and of Dirofilaria immitis antigen in dogs from Costa Rica.

    PubMed

    Montenegro, Víctor M; Bonilla, Marta C; Kaminsky, Darwin; Romero-Zúñiga, Juan José; Siebert, Susanne; Krämer, Friederike

    2017-03-15

    In a study in Costa Rica 314 serum samples from dogs throughout all seven provinces were tested using a commercial kit for the detection of circulating antibodies against Anaplasma spp., Borrelia burgdorferi sensu lato and Ehrlichia canis, and of circulating antigen of Dirofilaria immitis. A total of 6.4% (20/314) and 38.2% (120/314) were positive for Anaplasma spp. (An) and E. canis (Ec) antibodies. Overall, 8.0% (25/314) were positive for D. immitis (Di) antigen. One single dog reacted positive with B. burgdorferi s.l. (Bb) antigen (0.3%, 1/314). E. canis positive dogs were detected in all provinces (highest percentages in Guanacaste, Puntarenas [both significantly different compared to the overall] and Limón). Guanacaste and Puntarenas also showed the highest prevalences of Anaplasma spp. (both significantly different compared to the overall). The highest prevalence of D. immitis was detected in Puntarenas (significantly different compared to the overall). Double pathogen exposure (Ec plus An; Ec plus Di; Ec plus Bb) were recorded in 8.9% (28/314). Two dogs showed a triple pathogen exposure (0.6%, 2/314; An, Ec and Di). There was a significant difference between male (11.5%, 18/156) and female (4.4%, 7/158) animals for D. immitis positive results. There was also a significant difference between breed and no breed dogs regarding the characteristics of a general positive test, as well as seropositivity to the single pathogens of Anaplasma spp., E. canis and D. immitis. Finally there was a significant difference in the presence of clinical signs again regarding the characteristics of a general positive test, as well as seropositivity to Anaplasma spp., E. canis and D. immitis. Practitioners in Costa Rica should be aware of the canine vector-borne diseases mentioned as dogs are at risk of becoming infected. Concerning the positive B. burgdorferi s.l. dog, an autochthonous occurrence cannot be confirmed due to a history of adoption and an unusual tattoo number

  18. PCR screening of tick-borne agents in sensitive conservation areas, Southeast Portugal.

    PubMed

    Santos-Silva, Maria Margarida; Melo, Pedro; Santos, Nuno; Antunes, Sandra; Duarte, Luís Raposo; Ferrolho, Joana; Milhano, Natacha; Santos, Patrícia Tavares; Domingos, Ana; Santos, Ana Sofia

    2017-02-01

    The Southeast region of Portugal, particularly the Guadiana valley, is currently the reintroduction territory of Lynx pardinus (Iberian lynx), one of the most endangered felids in the world that is only found in the Iberian Peninsula. Over the last century, populations have declined, placing L. pardinus at extremely high risk of extinction in the wild and relying on reintroduction projects. Among the aspects taken into account in the establishment of new populations is the sanitary status of the selected habitats, especially concerning infectious diseases, including tick-borne pathogens (TBPs). This study presents the results of TBPs survey on ticks collected at sensitive conservation areas of Southeast Portugal. From 2012 to 2014, 231 ticks obtained from vegetation, sympatric domestic and wild animals were submitted for analysis. The presence of Babesia spp., Cytauxzoon spp., Theileria spp., Hepatozoon spp., Anaplasma spp., Ehrlichia spp., Candidatus Neoehrlichia mikurensis, among other Anaplasmataceae, and Coxiella burnetii were investigated by PCR. Six tick species were recorded, Dermacentor marginatus (n = 13/5.6%), Hyalomma lusitanicum (n = 175/75.8%), Ixodes ricinus (n = 4/1.7%), Rhipicephalus bursa (n = 7/3.0%), R. pusillus (n = 21/9.1%) and R. sanguineus sensu lato (n = 11/4.8%). The molecular screening confirmed the presence of two tick-borne pathogens, C. burnetii (N = 34) and Anaplasma platys (N = 1), and one tick-endosymbiont, Candidatus Midichloria mitochondrii (N = 45). The results obtained provide new information on the circulation of ticks and TBPs with potential veterinary importance in Iberian lynx habitat.

  19. PALATAL DYSMORPHOGENESIS: QUANTITATIVE RT-PCR

    EPA Science Inventory

    ABSTRACT

    Palatal Dysmorphogenesis : Quantitative RT-PCR

    Gary A. Held and Barbara D. Abbott

    Reverse transcription PCR (RT-PCR) is a very sensitive method for detecting mRNA in tissue samples. However, as it is usually performed it is does not yield quantitativ...

  20. Real-time PCR in Food Science: PCR Diagnostics.

    PubMed

    Rodriguez-Lazaro, David; Cook, Nigel; Hernandez, Marta

    2013-01-01

    A principal consumer demand is a guarantee of the safety and quality of food. The presence of foodborne pathogens and their potential hazard, the use of genetically modified organisms (GMOs) in food production, and the correct labelling in foods suitable for vegetarians are among the subjects where society demands total transparency. The application of controls within the quality assessment programmes of the food industry is a way to satisfy these demands, and is necessary to ensure efficient analytical methodologies are possessed and correctly applied by the Food Sector. The use of real-time PCR has become a promising alternative approach in food diagnostics. It possesses a number of advantages over conventional culturing approaches, including rapidity, excellent analytical sensitivity and selectivity, and potential for quantification. However, the use of expensive equipment and reagents, the need for qualified personnel, and the lack of standardized protocols are impairing its practical implementation for food monitoring and control.

  1. Comparison of next-generation droplet digital PCR (ddPCR) with quantitative PCR (qPCR) for enumeration of Cryptosporidium oocysts in faecal samples.

    PubMed

    Yang, Rongchang; Paparini, Andrea; Monis, Paul; Ryan, Una

    2014-12-01

    Clinical microbiology laboratories rely on quantitative PCR for its speed, sensitivity, specificity and ease-of-use. However, quantitative PCR quantitation requires the use of a standard curve or normalisation to reference genes. Droplet digital PCR provides absolute quantitation without the need for calibration curves. A comparison between droplet digital PCR and quantitative PCR-based analyses was conducted for the enteric parasite Cryptosporidium, which is an important cause of gastritis in both humans and animals. Two loci were analysed (18S rRNA and actin) using a range of Cryptosporidium DNA templates, including recombinant plasmids, purified haemocytometer-counted oocysts, commercial flow cytometry-counted oocysts and faecal DNA samples from sheep, cattle and humans. Each method was evaluated for linearity, precision, limit of detection and cost. Across the same range of detection, both methods showed a high degree of linearity and positive correlation for standards (R(2)⩾0.999) and faecal samples (R(2)⩾0.9750). The precision of droplet digital PCR, as measured by mean Relative Standard Deviation (RSD;%), was consistently better compared with quantitative PCR, particularly for the 18S rRNA locus, but was poorer as DNA concentration decreased. The quantitative detection of quantitative PCR was unaffected by DNA concentration, but droplet digital PCR quantitative PCR was less affected by the presence of inhibitors, compared with quantitative PCR. For most templates analysed including Cryptosporidium-positive faecal DNA, the template copy numbers, as determined by droplet digital PCR, were consistently lower than by quantitative PCR. However, the quantitations obtained by quantitative PCR are dependent on the accuracy of the standard curve and when the quantitative PCR data were corrected for pipetting and DNA losses (as determined by droplet digital PCR), then the sensitivity of both methods was comparable. A cost analysis based on 96 samples revealed that

  2. Real-Time PCR (qPCR) Primer Design Using Free Online Software

    ERIC Educational Resources Information Center

    Thornton, Brenda; Basu, Chhandak

    2011-01-01

    Real-time PCR (quantitative PCR or qPCR) has become the preferred method for validating results obtained from assays which measure gene expression profiles. The process uses reverse transcription polymerase chain reaction (RT-PCR), coupled with fluorescent chemistry, to measure variations in transcriptome levels between samples. The four most…

  3. Direct in situ rt-PCR.

    PubMed

    Lossi, Laura; Gambino, Graziana; Salio, Chiara; Merighi, Adalberto

    2011-01-01

    In situ polymerase chain reaction (PCR) is a histological technique that exploits the advantages of PCR for detection of mRNA directly in tissue sections. It somehow conjugates together PCR and in situ hybridization that is more traditionally employed for mRNA localization in cell organelles, intact cells, or tissue sections. This chapter describes the application of in situ PCR for neuropeptide mRNA localization. We provide here a detailed protocol for direct in situ reverse transcription (RT) PCR (RT-PCR) with nonradioactive probes after fixation and paraffin embedding or cryosectioning. Digoxigenin-labeled nucleotides (digoxigenin-11-dUTP) are incorporated in the PCR product after RT and subsequently detected with an anti-digoxigenin antibody conjugated with alkaline phosphatase. The procedure can be modified for use with fluorescent probes and employed in combination with enzyme/fluorescence immunocytochemical labeling.

  4. Multiplexed Primer Prediction for PCR

    SciTech Connect

    2007-07-23

    MPP predicts sets of multiplex-compatible primers for Polymerase Chain Reaction (PCR), finding a near minimal set of primers such that at least one amplicon will be generated from every target sequence in the input file. The code finds highly conserved oligos that are suitable as primers, according to user-specified desired primer characteristics such as length, melting temperature, and amplicon length. The primers are predicted not to form unwanted dimer or hairpin structures. The target sequences used as input can be diverse, since no multiple sequence alighment is required. The code is scalable, taking up to tens of thousands of sequences as input, and works, for example, to find a "universal primer set" for all viral genomes provided as a single input file. The code generates a periodic check-point file, thus in the event of premature execution termination, the application can be restarted from the last check-point file.

  5. Study of the bacterial diversity of foods: PCR-DGGE versus LH-PCR.

    PubMed

    Garofalo, Cristiana; Bancalari, Elena; Milanović, Vesna; Cardinali, Federica; Osimani, Andrea; Sardaro, Maria Luisa Savo; Bottari, Benedetta; Bernini, Valentina; Aquilanti, Lucia; Clementi, Francesca; Neviani, Erasmo; Gatti, Monica

    2017-02-02

    The present study compared two culture-independent methods, polymerase chain reaction denaturing gradient gel electrophoresis (PCR-DGGE) and length-heterogeneity polymerase chain reaction (LH-PCR), for their ability to reveal food bacterial microbiota. Total microbial DNA and RNA were extracted directly from fourteen fermented and unfermented foods, and domain A of the variable regions V1 and V2 of the 16S rRNA gene was analyzed through LH-PCR and PCR-DGGE. Finally, the outline of these analyses was compared with bacterial viable counts obtained after bacterial growth on suitable selective media. For the majority of the samples, RNA-based PCR-DGGE revealed species that the DNA-based PCR-DGGE was not able to highlight. When analyzing either DNA or RNA, LH-PCR identified several lactic acid bacteria (LAB) and coagulase negative cocci (CCN) species that were not identified by PCR-DGGE. This phenomenon was particularly evident in food samples with viable loads<5.0 Logcfug(-1). Furthermore, LH-PCR was able to detect a higher number of peaks in the analyzed food matrices relative to species identified by PCR-DGGE. In light of these findings, it may be suggested that LH-PCR shows greater sensitivity than PCR-DGGE. However, PCR-DGGE detected some other species (LAB included) that were not detected by LH-PCR. Therefore, certain LH-PCR peaks not attributed to known species within the LH-PCR database could be solved by comparing them with species identified by PCR-DGGE. Overall, this study also showed that LH-PCR is a promising method for use in the food microbiology field, indicating the necessity to expand the LH-PCR database, which is based, up to now, mainly on LAB isolates from dairy products.

  6. Pitfalls in PCR troubleshooting: Expect the unexpected?

    PubMed Central

    Schrick, Livia; Nitsche, Andreas

    2015-01-01

    PCR is a well-understood and established laboratory technique often used in molecular diagnostics. Huge experience has been accumulated over the last years regarding the design of PCR assays and their set-up, including in-depth troubleshooting to obtain the optimal PCR assay for each purpose. Here we report a PCR troubleshooting that came up with a surprising result never observed before. With this report we hope to sensitize the reader to this peculiar problem and to save troubleshooting efforts in similar situations, especially in time-critical and ambitious diagnostic settings. PMID:27077041

  7. Real-time PCR in microfluidic devices

    NASA Astrophysics Data System (ADS)

    Becker, Holger; Hlawatsch, Nadine; Klemm, Richard; Moche, Christian; Hansen-Hagge, Thomas; Gärtner, Claudia

    2014-03-01

    A central method in a standard biochemical laboratory is represented by the polymerase chain reaction (PCR), therefore many attempts have been performed so far to implement this technique in lab-on-a-chip (LOC) devices. PCR is an ideal candidate for miniaturization because of a reduction of assay time and decreased costs for expensive bio-chemicals. In case of the "classical" PCR, detection is done by identification of DNA fragments electrophoretically separated in agarose gels. This method is meanwhile frequently replaced by the so-called Real-Time-PCR because here the exponential increase of amplificates can be observed directly by measurement of DNA interacting fluorescent dyes. Two main methods for on-chip PCRs are available: traditional "batch" PCR in chambers on a chip using thermal cycling, requiring about 30 minutes for a typical PCR protocol and continuous-flow PCR, where the liquid is guided over stationary temperature zones. In the latter case, the PCR protocol can be as fast as 5 minutes. In the presented work, a proof of concept is demonstrated for a real-time-detection of PCR products in microfluidic systems.

  8. Absolute quantification by droplet digital PCR versus analog real-time PCR

    PubMed Central

    Hindson, Christopher M; Chevillet, John R; Briggs, Hilary A; Gallichotte, Emily N; Ruf, Ingrid K; Hindson, Benjamin J; Vessella, Robert L; Tewari, Muneesh

    2014-01-01

    Nanoliter-sized droplet technology paired with digital PCR (ddPCR) holds promise for highly precise, absolute nucleic acid quantification. Our comparison of microRNA quantification by ddPCR and real-time PCR revealed greater precision (coefficients of variation decreased by 37–86%) and improved day-to-day reproducibility (by a factor of seven) of ddPCR but with comparable sensitivity. When we applied ddPCR to serum microRNA biomarker analysis, this translated to superior diagnostic performance for identifying individuals with cancer. PMID:23995387

  9. Evaluation of AMPLICOR Neisseria gonorrhoeae PCR using cppB nested PCR and 16S rRNA PCR.

    PubMed

    Farrell, D J

    1999-02-01

    Certain strains of Neisseria subflava and Neisseria cinerea are known to produce false-positive results with the AMPLICOR Neisseria gonorrhoeae PCR (Roche Diagnostic Systems, Branchburg, N.J.). The analytical sensitivity and analytical specificity of three PCR tests were assessed with 3 geographically diverse N. gonorrhoeae strains and 30 non-N. gonorrhoeae Neisseria spp. The sensitivities of the in-house nested cppB gene and the 16S rRNA PCR methods were greater than that of the AMPLICOR N. gonorrhoeae PCR with purified DNA from all 3 N. gonorrhoeae strains. Six of 14 clinical strains of N. subflava (1 from a vaginal swab, 5 from respiratory sites) produced false-positive AMPLICOR N. gonorrhoeae PCR results and were negative by the two other PCR methods. When applied to 207 clinical specimens selected from a population with a high prevalence ( approximately 9%) of infection, the results for 15 of 96 (15.6%) AMPLICOR-positive specimens and 14 of 17 (82.3%) AMPLICOR-equivocal specimens were not confirmed by the more sensitive nested cppB PCR method. Only 2 of 94 (2.1%) of AMPLICOR N. gonorrhoeae PCR-negative specimens from the same population tested positive by the nested cppB method. These results suggest that for this population the AMPLICOR N. gonorrhoeae PCR test is suitable as a screening test only and all positive results should be confirmed by a PCR method that is more specific and at least as sensitive. This study also illustrates that caution should be used when introducing commercially available nucleic acid amplification-based diagnostic tests into the regimens of tests used for populations not previously tested with these products.

  10. Digital PCR for detection of citrus pathogens

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Citrus trees are often infected with multiple pathogens of economic importance, especially those with insect or mite vectors. Real-time/quantitative PCR (qPCR) has been used for high-throughput detection and relative quantification of pathogens; however, target reference or standards are required. I...

  11. Testing for Genetically Modified Foods Using PCR

    ERIC Educational Resources Information Center

    Taylor, Ann; Sajan, Samin

    2005-01-01

    The polymerase chain reaction (PCR) is a Nobel Prize-winning technique that amplifies a specific segment of DNA and is commonly used to test for the presence of genetic modifications. Students use PCR to test corn meal and corn-muffin mixes for the presence of a promoter commonly used in genetically modified foods, the cauliflower mosaic virus 35S…

  12. Real-time PCR and PCR-tandem Mass Spectrometry for Biodetection

    DTIC Science & Technology

    2005-10-01

    Real - time PCR and PCR- tandem mass spectrometry for biodetection Alvin Fox, University of South Carolina, School of Medicine Report Documentation...TYPE N/A 3. DATES COVERED - 4. TITLE AND SUBTITLE Real - time PCR and PCRtandem mass spectrometry for biodetection 5a. CONTRACT NUMBER 5b...interspace region Bacillus subtilis W23 standard Blank Barn dust House dust Cycle Real - time PCR (16s rRNA) - environmental samples Real - time

  13. Pre-PCR processing: strategies to generate PCR-compatible samples.

    PubMed

    Rådström, Peter; Knutsson, Rickard; Wolffs, Petra; Lövenklev, Maria; Löfström, Charlotta

    2004-02-01

    Polymerase chain reaction (PCR) is recognized as a rapid, sensitive, and specific molecular diagnostic tool for the analysis of nucleic acids. However, the sensitivity and kinetics of diagnostic PCR may be dramatically reduced when applied directly to biological samples, such as blood and feces, owing to PCR-inhibitory components. As a result, pre-PCR processing procedures have been developed to remove or reduce the effects of PCR inhibitors. Pre-PCR processing comprises all steps prior to the detection of PCR products, that is, sampling, sample preparation, and deoxyribonucleic acid (DNA) amplification. The aim of pre-PCR processing is to convert a complex biological sample with its target nucleic acids/cells into PCR-amplifiable samples by combining sample preparation and amplification conditions. Several different pre-PCR processing strategies are used: (1) optimization of the DNA amplification conditions by the use of alternative DNA polymerases and/or amplification facilitators, (2) optimization of the sample preparation method, (3) optimization of the sampling method, and (4) combinations of the different strategies. This review describes different pre-PCR processing strategies to circumvent PCR inhibition to allow accurate and precise DNA amplification.

  14. A new PCR method: one primer amplification of PCR-CTPP products.

    PubMed

    Yin, Guang; Mitsuda, Yoko; Ezaki, Takayuki; Hamajima, Nobuyuki

    2012-10-01

    Polymerase chain reaction with confronting two-pair primers (PCR-CTPP) is a convenient method for genotyping single nucleotide polymorphisms, saving time, and costs. It uses four primers for PCR; F1 and R1 for one allele, and F2 and R2 for the other allele, by which three different sizes of DNA are amplified; between F1 and R1, between F2 and R2, and between F1 and R2. To date, we have applied PCR-CTPP successfully for genotyping more than 60 polymorphisms. However, it is not rare that PCR does not produce balanced amplification of allele specific bands. Accordingly, the method was modified by attaching a common sequence at the 5' end of two-pair primers and adding another primer with the common sequence in PCR, in total five different primers in a tube for PCR. The modification allowed one primer amplification for the products of initial PCR with confronting two-pair primers, named as one primer amplification of PCR-CTPP products (OPA-CTPP). This article demonstrates an example for an A/G polymorphism of paraoxonase 1 (PON1) Gln192Arg (rs662). PCR-CTPP failed clear genotyping for the polymorphism, while OPA-CTPP successfully produced PCR products corresponding to the allele. The present example indicated that the OPA-CTPP would be useful in the case that PCR-CTPP failed to produce balanced PCR products specific to each allele.

  15. Real-Time PCR (RT-PCR) Assays for Burkholderia mallei and B. pseudomallei

    DTIC Science & Technology

    2005-10-01

    1 Real - time PCR (RT-PCR) Assays for Burkholderia mallei and B. pseudomallei Vipin K. Rastogi1, Tu-chen Cheng1, Lisa Collins1 and Jennifer Bagley2 1...A 3. DATES COVERED - 4. TITLE AND SUBTITLE Real - time PCR (RT-PCR) Assays for Burkholderia mallei and B.pseudomallei 5a. CONTRACT NUMBER 5b...risk. There is currently no real - time PCR assay for detection of both of these pathogens. Primers and probes corresponding to specific genomic regions

  16. Quantitative real-time PCR eliminates false-positives in colony screening PCR.

    PubMed

    Skarratt, Kristen K; Fuller, Stephen J

    2014-01-01

    We report an alternative approach to colony screening using real-time PCR (qPCR) which can be used instead of the traditional end-point PCR to eliminate false-positives and reduce processing times. False-positive transformants can easily be distinguished from true-positives by comparing Ct values derived from qPCR amplification curves. In addition, the use of qPCR allows for more efficient processing since a gel electrophoresis step is not required and the screening process is no longer limited by the capacity of the gel apparatus.

  17. PCR for capsular typing of Haemophilus influenzae.

    PubMed Central

    Falla, T J; Crook, D W; Brophy, L N; Maskell, D; Kroll, J S; Moxon, E R

    1994-01-01

    A PCR method for the unequivocal assignment of Haemophilus influenzae capsular type (types a to f) was developed. PCR primers were designed from capsule type-specific DNA sequences cloned from the capsular gene cluster of each of the six capsular types. PCR product was amplified only from the capsular type for which the primers were designed. Product was confirmed by using either an internal oligonucleotide or restriction endonuclease digestion. A total of 172 H. influenzae strains of known capsular type (determined genetically) comprising all capsular types and noncapsulate strains were tested by PCR capsular typing. In all cases the PCR capsular type corresponded to the capsular genotype determined by restriction fragment length polymorphism analysis of the cap region. When used in conjunction with PCR primers derived from the capsular gene bexA, capsulate, noncapsulate, and capsule-deficient type b mutant strains could be differentiated. PCR capsular typing overcomes the problems of cross-reaction and autoagglutination associated with the serotyping of H. influenzae strains. The rapid and unequivocal capsular typing method that is described will be particularly important for typing invasive H. influenzae strains isolated from recipients of H. influenzae type b vaccine. Images PMID:7814470

  18. Quantitative DNA Analysis Using Droplet Digital PCR.

    PubMed

    Vossen, Rolf H A M; White, Stefan J

    2017-01-01

    Droplet digital PCR (ddPCR) is based on the isolated amplification of thousands of individual DNA molecules simultaneously, with each molecule compartmentalized in a droplet. The presence of amplified product in each droplet is indicated by a fluorescent signal, and the proportion of positive droplets allows the precise quantification of a given sequence. In this chapter we briefly outline the basis of ddPCR, and describe two different applications using the Bio-Rad QX200 system: genotyping copy number variation and quantification of Illumina sequencing libraries.

  19. The potential advantages of digital PCR for clinical virology diagnostics.

    PubMed

    Hall Sedlak, Ruth; Jerome, Keith R

    2014-05-01

    Digital PCR (dPCR), a new nucleic acid amplification technology, offers several potential advantages over real-time or quantitative PCR (qPCR), the current workhorse of clinical molecular virology diagnostics. Several studies have demonstrated dPCR assays for human cytomegalovirus or HIV, which give more precise and reproducible results than qPCR assays without sacrificing sensitivity. Here we review the literature comparing dPCR and qPCR performance in viral molecular diagnostic assays and offer perspective on the future of dPCR in clinical virology diagnostics.

  20. Recent advances in quantitative PCR (qPCR) applications in food microbiology.

    PubMed

    Postollec, Florence; Falentin, Hélène; Pavan, Sonia; Combrisson, Jérôme; Sohier, Danièle

    2011-08-01

    Molecular methods are being increasingly applied to detect, quantify and study microbial populations in food or during food processes. Among these methods, PCR-based techniques have been the subject of considerable focus and ISO guidelines have been established for the detection of food-borne pathogens. More particularly, real-time quantitative PCR (qPCR) is considered as a method of choice for the detection and quantification of microorganisms. One of its major advantages is to be faster than conventional culture-based methods. It is also highly sensitive, specific and enables simultaneous detection of different microorganisms. Application of reverse-transcription-qPCR (RT-qPCR) to study population dynamics and activities through quantification of gene expression in food, by contrast with the use of qPCR, is just beginning. Provided that appropriate controls are included in the analyses, qPCR and RT-qPCR appear to be highly accurate and reliable for quantification of genes and gene expression. This review addresses some important technical aspects to be considered when using these techniques. Recent applications of qPCR and RT-qPCR in food microbiology are given. Some interesting applications such as risk analysis or studying the influence of industrial processes on gene expression and microbial activity are reported.

  1. Sensitive and accurate quantification of human malaria parasites using droplet digital PCR (ddPCR)

    PubMed Central

    Koepfli, Cristian; Nguitragool, Wang; Hofmann, Natalie E.; Robinson, Leanne J.; Ome-Kaius, Maria; Sattabongkot, Jetsumon; Felger, Ingrid; Mueller, Ivo

    2016-01-01

    Accurate quantification of parasite density in the human host is essential for understanding the biology and pathology of malaria. Semi-quantitative molecular methods are widely applied, but the need for an external standard curve makes it difficult to compare parasite density estimates across studies. Droplet digital PCR (ddPCR) allows direct quantification without the need for a standard curve. ddPCR was used to diagnose and quantify P. falciparum and P. vivax in clinical patients as well as in asymptomatic samples. ddPCR yielded highly reproducible measurements across the range of parasite densities observed in humans, and showed higher sensitivity than qPCR to diagnose P. falciparum, and equal sensitivity for P. vivax. Correspondence in quantification was very high (>0.95) between qPCR and ddPCR. Quantification between technical replicates by ddPCR differed 1.5–1.7-fold, compared to 2.4–6.2-fold by qPCR. ddPCR facilitates parasite quantification for studies where absolute densities are required, and will increase comparability of results reported from different laboratories. PMID:27982132

  2. Transgene detection by digital droplet PCR.

    PubMed

    Moser, Dirk A; Braga, Luca; Raso, Andrea; Zacchigna, Serena; Giacca, Mauro; Simon, Perikles

    2014-01-01

    Somatic gene therapy is a promising tool for the treatment of severe diseases. Because of its abuse potential for performance enhancement in sports, the World Anti-Doping Agency (WADA) included the term 'gene doping' in the official list of banned substances and methods in 2004. Several nested PCR or qPCR-based strategies have been proposed that aim at detecting long-term presence of transgene in blood, but these strategies are hampered by technical limitations. We developed a digital droplet PCR (ddPCR) protocol for Insulin-Like Growth Factor 1 (IGF1) detection and demonstrated its applicability monitoring 6 mice injected into skeletal muscle with AAV9-IGF1 elements and 2 controls over a 33-day period. A duplex ddPCR protocol for simultaneous detection of Insulin-Like Growth Factor 1 (IGF1) and Erythropoietin (EPO) transgenic elements was created. A new DNA extraction procedure with target-orientated usage of restriction enzymes including on-column DNA-digestion was established. In vivo data revealed that IGF1 transgenic elements could be reliably detected for a 33-day period in DNA extracted from whole blood. In vitro data indicated feasibility of IGF1 and EPO detection by duplex ddPCR with high reliability and sensitivity. On-column DNA-digestion allowed for significantly improved target detection in downstream PCR-based approaches. As ddPCR provides absolute quantification, it ensures excellent day-to-day reproducibility. Therefore, we expect this technique to be used in diagnosing and monitoring of viral and bacterial infection, in detecting mutated DNA sequences as well as profiling for the presence of foreign genetic material in elite athletes in the future.

  3. A naked-eye colorimetric "PCR developer"

    NASA Astrophysics Data System (ADS)

    Valentini, Paola; Pompa, Pier Paolo

    2016-04-01

    Despite several advances in molecular biology and diagnostics, Polymerase Chain Reaction (PCR) is currently the gold standard for nucleic acids amplification and detection, due to its versatility, low-cost and universality, with estimated <10 billion reactions per year and a worldwide market of several billion dollars/year. Nevertheless, PCR still relies on the laborious, time-consuming, and multi-step gel electrophoresis-based detection, which includes gel casting, electrophoretic run, gel staining, and gel visualization. In this work, we propose a "PCR developer", namely a universal one-step, one-tube method, based on controlled aggregation of gold nanoparticles (AuNPs), to detect PCR products by naked eye in few minutes, with no need for any instrumentation. We demonstrated the specificity and sensitivity of the PCR developer on different model targets, suitable for a qualitative detection in real-world diagnostics (i.e., gene rearrangements, genetically modified organisms, and pathogens). The PCR developer proved to be highly specific and ultra-sensitive, discriminating down to few copies of HIV viral DNA, diluted in an excess of interfering human genomic DNA, which is a clinically relevant viral load. Hence, it could be a valuable tool for both academic research and clinical applications.

  4. A method for amplification of unknown flanking sequences based on touchdown PCR and suppression-PCR.

    PubMed

    Gao, Song; He, Dan; Li, Guangquan; Zhang, Yanhua; Lv, Huiying; Wang, Li

    2016-09-15

    Thermal asymmetric staggered PCR is the most widely used technique to obtain the flanking sequences. However, it has some limitations, including a low rate of positivity, and complex operation. In this study, a improved method of it was made based on suppression-PCR and touchdown PCR. The PCR fragment obtained by the amplification was used directly for sequencing after gel purification. Using this improved method, the positive rate of amplified flanking sequences of the ATMT mutants reached 99%. In addition, the time from DNA extraction to flanking sequence analysis was shortened to 2 days with about 6 dollars each sample.

  5. Evaluation of Altona Diagnostics RealStar Zika Virus RT-PCR Test Kit for Zika virus PCR testing.

    PubMed

    L'Huillier, Arnaud G; Lombos, Ernesto; Tang, Elaine; Perusini, Stephen; Eshaghi, Alireza; Nagra, Sandeep; Frantz, Christine; Olsha, Romy; Kristjanson, Erik; Dimitrova, Kristina; Safronetz, David; Drebot, Mike; Gubbay, Jonathan B

    2017-03-15

    Background: With the emerging ZIKA virus (ZIKV) epidemic, accessible real-time reverse-transcription PCR (rRT-PCR) assays are needed to streamline testing. The commercial Altona Diagnostics RealStar ZIKV rRT-PCR Test Kit has been approved for Emergency Use Authorization by the FDA. Our aim was to verify Altona-PCR, by comparing it to the CDC-designed dual target ZIKV virus rRT-PCR reference assay (Reference-PCR), and describe demographics of patients tested for ZIKV by rRT-PCR in Ontario, Canada.Methods: A large set of clinical specimens were tested for ZIKV by Altona-PCR and Reference-PCR. Positive or equivocal specimens underwent PCR and Sanger sequencing targeting ZIKV NS5 gene.Results: 671 serum specimens were tested by Reference-PCR: 58 (8.6%) were positive, 193 (28.8%) equivocal and 420 (62.6%) negative. Ninety percent of Reference-PCR positive patients were tested in the first 5 days after symptom onset. Altona-PCR was performed on 284/671 tested specimens by Reference-PCR. Altona-PCR was positive in 53/58 (91%) Reference-PCR positive and 16/193 (8%) Reference-PCR equivocal specimens; ZIKV NS5 PCR was positive in all 68 Altona-PCR positive specimens, and negative in all 181 Altona-PCR negative specimens that underwent NS5 PCR.Conclusion: Most ZIKV PCR positive cases are detected in the first five days of illness. Altona-PCR has very good sensitivity (91%) and specificity (97%) compared to Reference-PCR. Altona-PCR can be used for ZIKV diagnostic testing, with less extensive verification requirements compared to a laboratory developed test.

  6. PCR+ In Diesel Fuels and Emissions Research

    SciTech Connect

    McAdams, H.T.

    2002-04-15

    In past work for the U.S. Department of Energy (DOE) and Oak Ridge National Laboratory (ORNL), PCR+ was developed as an alternative methodology for building statistical models. PCR+ is an extension of Principal Components Regression (PCR), in which the eigenvectors resulting from Principal Components Analysis (PCA) are used as predictor variables in regression analysis. The work was motivated by the observation that most heavy-duty diesel (HDD) engine research was conducted with test fuels that had been ''concocted'' in the laboratory to vary selected fuel properties in isolation from each other. This approach departs markedly from the real world, where the reformulation of diesel fuels for almost any purpose leads to changes in a number of interrelated properties. In this work, we present new information regarding the problems encountered in the conventional approach to model-building and how the PCR+ method can be used to improve research on the relationship between fuel characteristics and engine emissions. We also discuss how PCR+ can be applied to a variety of other research problems related to diesel fuels.

  7. Development of SCAR markers and UP-PCR cross-hybridization method for specific detection of four major subgroups of Rhizoctonia from infected turfgrasses

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Several species and hyphal anastomosis groups (AG) of Rhizoctonia solani (sensu lato) cause brown patch diseases of turfgrasses. Conventional methods of identification of Rhizoctonia pathogens are time consuming and often inaccurate. A rapid identification assay for Waitea circinata (anamorph: Rhizo...

  8. 16S rRNA Gene-Based Identification of Midgut Bacteria from Field-Caught Anopheles gambiae Sensu Lato and A. funestus Mosquitoes Reveals New Species Related to Known Insect Symbionts

    PubMed Central

    Lindh, Jenny M.; Terenius, Olle; Faye, Ingrid

    2005-01-01

    Field-collected mosquitoes of the two main malaria vectors in Africa, Anopheles gambiae sensu lato and Anopheles funestus, were screened for their midgut bacterial contents. The midgut from each blood-fed mosquito was screened with two different detection pathways, one culture independent and one culture dependent. Bacterial species determination was achieved by sequence analysis of 16S rRNA genes. Altogether, 16 species from 14 genera were identified, 8 by each method. Interestingly, several of the bacteria identified are related to bacteria known to be symbionts in other insects. One isolate, Nocardia corynebacterioides, is a relative of the symbiont found in the vector for Chagas' disease that has been proven useful as a paratransgenic bacterium. Another isolate is a novel species within the γ-proteobacteria that could not be phylogenetically placed within any of the known orders in the class but is close to a group of insect symbionts. Bacteria representing three intracellular genera were identified, among them the first identifications of Anaplasma species from mosquitoes and a new mosquito-Spiroplasma association. The isolates will be further investigated for their suitability for a paratransgenic Anopheles mosquito. PMID:16269761

  9. 16S rRNA gene-based identification of midgut bacteria from field-caught Anopheles gambiae sensu lato and A. funestus mosquitoes reveals new species related to known insect symbionts.

    PubMed

    Lindh, Jenny M; Terenius, Olle; Faye, Ingrid

    2005-11-01

    Field-collected mosquitoes of the two main malaria vectors in Africa, Anopheles gambiae sensu lato and Anopheles funestus, were screened for their midgut bacterial contents. The midgut from each blood-fed mosquito was screened with two different detection pathways, one culture independent and one culture dependent. Bacterial species determination was achieved by sequence analysis of 16S rRNA genes. Altogether, 16 species from 14 genera were identified, 8 by each method. Interestingly, several of the bacteria identified are related to bacteria known to be symbionts in other insects. One isolate, Nocardia corynebacterioides, is a relative of the symbiont found in the vector for Chagas' disease that has been proven useful as a paratransgenic bacterium. Another isolate is a novel species within the gamma-proteobacteria that could not be phylogenetically placed within any of the known orders in the class but is close to a group of insect symbionts. Bacteria representing three intracellular genera were identified, among them the first identifications of Anaplasma species from mosquitoes and a new mosquito-Spiroplasma association. The isolates will be further investigated for their suitability for a paratransgenic Anopheles mosquito.

  10. A Proposal for a Genome Similarity-Based Taxonomy for Plant-Pathogenic Bacteria that Is Sufficiently Precise to Reflect Phylogeny, Host Range, and Outbreak Affiliation Applied to Pseudomonas syringae sensu lato as a Proof of Concept.

    PubMed

    Vinatzer, Boris A; Weisberg, Alexandra J; Monteil, Caroline L; Elmarakeby, Haitham A; Sheppard, Samuel K; Heath, Lenwood S

    2017-01-01

    Taxonomy of plant pathogenic bacteria is challenging because pathogens of different crops often belong to the same named species but current taxonomy does not provide names for bacteria below the subspecies level. The introduction of the host range-based pathovar system in the 1980s provided a temporary solution to this problem but has many limitations. The affordability of genome sequencing now provides the opportunity for developing a new genome-based taxonomic framework. We already proposed to name individual bacterial isolates based on pairwise genome similarity. Here, we expand on this idea and propose to use genome similarity-based codes, which we now call life identification numbers (LINs), to describe and name bacterial taxa. Using 93 genomes of Pseudomonas syringae sensu lato, LINs were compared with a P. syringae genome tree whereby the assigned LINs were found to be informative of a majority of phylogenetic relationships. LINs also reflected host range and outbreak association for strains of P. syringae pathovar actinidiae, a pathovar for which many genome sequences are available. We conclude that LINs could provide the basis for a new taxonomic framework to address the shortcomings of the current pathovar system and to complement the current taxonomic system of bacteria in general.

  11. pcrEfficiency: a Web tool for PCR amplification efficiency prediction

    PubMed Central

    2011-01-01

    Background Relative calculation of differential gene expression in quantitative PCR reactions requires comparison between amplification experiments that include reference genes and genes under study. Ignoring the differences between their efficiencies may lead to miscalculation of gene expression even with the same starting amount of template. Although there are several tools performing PCR primer design, there is no tool available that predicts PCR efficiency for a given amplicon and primer pair. Results We have used a statistical approach based on 90 primer pair combinations amplifying templates from bacteria, yeast, plants and humans, ranging in size between 74 and 907 bp to identify the parameters that affect PCR efficiency. We developed a generalized additive model fitting the data and constructed an open source Web interface that allows the obtention of oligonucleotides optimized for PCR with predicted amplification efficiencies starting from a given sequence. Conclusions pcrEfficiency provides an easy-to-use web interface allowing the prediction of PCR efficiencies prior to web lab experiments thus easing quantitative real-time PCR set-up. A web-based service as well the source code are provided freely at http://srvgen.upct.es/efficiency.html under the GPL v2 license. PMID:22014212

  12. Detection of Leishmania infantum in animals and their ectoparasites by conventional PCR and real time PCR.

    PubMed

    de Morais, Rayana Carla Silva; Gonçalves, Suênia da Cunha; Costa, Pietra Lemos; da Silva, Kamila Gaudêncio; da Silva, Fernando José; Silva, Rômulo Pessoa E; de Brito, Maria Edileuza Felinto; Brandão-Filho, Sinval Pinto; Dantas-Torres, Filipe; de Paiva-Cavalcanti, Milena

    2013-04-01

    Visceral leishmaniosis (VL) is a parasitic disease caused by Leishmania infantum, which is primarily transmitted by phlebotomine sandflies. However, there has been much speculation on the role of other arthropods in the transmission of VL. Thus, the aim of this study was to assess the presence of L. infantum in cats, dogs and their ectoparasites in a VL-endemic area in northeastern Brazil. DNA was extracted from blood samples and ectoparasites, tested by conventional PCR (cPCR) and quantitative real time PCR (qPCR) targeting the L. infantum kinetoplast DNA. A total of 280 blood samples (from five cats and 275 dogs) and 117 ectoparasites from dogs were collected. Animals were apparently healthy and not previously tested by serological or molecular diagnostic methods. Overall, 213 (76.1 %) animals and 51 (43.6 %) ectoparasites were positive to L. infantum, with mean parasite loads of 795.2, 31.9 and 9.1 fg in dogs, cats and ectoparasites, respectively. Concerning the positivity between dogs and their ectoparasites, 32 (15.3 %) positive dogs were parasitized by positive ectoparasites. The overall concordance between the PCR protocols used was 59.2 %, with qPCR being more efficient than cPCR; 34.1 % of all positive samples were exclusively positive by qPCR. The high number of positive animals and ectoparasites also indicates that they could serve as sentinels or indicators of the circulation of L. infantum in risk areas.

  13. Propidium monoazide reverse transcription PCR and RT-qPCR for detecting infectious enterovirus and norovirus

    EPA Science Inventory

    Presently there is no established cell line or small animal model that allows for the detection of infectious human norovirus. Current methods based on RT-PCR and RT-qPCR detect both infectious and non-infectious virus and thus the conclusions that may be drawn regarding the publ...

  14. Real-time PCR: Advanced technologies and applications

    Technology Transfer Automated Retrieval System (TEKTRAN)

    This book brings together contributions from 20 experts in the field of PCR, providing a broad perspective of the applications of quantitative real-time PCR (qPCR). The editors state in the preface that the aim is to provide detailed insight into underlying principles and methods of qPCR to provide ...

  15. PCR Techniques in Next-Generation Sequencing.

    PubMed

    Goswami, Rashmi S

    2016-01-01

    With the advent of next-generation sequencing and its prolific use in the clinical realm, it would appear that techniques such as PCR would not be in high demand. This is not the case however, as PCR techniques play an important role in the success of NGS technology. Although NGS has rapidly become an important part of clinical molecular diagnostics, whole genome sequencing is still difficult to implement in a clinical laboratory due to high costs of sequencing, as well as issues surrounding data processing, analysis, and data storage, which can reduce efficiency and increase turnaround times. As a result, targeted sequencing is often used in clinical diagnostics, due to its increased efficiency. PCR techniques play an integral role in targeted NGS sequencing, allowing for the generation of multiple NGS libraries and the sequencing of multiple targeted regions simultaneously. We will outline the methods we employ in PCR amplification of targeted genomic regions for cancer mutation hotspots using the Ampliseq Cancer Hotspot v2 panel (Life Technologies, Carlsbad, CA).

  16. Manufacturing DNA microarrays from unpurified PCR products

    PubMed Central

    Diehl, Frank; Beckmann, Boris; Kellner, Nadine; Hauser, Nicole C.; Diehl, Susanne; Hoheisel, Jörg D.

    2002-01-01

    For the production of DNA microarrays from PCR products, purification of the the DNA fragments prior to spotting is a major expense in cost and time. Also, a considerable amount of material is lost during this process and contamination might occur. Here, a protocol is presented that permits the manufacture of microarrays from unpurified PCR products on aminated surfaces such as glass slides coated with the widely used poly(l-lysine) or aminosilane. The presence of primer molecules in the PCR sample does not increase the non-specific signal upon hybridisation. Overall, signal intensity on arrays made of unpurified PCR products is 94% of the intensity obtained with the respective purified molecules. This slight loss in signal, however, is offset by a reduced variation in the amount of DNA present at the individual spot positions across an array, apart from the considerable savings in time and cost. In addition, a larger number of arrays can be made from one batch of amplification products. PMID:12177307

  17. Miniaturized technology for DNA typing: cassette PCR.

    PubMed

    Manage, Dammika P; Pilarski, Linda M

    2015-01-01

    With the smaller size, low cost, and rapid testing capabilities, miniaturized lab-on-a-chip devices can change the way medical diagnostics are currently performed in the health-care system. We have demonstrated such a device that is self-contained, simple, disposable, and inexpensive. It is capable of performing DNA amplification on an inexpensive instrument suitable for near point of care settings. This technology will enable on the spot evaluation of patients in the clinic for faster medical decision-making and more informed therapeutic choices. Our device, a gel capillary cassette, termed cassette PCR, contains capillary reaction units each holding a defined primer set, with arrays of capillary reaction units for simultaneously detecting multiple targets. With the exception of the sample to be tested, each capillary reaction unit holds all the reagents needed for PCR in a desiccated form that can be stored at room temperature for up to 3 months and even longer in colder conditions. It relies on capillary forces for sample delivery of microliter volumes through capillaries, hence avoiding the need for pumps or valves. In the assembled cassette, the wax architecture supporting the capillaries melts during the PCR and acts as a vapor barrier as well as segregating capillaries with different primer sets. No other chip sealing techniques are required. Cassette PCR accepts raw samples such as urine, genital swabs, and blood. The cassette is made with off-the-shelf components and contains integrated positive and negative controls.

  18. The Power of Real-Time PCR

    ERIC Educational Resources Information Center

    Valasek, Mark A.; Repa, Joyce J.

    2005-01-01

    In recent years, real-time polymerase chain reaction (PCR) has emerged as a robust and widely used methodology for biological investigation because it can detect and quantify very small amounts of specific nucleic acid sequences. As a research tool, a major application of this technology is the rapid and accurate assessment of changes in gene…

  19. New PCR systems to confirm real-time PCR detection of Mycobacterium avium subsp. paratuberculosis

    PubMed Central

    Herthnek, David; Bölske, Göran

    2006-01-01

    Background Johne's disease, a serious chronic form of enteritis in ruminants, is caused by Mycobacterium avium subsp. paratuberculosis (MAP). As the organism is very slow-growing and fastidious, several PCR-based methods for detection have been developed, based mainly on the MAP-specific gene IS900. However, because this gene is similar to genes in other mycobacteria, there is a need for sensitive and reliable methods to confirm the presence of MAP. As described here, two new real-time PCR systems on the IS900 gene and one on the F57 gene were developed and carefully validated on 267 strains and 56 positive clinical faecal samples. Results Our confirmatory PCR systems on IS900 were found sensitive and specific, only yielding weak false positive reactions in one strain for each system. The PCR system on F57 did not elicit any false positives and was only slightly less sensitive than our primary IS900-system. DNA from both naturally infected and spiked faeces that tested positive with our primary system could be confirmed with all new systems, except one low-level infected sample that tested negative with the F57 system. Conclusion We recommend using the newly constructed DH3 PCR system on the F57 gene as the primary confirmatory test for PCR positives, but should it fail due to its lower sensitivity, the DH1 and DH2 PCR systems should be used. PMID:17020599

  20. Quantitative PCR and Digital PCR for Detection of Ascaris lumbricoides Eggs in Reclaimed Water

    PubMed Central

    Santísima-Trinidad, Ana Belén; Bornay-Llinares, Fernando Jorge; Martín González, Marcos; Pascual Valero, José Antonio; Ros Muñoz, Margarita

    2017-01-01

    The reuse of reclaimed water from wastewater depuration is a widespread and necessary practice in many areas around the world and must be accompanied by adequate and continuous quality control. Ascaris lumbricoides is one of the soil-transmitted helminths (STH) with risk for humans due to its high infectivity and an important determinant of transmission is the inadequacy of water supplies and sanitation. The World Health Organization (WHO) recommends a limit equal to or lower than one parasitic helminth egg per liter, to reuse reclaimed water for unrestricted irrigation. We present two new protocols of DNA extraction from large volumes of reclaimed water. Quantitative PCR (qPCR) and digital PCR (dPCR) were able to detect low amounts of A. lumbricoides eggs. By using the first extraction protocol, which processes 500 mL of reclaimed water, qPCR can detect DNA concentrations as low as one A. lumbricoides egg equivalent, while dPCR can detect DNA concentrations as low as five A. lumbricoides egg equivalents. By using the second protocol, which processes 10 L of reclaimed water, qPCR was able to detect DNA concentrations equivalent to 20 A. lumbricoides eggs. This fact indicated the importance of developing new methodologies to detect helminth eggs with higher sensitivity and precision avoiding possible human infection risks. PMID:28377928

  1. Quantitative PCR and Digital PCR for Detection of Ascaris lumbricoides Eggs in Reclaimed Water.

    PubMed

    Acosta Soto, Lucrecia; Santísima-Trinidad, Ana Belén; Bornay-Llinares, Fernando Jorge; Martín González, Marcos; Pascual Valero, José Antonio; Ros Muñoz, Margarita

    2017-01-01

    The reuse of reclaimed water from wastewater depuration is a widespread and necessary practice in many areas around the world and must be accompanied by adequate and continuous quality control. Ascaris lumbricoides is one of the soil-transmitted helminths (STH) with risk for humans due to its high infectivity and an important determinant of transmission is the inadequacy of water supplies and sanitation. The World Health Organization (WHO) recommends a limit equal to or lower than one parasitic helminth egg per liter, to reuse reclaimed water for unrestricted irrigation. We present two new protocols of DNA extraction from large volumes of reclaimed water. Quantitative PCR (qPCR) and digital PCR (dPCR) were able to detect low amounts of A. lumbricoides eggs. By using the first extraction protocol, which processes 500 mL of reclaimed water, qPCR can detect DNA concentrations as low as one A. lumbricoides egg equivalent, while dPCR can detect DNA concentrations as low as five A. lumbricoides egg equivalents. By using the second protocol, which processes 10 L of reclaimed water, qPCR was able to detect DNA concentrations equivalent to 20 A. lumbricoides eggs. This fact indicated the importance of developing new methodologies to detect helminth eggs with higher sensitivity and precision avoiding possible human infection risks.

  2. Digital PCR to assess gene-editing frequencies (GEF-dPCR) mediated by designer nucleases.

    PubMed

    Mock, Ulrike; Hauber, Ilona; Fehse, Boris

    2016-03-01

    Genome editing using designer nucleases such as transcription activator-like effector nucleases (TALENs) or clustered regularly interspersed short palindromic repeats (CRISPR)-Cas9 nucleases is an emerging technology in basic and applied research. Whereas the application of editing tools, namely CRISPR-Cas9, has recently become very straightforward, quantification of resulting gene knockout rates still remains a bottleneck. This is particularly true if the product of a targeted gene is not easily detectable. To address this problem, we devised a novel gene-editing frequency digital PCR (GEF-dPCR) technique. GEF-dPCR exploits two differently labeled probes that are placed within one amplicon at the gene-editing target site to simultaneously detect wild-type and nonhomologous end-joining (NHEJ)-affected alleles. Taking advantage of the principle of dPCR, this enables concurrent quantification of edited and wild-type alleles in a given sample. We propose that our method is optimal for the monitoring of gene-edited cells in vivo, e.g., in clinical settings. Here we describe preparation, design of primers and probes, and setup and analysis of GEF-dPCR. The setup of GEF-dPCR requires up to 2 weeks (depending on the starting point); once the dPCR has been established, the protocol for sample analysis takes <1 d.

  3. Real-time PCR (qPCR) primer design using free online software.

    PubMed

    Thornton, Brenda; Basu, Chhandak

    2011-01-01

    Real-time PCR (quantitative PCR or qPCR) has become the preferred method for validating results obtained from assays which measure gene expression profiles. The process uses reverse transcription polymerase chain reaction (RT-PCR), coupled with fluorescent chemistry, to measure variations in transcriptome levels between samples. The four most commonly used fluorescent chemistries are SYBR® Green dyes and TaqMan®, Molecular Beacon or Scorpion probes. SYBR® Green is very simple to use and cost efficient. As SYBR® Green dye binds to any double-stranded DNA product, its success depends greatly on proper primer design. Many types of online primer design software are available, which can be used free of charge to design desirable SYBR® Green-based qPCR primers. This laboratory exercise is intended for those who have a fundamental background in PCR. It addresses the basic fluorescent chemistries of real-time PCR, the basic rules and pitfalls of primer design, and provides a step-by-step protocol for designing SYBR® Green-based primers with free, online software.

  4. How Many Microorganisms Are Present? Quantitative Reverse Transcription PCR (qRT-PCR)

    NASA Astrophysics Data System (ADS)

    Price, Andy; Álvarez, Laura Acuña; Whitby, Corinne; Larsen, Jan

    Quantitative reverse transcription PCR (qRT-PCR) is a variation of conventional quantitative or real-time PCR, whereby mRNA is first converted into the complementary DNA (cDNA) by reverse transcription, the cDNA is then subsequently quantified by qPCR. The use of mRNA as the initial template allows the quantification of gene transcripts, rather than gene copy numbers. mRNA is only produced by actively metabolising cells and is produced by its corresponding gene to provide a 'blueprint' in order for a cell to manufacture a specific protein. Conventional qPCR detects not only DNA present in actively metabolising cells but also inactive and dead cells. qRT-PCR has the advantage that only actively metabolising cells are detected, hence provides a more reliable measure of microbial activity in oilfield samples. When qRT-PCR is combined with primers and probes for specific genes, the activity of microbial processes important in the oilfield, such as sulphate reduction, methanogenesis and nitrate reduction can be monitored.

  5. A survey of tools for the analysis of quantitative PCR (qPCR) data.

    PubMed

    Pabinger, Stephan; Rödiger, Stefan; Kriegner, Albert; Vierlinger, Klemens; Weinhäusel, Andreas

    2014-09-01

    Real-time quantitative polymerase-chain-reaction (qPCR) is a standard technique in most laboratories used for various applications in basic research. Analysis of qPCR data is a crucial part of the entire experiment, which has led to the development of a plethora of methods. The released tools either cover specific parts of the workflow or provide complete analysis solutions. Here, we surveyed 27 open-access software packages and tools for the analysis of qPCR data. The survey includes 8 Microsoft Windows, 5 web-based, 9 R-based and 5 tools from other platforms. Reviewed packages and tools support the analysis of different qPCR applications, such as RNA quantification, DNA methylation, genotyping, identification of copy number variations, and digital PCR. We report an overview of the functionality, features and specific requirements of the individual software tools, such as data exchange formats, availability of a graphical user interface, included procedures for graphical data presentation, and offered statistical methods. In addition, we provide an overview about quantification strategies, and report various applications of qPCR. Our comprehensive survey showed that most tools use their own file format and only a fraction of the currently existing tools support the standardized data exchange format RDML. To allow a more streamlined and comparable analysis of qPCR data, more vendors and tools need to adapt the standardized format to encourage the exchange of data between instrument software, analysis tools, and researchers.

  6. Identification of bacterial plant pathogens using multilocus PCR and electrospray ionization-mass spectrometry (PCR/ESI-MS)

    Technology Transfer Automated Retrieval System (TEKTRAN)

    PCR/electrospray ionization-mass spectrometry (PCR/ESI-MS, previously known as “TIGER”) utilizes PCR with broad range primers to amplify products from wide array of organisms within a taxonomic group, followed by analysis of PCR amplicons using mass spectrometry. Computer analysis of precise masses ...

  7. Rapid PCR thermocycling using microscale thermal convection.

    PubMed

    Muddu, Radha; Hassan, Yassin A; Ugaz, Victor M

    2011-03-05

    Many molecular biology assays depend in some way on the polymerase chain reaction (PCR) to amplify an initially dilute target DNA sample to a detectable concentration level. But the design of conventional PCR thermocycling hardware, predominantly based on massive metal heating blocks whose temperature is regulated by thermoelectric heaters, severely limits the achievable reaction speed(1). Considerable electrical power is also required to repeatedly heat and cool the reagent mixture, limiting the ability to deploy these instruments in a portable format. Thermal convection has emerged as a promising alternative thermocycling approach that has the potential to overcome these limitations(2-9). Convective flows are an everyday occurrence in a diverse array of settings ranging from the Earth's atmosphere, oceans, and interior, to decorative and colorful lava lamps. Fluid motion is initiated in the same way in each case: a buoyancy driven instability arises when a confined volume of fluid is subjected to a spatial temperature gradient. These same phenomena offer an attractive way to perform PCR thermocycling. By applying a static temperature gradient across an appropriately designed reactor geometry, a continuous circulatory flow can be established that will repeatedly transport PCR reagents through temperature zones associated with the denaturing, annealing, and extension stages of the reaction (Figure 1). Thermocycling can therefore be actuated in a pseudo-isothermal manner by simply holding two opposing surfaces at fixed temperatures, completely eliminating the need to repeatedly heat and cool the instrument. One of the main challenges facing design of convective thermocyclers is the need to precisely control the spatial velocity and temperature distributions within the reactor to ensure that the reagents sequentially occupy the correct temperature zones for a sufficient period of time(10,11). Here we describe results of our efforts to probe the full 3-D velocity and

  8. A quadruplex PCR (qxPCR) assay for adulteration in dairy products.

    PubMed

    Agrimonti, Caterina; Pirondini, Andrea; Marmiroli, Marta; Marmiroli, Nelson

    2015-11-15

    This study describes the development of a quadruplex quantitative Real Time PCR (qxPCR) based on SYBR®GreenER chemistry, for rapid identification of DNA of cow, goat, sheep and buffalo in dairy products, and for quantification of cow DNA in these products. The platform was applied to: (i) mixes of milks at fixed percentages; (ii) cheeses prepared with the same mixes; (iii) commercial dairy products. The methodology enabled the detection of DNA from cow in mixes of milk and cheeses with a limit of detection (LOD) of 0.1%. When applied to commercial dairy products the qxPCR gave results comparable with each single-plex Real Time PCR. A good correlation (R(2)>0.9) between peaks' area of derivative of melting curves of amplicons and percentages of cow milk in milk mixes and cheeses, allows for an estimation of cow DNA in a dynamic range varying from 0.1-5% to 1-25%.

  9. Five-month comparative efficacy evaluation of three ectoparasiticides against adult cat fleas (Ctenocephalides felis), flea egg hatch and emergence, and adult brown dog ticks (Rhipicephalus sanguineus sensu lato) on dogs housed outdoors.

    PubMed

    Varloud, Marie; Hodgkins, Elizabeth

    2015-03-01

    This study was designed to compare the efficacy of three topical combinations on dogs in outdoor conditions against adult cat fleas (Ctenocephalides felis), flea egg hatch and emergence, and against adult brown dog ticks (Rhipicephalus sanguineus sensu lato). Treatment was performed on day 0 with a placebo; dinotefuran, pyriproxifen and permethrin (DPP); fipronil and (S)-methoprene (FM) or imidacloprid and permethrin (IP). Dogs (n = 32), housed outdoors for 7 months, were treated monthly for four consecutive months (on days 0, 30, 60 and 90) and infested with ~100 unfed adult fleas on days 14, 55, 74, 115 and 150 and with ~50 unfed adult ticks on days 28, 44, 88 and 104. Adult fleas were counted and removed 24 h after infestation. Immediately after flea removal, dogs were reinfested with ~100 new adult fleas 72 h prior to egg collection for up to 48 h. Flea eggs were incubated for 32 days, and newly emerged adults were counted. Ticks were counted and removed 48 h after each infestation. FM had >90 % efficacy against fleas at each time point and variable efficacy against ticks (38.0-99.6 %). Efficacy of IP was <90 % against fleas at day 64 and against ticks at day 30 of the first post-treatment. No flea eggs were laid in the treated groups until infestation was carried out >60 days after the last treatment. Despite challenging weather conditions, DPP was highly effective, providing >90 % efficacy against adult ticks as well as adult and immature fleas at every time point of the study.

  10. Replaceable Microfluidic Cartridges for a PCR Biosensor

    NASA Technical Reports Server (NTRS)

    Francis, Kevin; Sullivan, Ron

    2005-01-01

    The figure depicts a replaceable microfluidic cartridge that is a component of a miniature biosensor that detects target deoxyribonucleic acid (DNA) sequences. The biosensor utilizes (1) polymerase chain reactions (PCRs) to multiply the amount of DNA to be detected, (2) fluorogenic polynucleotide probe chemicals for labeling the target DNA sequences, and (3) a high-sensitivity epifluorescence-detection optoelectronic subsystem. Microfluidics is a relatively new field of device development in which one applies techniques for fabricating microelectromechanical systems (MEMS) to miniature systems for containing and/or moving fluids. Typically, microfluidic devices are microfabricated, variously, from silicon or polymers. The development of microfluidic devices for applications that involve PCR and fluorescence-based detection of PCR products poses special challenges

  11. A nanofluidic system for massively parallel PCR

    NASA Astrophysics Data System (ADS)

    Brenan, Colin; Morrison, Tom; Roberts, Douglas; Hurley, James

    2008-02-01

    Massively parallel nanofluidic systems are lab-on-a-chip devices where solution phase biochemical and biological analyses are implemented in high density arrays of nanoliter holes micro-machined in a thin platen. Polymer coatings make the interior surfaces of the holes hydrophilic and the exterior surface of the platen hydrophobic for precise and accurate self-metered loading of liquids into each hole without cross-contamination. We have created a "nanoplate" based on this concept, equivalent in performance to standard microtiter plates, having 3072 thirty-three nanoliter holes in a stainless steel platen the dimensions of a microscope slide. We report on the performance of this device for PCR-based single nucleotide polymorphism (SNP) genotyping or quantitative measurement of gene expression by real-time PCR in applications ranging from plant and animal diagnostics, agricultural genetics and human disease research.

  12. PCR (Polymerase Chain Reaction) Testing for Leishmaniasis

    DTIC Science & Technology

    1993-10-20

    reactions. Prior to temperature cycling, this moderately heat stable enzyme selectively excises uracil residues which have been incorporated into DNA... temperature . Despite these precautions, some non-specific annealing of PCR primers does occur, even with single copy gene detection, and more so with...Mallinckrodt) in slightly hypotonic saline and allowed to remain at ambient temperature for 5 minutes, during which time RBC’s are lysed. Centrifugation

  13. Digital PCR analysis of circulating nucleic acids.

    PubMed

    Hudecova, Irena

    2015-10-01

    Detection of plasma circulating nucleic acids (CNAs) requires the use of extremely sensitive and precise methods. The commonly used quantitative real-time polymerase chain reaction (PCR) poses certain technical limitations in relation to the precise measurement of CNAs whereas the costs of massively parallel sequencing are still relatively high. Digital PCR (dPCR) now represents an affordable and powerful single molecule counting strategy to detect minute amounts of genetic material with performance surpassing many quantitative methods. Microfluidic (chip) and emulsion (droplet)-based technologies have already been integrated into platforms offering hundreds to millions of nanoliter- or even picoliter-scale reaction partitions. The compelling observations reported in the field of cancer research, prenatal testing, transplantation medicine and virology support translation of this technology into routine use. Extremely sensitive plasma detection of rare mutations originating from tumor or placental cells among a large background of homologous sequences facilitates unraveling of the early stages of cancer or the detection of fetal mutations. Digital measurement of quantitative changes in plasma CNAs associated with cancer or graft rejection provides valuable information on the monitoring of disease burden or the recipient's immune response and subsequent therapy treatment. Furthermore, careful quantitative assessment of the viral load offers great value for effective monitoring of antiviral therapy for immunosuppressed or transplant patients. The present review describes the inherent features of dPCR that make it exceptionally robust in precise and sensitive quantification of CNAs. Moreover, I provide an insight into the types of potential clinical applications that have been developed by researchers to date.

  14. Comparison of Droplet Digital PCR and Quantitative PCR Assays for Quantitative Detection of Xanthomonas citri Subsp. citri

    PubMed Central

    Yin, Youping; Wang, Zhongkang

    2016-01-01

    Droplet digital polymerase chain reaction (ddPCR) is a novel molecular biology technique providing absolute quantification of target nucleic acids without the need for an external calibrator. Despite its emerging applications in medical diagnosis, there are few reports of its use for the detection of plant pathogens. This work was designed to assess the diagnosis potential of the ddPCR for absolute quantitative detection of Xanthomonas citri subsp. citri, a quarantine plant pathogenic bacterium that causes citrus bacterial canker in susceptible Citrus species. We transferred an established quantitative PCR (qPCR) assay for citrus bacterial canker diagnosis directly to the ddPCR format and compared the performance of the two methods. The qPCR assay has a broader dynamic range compared to the ddPCR assay and the ddPCR assay has a significantly higher degree of sensitivity compared to the qPCR assay. The influence of PCR inhibitors can be reduced considerably in the ddPCR assay because the collection of end-point fluorescent signals and the counting of binomial events (positive or negative droplets) are associated with a Poisson algorithm. The ddPCR assay also shows lower coefficient of variation compared to the qPCR assay especially in low target concentration. The linear association of the measurements by ddPCR and qPCR assays is strong (Pearson correlation = 0.8633; P<0.001). Receiver operating characteristic analysis indicates the ddPCR methodology is a more robust approach for diagnosis of citrus bacterial canker. In summary, the results demonstrated that the ddPCR assay has the potential for the quantitative detection of X. citri subsp. citri with high precision and accuracy as compared with the results from qPCR assay. Further studies are required to evaluate and validate the value of ddPCR technology in the diagnosis of plant disease and quarantine applications. PMID:27427975

  15. Microfluidic gradient PCR (MG-PCR): a new method for microfluidic DNA amplification.

    PubMed

    Zhang, Chunsun; Xing, Da

    2010-02-01

    This study develops a new microfluidic DNA amplification strategy for executing parallel DNA amplification in the microfluidic gradient polymerase chain reaction (MG-PCR) device. The developed temperature gradient microfluidic system is generated by using an innovative fin design. The device mainly consists of modular thermally conductive copper flake which is attached onto a finned aluminum heat sink with a small fan. In our microfluidic temperature gradient prototype, a non-linear temperature gradient is produced along the gradient direction. On the copper flake of length 45 mm, width 40 mm and thickness 4 mm, the temperature gradient easily spans the range from 97 to 52 degrees Celsius. By making full use of the hot (90-97 degrees Celsius) and cold (60-70 degrees Celsius) regions on the temperature gradient device, the parallel, two-temperature MG-PCR amplification is feasible. As a demonstration, the MG-PCR from three parallel reactions of 112-bp Escherichia coli DNA fragment is performed in a continuous-flow format, in which the flow of the PCR reagent in the closed loop is induced by the buoyancy-driven nature convection. Although the prototype is not optimized, the MG-PCR amplification can be completed in less than 45 min. However, the MG-PCR thermocycler presented herein can be further scaled-down, and thus the amplification times and reagent consumption can be further reduced. In addition, the currently developed temperature gradient technology can be applied onto other continuous-flow MG-PCR systems or used for other analytical purposes such as parallel and combination measurements, and fluorescent melting curve analysis.

  16. Overcoming inhibition in real-time diagnostic PCR.

    PubMed

    Hedman, Johannes; Rådström, Peter

    2013-01-01

    PCR is an important and powerful tool in several fields, including clinical diagnostics, food analysis, and forensic analysis. In theory, PCR enables the detection of one single cell or DNA molecule. However, the presence of PCR inhibitors in the sample affects the amplification efficiency of PCR, thus lowering the detection limit, as well as the precision of sequence-specific nucleic acid quantification in real-time PCR. In order to overcome the problems caused by PCR inhibitors, all the steps leading up to DNA amplification must be optimized for the sample type in question. Sampling and sample treatment are key steps, but most of the methods currently in use were developed for conventional diagnostic methods and not for PCR. Therefore, there is a need for fast, simple, and robust sample preparation methods that take advantage of the accuracy of PCR. In addition, the thermostable DNA polymerases and buffer systems used in PCR are affected differently by inhibitors. During recent years, real-time PCR has developed considerably and is now widely used as a diagnostic tool. This technique has greatly improved the degree of automation and reduced the analysis time, but has also introduced a new set of PCR inhibitors, namely those affecting the fluorescence signal. The purpose of this chapter is to view the complexity of PCR inhibition from different angles, presenting both molecular explanations and practical ways of dealing with the problem. Although diagnostic PCR brings together scientists from different diagnostic fields, end-users have not fully exploited the potential of learning from each other. Here, we have collected knowledge from archeological analysis, clinical diagnostics, environmental analysis, food analysis, and forensic analysis. The concept of integrating sampling, sample treatment, and the chemistry of PCR, i.e., pre-PCR processing, will be addressed as a general approach to overcoming real-time PCR inhibition and producing samples optimal for PCR

  17. TqPCR: A Touchdown qPCR Assay with Significantly Improved Detection Sensitivity and Amplification Efficiency of SYBR Green qPCR.

    PubMed

    Zhang, Qian; Wang, Jing; Deng, Fang; Yan, Zhengjian; Xia, Yinglin; Wang, Zhongliang; Ye, Jixing; Deng, Youlin; Zhang, Zhonglin; Qiao, Min; Li, Ruifang; Denduluri, Sahitya K; Wei, Qiang; Zhao, Lianggong; Lu, Shun; Wang, Xin; Tang, Shengli; Liu, Hao; Luu, Hue H; Haydon, Rex C; He, Tong-Chuan; Jiang, Li

    2015-01-01

    The advent of fluorescence-based quantitative real-time PCR (qPCR) has revolutionized the quantification of gene expression analysis in many fields, including life sciences, agriculture, forensic science, molecular diagnostics, and medicine. While SYBR Green-based qPCR is the most commonly-used platform due to its inexpensive nature and robust chemistry, quantifying the expression of genes with low abundance or RNA samples extracted from highly restricted or limited sources can be challenging because the detection sensitivity of SYBR Green-based qPCR is limited. Here, we develop a novel and effective touchdown qPCR (TqPCR) protocol by incorporating a 4-cycle touchdown stage prior to the quantification amplification stage. Using the same cDNA templates, we find that TqPCR can reduce the average Cq values for Gapdh, Rps13, and Hprt1 reference genes by 4.45, 5.47, and 4.94 cycles, respectively, when compared with conventional qPCR; the overall average Cq value reduction for the three reference genes together is 4.95. We further find that TqPCR can improve PCR amplification efficiency and thus increase detection sensitivity. When the quantification of Wnt3A-induced target gene expression in mesenchymal stem cells is analyzed, we find that, while both conventional qPCR and TqPCR can detect the up-regulation of the relatively abundant target Axin2, only TqPCR can detect the up-regulation of the lowly-expressed targets Oct4 and Gbx2. Finally, we demonstrate that the MRQ2 and MRQ3 primer pairs derived from mouse reference gene Tbp can be used to validate the RNA/cDNA integrity of qPCR samples. Taken together, our results strongly suggest that TqPCR may increase detection sensitivity and PCR amplification efficiency. Overall, TqPCR should be advantageous over conventional qPCR in expression quantification, especially when the transcripts of interest are lowly expressed, and/or the availability of total RNA is highly restricted or limited.

  18. TqPCR: A Touchdown qPCR Assay with Significantly Improved Detection Sensitivity and Amplification Efficiency of SYBR Green qPCR

    PubMed Central

    Zhang, Qian; Wang, Jing; Deng, Fang; Yan, Zhengjian; Xia, Yinglin; Wang, Zhongliang; Ye, Jixing; Deng, Youlin; Zhang, Zhonglin; Qiao, Min; Li, Ruifang; Denduluri, Sahitya K.; Wei, Qiang; Zhao, Lianggong; Lu, Shun; Wang, Xin; Tang, Shengli; Liu, Hao; Luu, Hue H.; Haydon, Rex C.; He, Tong-Chuan; Jiang, Li

    2015-01-01

    The advent of fluorescence-based quantitative real-time PCR (qPCR) has revolutionized the quantification of gene expression analysis in many fields, including life sciences, agriculture, forensic science, molecular diagnostics, and medicine. While SYBR Green-based qPCR is the most commonly-used platform due to its inexpensive nature and robust chemistry, quantifying the expression of genes with low abundance or RNA samples extracted from highly restricted or limited sources can be challenging because the detection sensitivity of SYBR Green-based qPCR is limited. Here, we develop a novel and effective touchdown qPCR (TqPCR) protocol by incorporating a 4-cycle touchdown stage prior to the quantification amplification stage. Using the same cDNA templates, we find that TqPCR can reduce the average Cq values for Gapdh, Rps13, and Hprt1 reference genes by 4.45, 5.47, and 4.94 cycles, respectively, when compared with conventional qPCR; the overall average Cq value reduction for the three reference genes together is 4.95. We further find that TqPCR can improve PCR amplification efficiency and thus increase detection sensitivity. When the quantification of Wnt3A-induced target gene expression in mesenchymal stem cells is analyzed, we find that, while both conventional qPCR and TqPCR can detect the up-regulation of the relatively abundant target Axin2, only TqPCR can detect the up-regulation of the lowly-expressed targets Oct4 and Gbx2. Finally, we demonstrate that the MRQ2 and MRQ3 primer pairs derived from mouse reference gene Tbp can be used to validate the RNA/cDNA integrity of qPCR samples. Taken together, our results strongly suggest that TqPCR may increase detection sensitivity and PCR amplification efficiency. Overall, TqPCR should be advantageous over conventional qPCR in expression quantification, especially when the transcripts of interest are lowly expressed, and/or the availability of total RNA is highly restricted or limited. PMID:26172450

  19. Viral diagnostics in the era of digital PCR

    PubMed Central

    Sedlak, Ruth Hall; Jerome, Keith R.

    2012-01-01

    Unlike quantitative PCR (qPCR), digital PCR (dPCR) achieves sensitive and accurate absolute quantitation of a DNA sample without the need for a standard curve. A single PCR reaction is divided into many separate reactions that each have a positive or negative signal. By applying Poisson statistics, the number of DNA molecules in the original sample is directly calculated from the number of positive and negative reactions. The recent availability of multiple commercial dPCR platforms has led to increased interest in clinical diagnostic applications, such as low viral load detection and low abundance mutant detection, where dPCR could be superior to traditional qPCR.Here we review current literature that demonstrates dPCR’s potential utility in viral diagnostics, particularly through absolute quantification of target DNA sequences and rare mutant allele detection. PMID:23182074

  20. Multiplex Amplification Refractory Mutation System PCR (ARMS-PCR) provides sequencing independent typing of canine parvovirus.

    PubMed

    Chander, Vishal; Chakravarti, Soumendu; Gupta, Vikas; Nandi, Sukdeb; Singh, Mithilesh; Badasara, Surendra Kumar; Sharma, Chhavi; Mittal, Mitesh; Dandapat, S; Gupta, V K

    2016-10-29

    Canine parvovirus-2 antigenic variants (CPV-2a, CPV-2b and CPV-2c) ubiquitously distributed worldwide in canine population causes severe fatal gastroenteritis. Antigenic typing of CPV-2 remains a prime focus of research groups worldwide in understanding the disease epidemiology and virus evolution. The present study was thus envisioned to provide a simple sequencing independent, rapid, robust, specific, user-friendly technique for detecting and typing of presently circulating CPV-2 antigenic variants. ARMS-PCR strategy was employed using specific primers for CPV-2a, CPV-2b and CPV-2c to differentiate these antigenic types. ARMS-PCR was initially optimized with reference positive controls in two steps; where first reaction was used to differentiate CPV-2a from CPV-2b/CPV-2c. The second reaction was carried out with CPV-2c specific primers to confirm the presence of CPV-2c. Initial validation of the ARMS-PCR was carried out with 24 sequenced samples and the results were matched with the sequencing results. ARMS-PCR technique was further used to screen and type 90 suspected clinical samples. Randomly selected 15 suspected clinical samples that were typed with this technique were sequenced. The results of ARMS-PCR and the sequencing matched exactly with each other. The developed technique has a potential to become a sequencing independent method for simultaneous detection and typing of CPV-2 antigenic variants in veterinary disease diagnostic laboratories globally.

  1. Multiplex Amplification Refractory Mutation System PCR (ARMS-PCR) provides sequencing independent typing of canine parvovirus.

    PubMed

    Chander, Vishal; Chakravarti, Soumendu; Gupta, Vikas; Nandi, Sukdeb; Singh, Mithilesh; Badasara, Surendra Kumar; Sharma, Chhavi; Mittal, Mitesh; Dandapat, S; Gupta, V K

    2016-12-01

    Canine parvovirus-2 antigenic variants (CPV-2a, CPV-2b and CPV-2c) ubiquitously distributed worldwide in canine population causes severe fatal gastroenteritis. Antigenic typing of CPV-2 remains a prime focus of research groups worldwide in understanding the disease epidemiology and virus evolution. The present study was thus envisioned to provide a simple sequencing independent, rapid, robust, specific, user-friendly technique for detecting and typing of presently circulating CPV-2 antigenic variants. ARMS-PCR strategy was employed using specific primers for CPV-2a, CPV-2b and CPV-2c to differentiate these antigenic types. ARMS-PCR was initially optimized with reference positive controls in two steps; where first reaction was used to differentiate CPV-2a from CPV-2b/CPV-2c. The second reaction was carried out with CPV-2c specific primers to confirm the presence of CPV-2c. Initial validation of the ARMS-PCR was carried out with 24 sequenced samples and the results were matched with the sequencing results. ARMS-PCR technique was further used to screen and type 90 suspected clinical samples. Randomly selected 15 suspected clinical samples that were typed with this technique were sequenced. The results of ARMS-PCR and the sequencing matched exactly with each other. The developed technique has a potential to become a sequencing independent method for simultaneous detection and typing of CPV-2 antigenic variants in veterinary disease diagnostic laboratories globally.

  2. Lab-on-a-chip PCR: real time PCR in miniaturized format for HLA diagnostics

    NASA Astrophysics Data System (ADS)

    Gaertner, Claudia; Becker, Holger; Hlawatsch, Nadine; Klemm, Richard; Moche, Christian; Sewart, René; Frank, Rainer; Willems, Andreas

    2014-05-01

    In case of transplantation or the identification of special metabolic diseases like coeliac disease, HLA typing has to be done fast and reliably with easy-to-handle devices by using limited amount of sample. Against this background a lab-on-a-chip device was realized enabling a fast HLA typing via miniaturized Real-time PCR. Hereby, two main process steps were combined, namely the extraction of DNA from whole blood and the amplification of the target DNA by Real-time PCR giving rise-to a semi-quantitative analysis. For the implementation of both processes on chip, a sample preparation and a real-time module were used. Sample preparation was carried out by using magnetic beads that were stored directly on chip as dry powder, together with all lysis reagents. After purification of the DNA by applying a special buffer regime, the sample DNA was transferred into the PCR module for amplification and detection. Coping with a massively increased surface-to-volume ratio, which results in a higher amount of unspecific binding on the chip surface, special additives needed to be integrated to compensate for this effect. Finally the overall procedure showed a sensitivity comparable to standard Real-time PCR but reduced the duration of analysis to significantly less than one hour. The presented work demonstrates that the combination of lab-on-a-chip PCR with direct optical read-out in a real-time fashion is an extremely promising tool for molecular diagnostics.

  3. Miniaturized detection system for handheld PCR assays

    NASA Astrophysics Data System (ADS)

    Richards, James B.; Benett, William J.; Stratton, Paul; Hadley, Dean R.; Nasarabadi, Shanavaz L.; Milanovich, Fred P.

    2000-12-01

    We have developed and delivered a four chamber, battery powered, handheld instrument referred to as the HANAA which monitors the polymerase chain reaction (PCR) process using a TaqMan based fluorescence assay. The detection system differs form standard configurations in two essential ways. First, the size is miniaturized, with a combined cycling and optics plug-in module for a duplex assay begin about the size of a small box of matches. Second, the detection/analysis system is designed to call a positive sample in real time.

  4. Determining Fungi rRNA Copy Number by PCR

    EPA Science Inventory

    The goal of this project is to improve the quantification of indoor fungal pollutants via the specific application of quantitative PCR (qPCR). Improvement will be made in the controls used in current qPCR applications. This work focuses on the use of two separate controls within ...

  5. Automated PCR setup for forensic casework samples using the Normalization Wizard and PCR Setup robotic methods.

    PubMed

    Greenspoon, S A; Sykes, K L V; Ban, J D; Pollard, A; Baisden, M; Farr, M; Graham, N; Collins, B L; Green, M M; Christenson, C C

    2006-12-20

    Human genome, pharmaceutical and research laboratories have long enjoyed the application of robotics to performing repetitive laboratory tasks. However, the utilization of robotics in forensic laboratories for processing casework samples is relatively new and poses particular challenges. Since the quantity and quality (a mixture versus a single source sample, the level of degradation, the presence of PCR inhibitors) of the DNA contained within a casework sample is unknown, particular attention must be paid to procedural susceptibility to contamination, as well as DNA yield, especially as it pertains to samples with little biological material. The Virginia Department of Forensic Science (VDFS) has successfully automated forensic casework DNA extraction utilizing the DNA IQ(trade mark) System in conjunction with the Biomek 2000 Automation Workstation. Human DNA quantitation is also performed in a near complete automated fashion utilizing the AluQuant Human DNA Quantitation System and the Biomek 2000 Automation Workstation. Recently, the PCR setup for casework samples has been automated, employing the Biomek 2000 Automation Workstation and Normalization Wizard, Genetic Identity version, which utilizes the quantitation data, imported into the software, to create a customized automated method for DNA dilution, unique to that plate of DNA samples. The PCR Setup software method, used in conjunction with the Normalization Wizard method and written for the Biomek 2000, functions to mix the diluted DNA samples, transfer the PCR master mix, and transfer the diluted DNA samples to PCR amplification tubes. Once the process is complete, the DNA extracts, still on the deck of the robot in PCR amplification strip tubes, are transferred to pre-labeled 1.5 mL tubes for long-term storage using an automated method. The automation of these steps in the process of forensic DNA casework analysis has been accomplished by performing extensive optimization, validation and testing of the

  6. Rapid direct PCR for ABO blood typing.

    PubMed

    Lee, Hwan Young; Park, Myung Jin; Kim, Na Young; Yang, Woo Ick; Shin, Kyoung-Jin

    2011-01-01

    Many different molecular typing methods have been reported to complement routine serological ABO blood typing in forensics. However, these ABO genotyping methods are often time-consuming and call for an initial DNA isolation step that requires the use of expensive kits or reagents. We report here a rapid direct ABO genotyping method that eliminates the need for DNA extraction from fresh blood, hair, and body fluid stains before PCR. Using a fast PCR instrument and an optimized polymerase, the genotyping method-which employs a multiplex allele-specific primer set for the simultaneous detection of three single-nucleotide polymorphism (SNP) sites (nucleotides 261, 526, and 803)-identifies A, B, O01/O02, O03, and cis-AB01 alleles in around 70 min from sample collection to electropherogram. Not only will this ABO genotyping method be efficiently used in forensic practice for rapid screening of samples before full-blown multilocus short tandem repeat profiling, but it will also demonstrate an example of rapid direct genotyping of SNPs that offers the advantages of time- and cost-efficiency, convenience, and reduced contamination during DNA analysis.

  7. A Guide to Using STITCHER for Overlapping Assembly PCR Applications.

    PubMed

    O'Halloran, Damien M

    2017-01-01

    Overlapping PCR is commonly used in many molecular applications that include stitching PCR fragments together, generating fluorescent transcriptional and translational fusions, inserting mutations, making deletions, and PCR cloning. Overlapping PCR is also used for genotyping and in detection experiments using techniques such as loop-mediated isothermal amplification (LAMP). STITCHER is a web tool providing a central resource for researchers conducting all types of overlapping assembly PCR experiments with an intuitive interface for automated primer design that's fast, easy to use, and freely available online.

  8. Infections and mixed infections with the selected species of Borrelia burgdorferi sensu lato complex in Ixodes ricinus ticks collected in eastern Poland: a significant increase in the course of 5 years.

    PubMed

    Wójcik-Fatla, Angelina; Zając, Violetta; Sawczyn, Anna; Sroka, Jacek; Cisak, Ewa; Dutkiewicz, Jacek

    2016-02-01

    In the years 2008-2009 and 2013-2014, 1620 and 1500 questing Ixodes ricinus ticks, respectively, were examined on the territory of the Lublin province (eastern Poland). The presence of three pathogenic species causing Lyme disease was investigated: Borrelia burgdorferi sensu stricto, B. afzelii and B. garinii. The proportion of I. ricinus ticks infected with B. burgdorferi sensu lato showed a highly significant increase between 2008-2009 and 2013-2014, from 6.0 to 15.3%. A significant increase was noted with regard to all types of infections with individual species: single (4.7-7.8%), dual (1.2-6.6%), and triple (0.1-0.9%). When expressed as the percent of all infections, the frequency of mixed infections increased from 21.4 to 49.2%. Statistical analysis performed with two methods (by calculating of odds ratios and by Fisher's exact test) showed that the frequencies of mixed infections in most cases proved to be significantly greater than expected. The strongest associations were found between B. burgdorferi s. s. and B. afzelii, and between B. burgdorferi s. s. and B. garinii. They appeared to be highly significant (P < 0.0001) when assessed by two methods for 2013-2014, and for the sum of findings for both time periods. The proportions of the individual species detected in the mixed infections in 2008-2009 and 2013-2014 revealed highly significant increases for B. burgdorferi s. s. and B. garinii (from 33.9 to 71.1% and from 18.2 to 82.9%, respectively), and an insignificant decrease for B. afzelii (from 51.4 to 41.6%). The proportions of the species B. burgdorferi s. s., B. afzelii and B. garinii (with combined single and mixed infections) for 2008-2009 and 2013-2014 were: 51.2/44.0 %, 30.6/24.9% and 18.2/31.1%, respectively. In conclusion, our results seem to indicate the detrimental trend of the increasing infection rate of I. ricinus ticks with B. burgdorferi s. l. in eastern Poland, and dramatic enhancement of mixed infections with individual species, which

  9. Accurate quantification of supercoiled DNA by digital PCR.

    PubMed

    Dong, Lianhua; Yoo, Hee-Bong; Wang, Jing; Park, Sang-Ryoul

    2016-04-11

    Digital PCR (dPCR) as an enumeration-based quantification method is capable of quantifying the DNA copy number without the help of standards. However, it can generate false results when the PCR conditions are not optimized. A recent international comparison (CCQM P154) showed that most laboratories significantly underestimated the concentration of supercoiled plasmid DNA by dPCR. Mostly, supercoiled DNAs are linearized before dPCR to avoid such underestimations. The present study was conducted to overcome this problem. In the bilateral comparison, the National Institute of Metrology, China (NIM) optimized and applied dPCR for supercoiled DNA determination, whereas Korea Research Institute of Standards and Science (KRISS) prepared the unknown samples and quantified them by flow cytometry. In this study, several factors like selection of the PCR master mix, the fluorescent label, and the position of the primers were evaluated for quantifying supercoiled DNA by dPCR. This work confirmed that a 16S PCR master mix avoided poor amplification of the supercoiled DNA, whereas HEX labels on dPCR probe resulted in robust amplification curves. Optimizing the dPCR assay based on these two observations resulted in accurate quantification of supercoiled DNA without preanalytical linearization. This result was validated in close agreement (101~113%) with the result from flow cytometry.

  10. Accurate quantification of supercoiled DNA by digital PCR

    PubMed Central

    Dong, Lianhua; Yoo, Hee-Bong; Wang, Jing; Park, Sang-Ryoul

    2016-01-01

    Digital PCR (dPCR) as an enumeration-based quantification method is capable of quantifying the DNA copy number without the help of standards. However, it can generate false results when the PCR conditions are not optimized. A recent international comparison (CCQM P154) showed that most laboratories significantly underestimated the concentration of supercoiled plasmid DNA by dPCR. Mostly, supercoiled DNAs are linearized before dPCR to avoid such underestimations. The present study was conducted to overcome this problem. In the bilateral comparison, the National Institute of Metrology, China (NIM) optimized and applied dPCR for supercoiled DNA determination, whereas Korea Research Institute of Standards and Science (KRISS) prepared the unknown samples and quantified them by flow cytometry. In this study, several factors like selection of the PCR master mix, the fluorescent label, and the position of the primers were evaluated for quantifying supercoiled DNA by dPCR. This work confirmed that a 16S PCR master mix avoided poor amplification of the supercoiled DNA, whereas HEX labels on dPCR probe resulted in robust amplification curves. Optimizing the dPCR assay based on these two observations resulted in accurate quantification of supercoiled DNA without preanalytical linearization. This result was validated in close agreement (101~113%) with the result from flow cytometry. PMID:27063649

  11. Droplet digital PCR for absolute quantification of pathogens.

    PubMed

    Gutiérrez-Aguirre, Ion; Rački, Nejc; Dreo, Tanja; Ravnikar, Maja

    2015-01-01

    The recent advent of different digital PCR (dPCR) platforms is enabling the expansion of this technology for research and diagnostic applications worldwide. The main principle of dPCR, as in other PCR-based methods including quantitative PCR (qPCR), is the specific amplification of a nucleic acid target. The distinctive feature of dPCR is the separation of the reaction mixture into thousands to millions of partitions which is followed by a real time or end point detection of the amplification. The distribution of target sequences into partitions is described by the Poisson distribution, thus allowing accurate and absolute quantification of the target from the ratio of positive against all partitions at the end of the reaction. This omits the need to use reference materials with known target concentrations and increases the accuracy of quantification at low target concentrations compared to qPCR. dPCR has also shown higher resilience to inhibitors in a number of different types of samples. In this chapter we describe the droplet digital PCR (ddPCR) workflow for the detection and quantification of pathogens using the droplet digital Bio-Rad platform QX100. We present as an example the quantification of the quarantine plant pathogenic bacterium, Erwinia amylovora.

  12. Application of real time PCR for diagnosis of Swine Dysentery.

    PubMed

    Akase, Satoru; Uchitani, Yumi; Sohmura, Yoshiko; Tatsuta, Keikichi; Sadamasu, Kenji; Adachi, Yoshikazu

    2009-03-01

    Evaluation of a genetic diagnostic technique using real time PCR of Swine Dysentery (SD) was performed using nox primers. Culture, ordinary PCR and real time PCR were compared in this experiment. Sixty-seven specimens from pigs with clinical signs of SD brought to a slaughterhouse in Shibaura, Tokyo, were used. B. hyodysenteriae was isolated from 49 of the pigs, was detected by ordinary PCR in 49 of the pigs and was detected by real time PCR in 54 of the pigs. Furthermore, we were able to determine the numbers of B. hyodysenteriae cells in all positive specimens by real time PCR. The rapid diagnostic technique established in this experiment was useful for detection of B. hyodysenteriae because it was more effective than ordinary PCR and culture.

  13. cDNA amplification by SMART-PCR and suppression subtractive hybridization (SSH)-PCR.

    PubMed

    Hillmann, Andrew; Dunne, Eimear; Kenny, Dermot

    2009-01-01

    The comparison of two RNA populations that differ from the effects of a single-independent variable, such as a drug treatment or a specific genetic defect, can identify differences in the abundance of specific transcripts that vary in a population-dependent manner. There are a variety of methods for identifying differentially expressed genes, including microarray, SAGE, qRT-PCR, and DDGE. This protocol describes a potentially less sensitive yet relatively easy and cost-effective alternative that does not require prior knowledge of the transcriptomes under investigation and is particularly applicable when minimal levels of starting material, RNA, are available. RNA input can often be a limiting factor when analyzing RNA from, for example, rigorously purified blood cells. This protocol describes the use of SMART-PCR to amplify cDNA from sub-microgram levels of RNA. The amplified cDNA populations under comparison are then subjected to suppression subtractive hybridization (SSH-PCR), a technique that couples subtractive hybridization with suppression PCR to selectively amplify fragments of differentially expressed genes. The final products are cDNA populations enriched for significantly over-represented transcripts in either of the two input RNA preparations. These cDNA populations may then be cloned to make subtracted cDNA libraries and/or used as probes to screen subtracted cDNA, global cDNA, or genomic DNA libraries.

  14. Simultaneous detection of ricin and abrin DNA by real-time PCR (qPCR).

    PubMed

    Felder, Eva; Mossbrugger, Ilona; Lange, Mirko; Wölfel, Roman

    2012-09-01

    Ricin and abrin are two of the most potent plant toxins known and may be easily obtained in high yield from the seeds using rather simple technology. As a result, both toxins are potent and available toxins for criminal or terrorist acts. However, as the production of highly purified ricin or abrin requires sophisticated equipment and knowledge, it may be more likely that crude extracts would be used by non-governmental perpetrators. Remaining plant-specific nucleic acids in these extracts allow the application of a real-time PCR (qPCR) assay for the detection and identification of abrin or ricin genomic material. Therefore, we have developed a duplex real-time PCR assays for simultaneous detection of ricin and abrin DNA based on the OmniMix HS bead PCR reagent mixture. Novel primers and hybridization probes were designed for detection on a SmartCycler instrument by using 5'-nuclease technology. The assay was thoroughly optimized and validated in terms of analytical sensitivity. Evaluation of the assay sensitivity by probit analysis demonstrated a 95% probability of detection at 3 genomes per reaction for ricin DNA and 1.2 genomes per reaction for abrin DNA. The suitability of the assays was exemplified by detection of ricin and abrin contaminations in a food matrix.

  15. Rapid micro-PCR system for hepatitis C virus amplification

    NASA Astrophysics Data System (ADS)

    Lin, Yu-Cheng; Huang, Ming-Yuan; Young, Kung-Chia; Chang, Ting-Tsung; Wu, Ching-Yi

    2000-08-01

    A rapid micro-polymerase chain reaction ((mu) -PCR) system was integrated to amplify the complementary DNA (cDNA) molecules of hepatitis C virus (HCV). This system consists of a rapid thermal cycling system and a (mu) PCR chip fabricated by MEMS fabrication techniques. This rapid (mu) PCR system is verified by using serum samples from patients with chronic hepatitis C. The HCV amplicon of the rapid (mu) PCR system was analyzed by slab gel electrophoresis with separation of DNA marker in parallel. The (mu) PCR chip was fabricated on silicon wafer and Pyrex glass using photolithography, wet etching, and anodic bonding methods. Using silicon material to fabricate the raction well improves the temperature uniformity of sample and helps to reach the desired temperature faster. The rapid close loop thermal cycling system comprises power supplies, a thermal generator, a computer control PID controller, and a data acquisition subsystem. The thermoelectric (T.E.) cooler is used to work as the thermal generator and a heat sink by controlling the polarity of supplied power. The (mu) PCR system was verified with traditional PCR equipment by loading the same PCR mixture with HCV cDNA and running the same cycle numbers, then comparing both HCV amplicon slab gel electrophoresis. The HCV amplicon from the (mu) PCR system shows a DNA fragment with an expected size of 145 base pairs. The background is lower with the (mu) PCR system than that with the tradional PCR equipment. Comparing the traditional PCR equipment which spends 5.5 hours for 30 cycles to gain the detectable amount of HCV amplicon in slab gel separation, this (mu) PCR system takes 30 minutes to finish the 30 thermal cycles. This work has demonstrated that this rapid (mu) PCR system can provide rapid heat generation and dissipation, improved temperature uniformity in DNA amplification.

  16. STS-102 MPLM Leonardo moves into PCR

    NASA Technical Reports Server (NTRS)

    2001-01-01

    KENNEDY SPACE CENTER, Fla. -- In the payload changeout room on the Rotating Service Structure, Launch Pad 39B, workers move the Multi-Purpose Logistics Module Leonardo out of the payload canister. From the PCR Leonardo then will be transferred into Space Shuttle Discovery'''s payload bay. One of Italy'''s major contributions to the International Space Station program, Leonardo is a reusable logistics carrier. It is the primary delivery system used to resupply and return Station cargo requiring a pressurized environment. Leonardo is the primary payload on mission STS-102 and will deliver up to 10 tons of laboratory racks filled with equipment, experiments and supplies for outfitting the newly installed U.S. Laboratory Destiny. STS-102 is scheduled to launch March 8 at 6:45 a.m. EST.

  17. Comparison of droplet digital PCR to real-time PCR for quantification of hepatitis B virus DNA.

    PubMed

    Tang, Hui; Cai, Qingchun; Li, Hu; Hu, Peng

    2016-06-16

    Quantitative real-time PCR (qPCR) has been widely implemented for clinical hepatitis B viral load testing, but a lack of standardization and relatively poor precision hinder its usefulness. Droplet digital PCR (ddPCR) is a promising tool that offers high precision and direct quantification. In this study, we compared the ddPCR QX100 platform by Bio-Rad with the CFX384 Touch Real-Time PCR Detection System (Bio-Rad, USA) to detect serial plasmid DNA dilutions of known concentrations as well as HBV DNA extracted from patient serum samples. Both methods showed a high degree of linearity and quantitative correlation. However, ddPCR assays generated more reproducible results and detected lower copy numbers than qPCR assays. Patient sample quantifications by ddPCR and qPCR were highly agreeable based on the Bland-Altman analysis. Collectively, our findings demonstrate that ddPCR offers improved analytical sensitivity and specificity for HBV measurements and is suitable for clinical HBV detection.

  18. Sensitivity of PCR and real-time PCR for the diagnosis of human visceral leishmaniasis using peripheral blood

    PubMed Central

    da Costa Lima, Manoel Sebastião; Zorzenon, Denielly Christina Rodrigues; Dorval, Maria Elizabeth Cavalheiros; Pontes, Elenir Rose Jardim Cury; Oshiro, Elisa Teruya; Cunha, Rodrigo; Andreotti, Renato; Matos, Maria de Fatima Cepa

    2013-01-01

    Objective To evaluate the effectiveness of PCR and real-time PCR for the diagnosis of human visceral leishmaniasis using peripheral blood samples. Methods DNA extraction was performed using Promega Wizard® Genomic kits. PCR employing RV1/RV2 primers yielded 145-bp amplicons. Real-time PCR was performed with the same primers and SYBR Green ROX Plus mix. These techniques were used to analyze 100 peripheral blood samples from patients with clinical signs of the disease. Results The sensitivity and specificity levels were 91,3%% and 29,6%, respectively, for real-time PCR and 97.78% and 61.82%, respectively, for PCR. Conclusions Real-time PCR proved to be a satisfactory method for the diagnosis of human visceral leishmaniasis.

  19. COMPARISON OF 16S rRNA-PCR-RFLP, LipL32-PCR AND OmpL1-PCR METHODS IN THE DIAGNOSIS OF LEPTOSPIROSIS

    PubMed Central

    GÖKMEN, Tülin GÜVEN; SOYAL, Ayben; KALAYCI, Yıldız; ÖNLEN, Cansu; KÖKSAL, Fatih

    2016-01-01

    SUMMARY Leptospirosis is still one of the most important health problems in developing countries located in humid tropical and subtropical regions. Human infections are generally caused by exposure to water, soil or food contaminated with the urine of infected wild and domestic animals such as rodents and dogs. The clinical course of leptospirosis is variable and may be difficult to distinguish from many other infectious diseases. The dark-field microscopy (DFM), serology and nucleic acid amplification techniques are used to diagnose leptospirosis, however, a distinctive standard reference method is still lacking. Therefore, in this study, we aimed to determine the presence of Leptospira spp., to differentiate the pathogenic L. interrogans and the non-pathogenic L. biflexa, and also to determine the sensitivity and specificity values of molecular methods as an alternative to conventional ones. A total of 133 serum samples, from 47 humans and 86 cattle were evaluated by two conventional tests: the Microagglutination Test (MAT) and the DFM, as well as three molecular methods, the 16S rRNA-PCR followed by Restriction Fragment Lenght Polymorphism (RFLP) of the amplification products 16S rRNA-PCR-RFLP, LipL32-PCR and OmpL1-PCR. In this study, for L. interrogans, the specificity and sensitivity rates of the 16S rRNA-PCR and the LipL32-PCR were considered similar (100% versus 98.25% and 100% versus 98.68%, respectively). The OmpL1-PCR was able to classify L. interrogans into two intergroups, but this PCR was less sensitive (87.01%) than the other two PCR methods. The 16S rRNA-PCR-RFLP could detect L. biflexa DNA, but LipL32-PCR and OmpL1-PCR could not. The 16S rRNA-PCR-RFLP provided an early and accurate diagnosis and was able to distinguish pathogenic and non-pathogenic Leptospira species, hence it may be used as an alternative method to the conventional gold standard techniques for the rapid disgnosis of leptospirosis. PMID:27680169

  20. Multiplex enrichment quantitative PCR (ME-qPCR): a high-throughput, highly sensitive detection method for GMO identification.

    PubMed

    Fu, Wei; Zhu, Pengyu; Wei, Shuang; Zhixin, Du; Wang, Chenguang; Wu, Xiyang; Li, Feiwu; Zhu, Shuifang

    2017-04-01

    Among all of the high-throughput detection methods, PCR-based methodologies are regarded as the most cost-efficient and feasible methodologies compared with the next-generation sequencing or ChIP-based methods. However, the PCR-based methods can only achieve multiplex detection up to 15-plex due to limitations imposed by the multiplex primer interactions. The detection throughput cannot meet the demands of high-throughput detection, such as SNP or gene expression analysis. Therefore, in our study, we have developed a new high-throughput PCR-based detection method, multiplex enrichment quantitative PCR (ME-qPCR), which is a combination of qPCR and nested PCR. The GMO content detection results in our study showed that ME-qPCR could achieve high-throughput detection up to 26-plex. Compared to the original qPCR, the Ct values of ME-qPCR were lower for the same group, which showed that ME-qPCR sensitivity is higher than the original qPCR. The absolute limit of detection for ME-qPCR could achieve levels as low as a single copy of the plant genome. Moreover, the specificity results showed that no cross-amplification occurred for irrelevant GMO events. After evaluation of all of the parameters, a practical evaluation was performed with different foods. The more stable amplification results, compared to qPCR, showed that ME-qPCR was suitable for GMO detection in foods. In conclusion, ME-qPCR achieved sensitive, high-throughput GMO detection in complex substrates, such as crops or food samples. In the future, ME-qPCR-based GMO content identification may positively impact SNP analysis or multiplex gene expression of food or agricultural samples. Graphical abstract For the first-step amplification, four primers (A, B, C, and D) have been added into the reaction volume. In this manner, four kinds of amplicons have been generated. All of these four amplicons could be regarded as the target of second-step PCR. For the second-step amplification, three parallels have been taken for

  1. Comparison of the immunofluorescence assay with RT-PCR and nested PCR in the diagnosis of canine distemper.

    PubMed

    Jóźwik, A; Frymus, T

    2005-05-01

    Two pairs of primers were prepared, both localized within the sequences of the nucleoprotein gene (NP) of canine distemper virus (CDV). A number of experiments were done to optimize the conditions of RT-PCR and nested PCR methods. The nucleic acids of the Onderstepoort, Rockborn, Snyder Hill and Lederle strains of CDV could be detected with these primers. However, they did not react with the sequences of the Edmonston strain of the measles virus. The detection limit for RT-PCR was 10 TCID50 and for nested PCR 0.1 TCID50 of CDV. The RT-PCR was able to demonstrate the nucleic acid of CDV in the blood of all seven puppies vaccinated with a modified live virus. Blood samples of 23 dogs clinically suspected of distemper were examined by RT-PCR combined with nested PCR, and the results were compared with the detection of the CDV antigen in the smears from the mucous membranes by the direct immunofluorescence (IF) test. Of the 23 dogs, 12 were positive in nested PCR, six in the IF assay, and only two in single RT-PCR. It is concluded that nested PCR seems to be the most sensitive method for ante-mortem diagnosis of canine distemper, especially in its subacute or chronic forms.

  2. Microfluidics-Based PCR for Fusion Transcript Detection.

    PubMed

    Chen, Hui

    2016-01-01

    The microfluidic technology allows the production of network of submillimeter-size fluidic channels and reservoirs in a variety of material systems. The microfluidic-based polymerase chain reaction (PCR) allows automated multiplexing of multiple samples and multiple assays simultaneously within a network of microfluidic channels and chambers that are co-ordinated in controlled fashion by the valves. The individual PCR reaction is performed in nanoliter volume, which allows testing on samples with limited DNA and RNA. The microfluidics devices are used in various types of PCR such as digital PCR and single molecular emulsion PCR for genotyping, gene expression, and miRNA expression. In this chapter, the use of a microfluidics-based PCR for simultaneous screening of 14 known fusion transcripts in patients with leukemia is described.

  3. Misuse of PCR assay for diagnosis of mollusc protistan infections.

    PubMed

    Burreson, Eugene M

    2008-06-19

    Polymerase chain reaction (PCR) assays are useful tools for pathogen surveillance, but they are only proxy indications of pathogen presence in that they detect a DNA sequence. To be useful for detection of actual infections, PCR assays must be thoroughly tested for sensitivity and specificity, and ultimately validated against a technique, typically histology, which allows visualization of the parasite in host tissues. There is growing use of PCR assays for pathogen surveillance, but too often the assumption is made that a positive PCR result verifies an infection in a tested host. This assumption is valid only if the assay has been properly validated for the geographic area and for the hosts examined. Researchers should interpret unvalidated PCR assay results with caution, and editors and reviewers should insist that robust validations support all assertions that PCR results confirm infections.

  4. Combined Overlap Extension PCR Method for Improved Site Directed Mutagenesis

    PubMed Central

    Chong, Nikson Fatt-Ming

    2016-01-01

    The combined overlap extension PCR (COE-PCR) method developed in this work combines the strengths of the overlap extension PCR (OE-PCR) method with the speed and ease of the asymmetrical overlap extension (AOE-PCR) method. This combined method allows up to 6 base pairs to be mutated at a time and requires a total of 40–45 PCR cycles. A total of eight mutagenesis experiments were successfully carried out, with each experiment mutating between two to six base pairs. Up to four adjacent codons were changed in a single experiment. This method is especially useful for codon optimization, where doublet or triplet rare codons can be changed using a single mutagenic primer set, in a single experiment. PMID:27995143

  5. Optimized MOL-PCR for Characterization of Microbial Pathogens.

    PubMed

    Wuyts, Véronique; Roosens, Nancy H C; Bertrand, Sophie; Marchal, Kathleen; De Keersmaecker, Sigrid C J

    2016-01-06

    Characterization of microbial pathogens is necessary for surveillance, outbreak detection, and tracing of outbreak sources. This unit describes a multiplex oligonucleotide ligation-PCR (MOL-PCR) optimized for characterization of microbial pathogens. With MOL-PCR, different types of markers, like unique sequences, single-nucleotide polymorphisms (SNPs) and indels, can be simultaneously analyzed in one assay. This assay consists of a multiplex ligation for detection of the markers, a singleplex PCR for signal amplification, and hybridization to MagPlex-TAG beads for readout on a Luminex platform after fluorescent staining. The current protocol describes the MOL-PCR, as well as methods for DNA isolation, probe design, and data interpretation and it is based on an optimized MOL-PCR assay for subtyping of Salmonella Typhimurium.

  6. Forensic implications of PCR inhibition--A review.

    PubMed

    Alaeddini, Reza

    2012-05-01

    Polymerase chain reaction (PCR) is currently the method of choice for the identification of human remains in forensic coursework. DNA samples from crime scenes often contain co-purified impurities which inhibit PCR. PCR inhibition is the most common cause of PCR failure when adequate copies of DNA are present. Inhibitors have been routinely reported in forensic investigations of DNA extracted from a variety of templates. Humic compounds, a series of substances produced during decay process have been considered as the materials contaminating DNA in soil, natural waters and recent sediments. Those compounds have been frequently assigned as PCR inhibitors. The current report reviews the characteristics of PCR inhibition, including the proposed mechanisms of inhibition, detection methods and the available technologies to remove or overcome the inhibitory activities.

  7. Blood grouping based on PCR methods and agarose gel electrophoresis.

    PubMed

    Sell, Ana Maria; Visentainer, Jeane Eliete Laguila

    2015-01-01

    The study of erythrocyte antigens continues to be an intense field of research, particularly after the development of molecular testing methods. More than 300 specificities have been described by the International Society for Blood Transfusion as belonging to 33 blood group systems. The polymerase chain reaction (PCR) is a central tool for red blood cells (RBC) genotyping. PCR and agarose gel electrophoresis are low cost, easy, and versatile in vitro methods for amplifying defined target DNA (RBC polymorphic region). Multiplex-PCR, AS-PCR (Specific Allele Polymerase Chain Reaction), and RFLP-PCR (Restriction Fragment Length Polymorphism-Polymerase Chain Reaction) techniques are usually to identify RBC polymorphisms. Furthermore, it is an easy methodology to implement. This chapter describes the PCR methodology and agarose gel electrophoresis to identify the polymorphisms of the Kell, Duffy, Kidd, and MNS blood group systems.

  8. Direct PCR Improves the Recovery of DNA from Various Substrates.

    PubMed

    Templeton, Jennifer E L; Taylor, Duncan; Handt, Oliva; Skuza, Pawel; Linacre, Adrian

    2015-11-01

    This study reports on the comparison of a standard extraction process with the direct PCR approach of processing low-level DNA swabs typical in forensic investigations. Varying concentrations of control DNA were deposited onto three commonly encountered substrates, brass, plastic, and glass, left to dry, and swabbed using premoistened DNA-free nylon FLOQswabs(™) . Swabs (n = 90) were either processed using the DNA IQ(™) kit or, for direct PCR, swab fibers (~2 mm(2) ) were added directly to the PCR with no prior extraction. A significant increase in the height of the alleles (p < 0.005) was observed when using the direct PCR approach over the extraction methodology when controlling for surface type and mass of DNA deposited. The findings indicate the potential use of direct PCR for increasing the PCR product obtained from low-template DNA samples in addition to minimizing contamination and saving resources.

  9. Biofilm formation by Borrelia burgdorferi sensu lato.

    PubMed

    Timmaraju, Venkata Arun; Theophilus, Priyanka A S; Balasubramanian, Kunthavai; Shakih, Shafiq; Luecke, David F; Sapi, Eva

    2015-08-01

    Bacterial biofilms are microbial communities held together by an extracellular polymeric substance matrix predominantly composed of polysaccharides, proteins and nucleic acids. We had previously shown that Borrelia burgdorferi sensu stricto, the causative organism of Lyme disease in the United States is capable of forming biofilms in vitro. Here, we investigated biofilm formation by B. afzelii and B. garinii, which cause Lyme disease in Europe. Using various histochemistry and microscopy techniques, we show that B. afzelii and B. garinii form biofilms, which resemble biofilms formed by B. burgdorferi sensu stricto. High-resolution atomic force microscopy revealed similarities in the ultrastructural organization of the biofilms form by three Borrelia species. Histochemical experiments revealed a heterogeneous organization of exopolysaccharides among the three Borrelia species. These results suggest that biofilm formation might be a common trait of Borrelia genera physiology.

  10. Specific detection of chikungunya virus using a RT-PCR/nested PCR combination.

    PubMed

    Pfeffer, M; Linssen, B; Parke, M D; Kinney, R M

    2002-02-01

    Chikungunya (CHIK) virus is enzootic in many countries in Asia and throughout tropical Africa. In Asia the virus is transmitted from primates to humans almost exclusively by Aedes aegypti, while various aedine mosquito species are responsible for human infections in Africa. The clinical picture is characterized by a sudden onset of fever, rash and severe pain in the joints which may persist in a small proportion of cases. Although not listed as a haemorrhagic fever virus, illness caused by CHIK virus can be confused with diseases such as dengue or yellow fever, based on the similarity of the symptoms. Thus, laboratory confirmation of suspected cases is required to launch control measures during an epidemic. CHIK virus diagnosis based on virus isolation is very sensitive, yet requires at least a week in conjunction with virus identification using monovalent sera. We developed a reverse transcription-polymerase chain reaction (RT-PCR) assay which amplifies a 427-bp fragment of the E2 gene. Specificity was confirmed by testing representative strains of all known alphavirus species. To verify further the viral origin of the amplicon and to enhance sensitivity, a nested PCR was performed subsequently. This RT-PCR/nested PCR combination was able to amplify a CHIK virus-specific 172-bp amplicon from a sample containing as few as 10 genome equivalents. This assay was successfully applied to four CHIK virus isolates from Asia and Africa as well as to a vaccine strain developed by USAMRIID. Our method can be completed in less than two working days and may serve as a sensitive alternative in CHIK virus diagnosis.

  11. A comparison of DNA methylation specific droplet digital PCR (ddPCR) and real time qPCR with flow cytometry in characterizing human T cells in peripheral blood.

    PubMed

    Wiencke, John K; Bracci, Paige M; Hsuang, George; Zheng, Shichun; Hansen, Helen; Wrensch, Margaret R; Rice, Terri; Eliot, Melissa; Kelsey, Karl T

    2014-10-01

    Quantitating the copy number of demethylated CpG promoter sites of the CD3Z gene can be used to estimate the numbers and proportions of T cells in human blood and tissue. Quantitative methylation specific PCR (qPCR) is useful for studying T cells but requires extensive calibration and is imprecise at low copy numbers. Here we compared the performance of a new digital PCR platform (droplet digital PCR or ddPCR) to qPCR using bisulfite converted DNA from 157 blood specimens obtained from ambulatory care controls and patients with primary glioma. We compared both ddPCR and qPCR with conventional flow cytometry (FACS) evaluation of CD3 positive T cells. Repeated measures on the same blood sample revealed ddPCR to be less variable than qPCR. Both qPCR and ddPCR correlated significantly with FACS evaluation of peripheral blood CD3 counts and CD3/total leukocyte values. However, statistical measures of agreement showed that linear concordance was stronger for ddPCR than for qPCR and the absolute values were closer to FACS for ddPCR. Both qPCR and ddPCR could distinguish clinically significant differences in T cell proportions and performed similarly to FACS. Given the higher precision, greater accuracy, and technical simplicity of ddPCR, this approach appears to be a superior DNA methylation based method than conventional qPCR for the assessment of T cells.

  12. Loop-linker PCR: an advanced PCR technique for genome walking.

    PubMed

    Trinh, Quoclinh; Shi, Hui; Xu, Wentao; Hao, Junran; Luo, Yunbo; Huang, Kunlun

    2012-10-01

    In this article, we developed a novel PCR method, termed loop-linker PCR, to isolate flanking sequences in transgenic crops. The novelty of this approach is its use of a stem-loop structure to design a loop-linker adapter. The adapter is designed to form a nick site when ligated with restricted DNA. This modification not only can prevent the self-ligation of adapters but also promotes the elongation of the 3' end of the loop-linker adapter to generate a stem-loop structure in the ligation products. Moreover, the suppressive effect of the stem-loop structure decreases nonspecific amplification and increases the success rate of the approach; all extension products will suppress exponential amplification except from the ligation product that contains the specific primer binding site. Using this method, 442, 1830, 107, and 512 bp left border flanking sequences were obtained from the transgenic maizes LY038, DAS-59122-7, Event 3272, and the transgenic soybean MON89788, respectively. The experimental results demonstrated that loop-linker PCR is an efficient, reliable, and cost-effective method for identifying flanking sequences in transgenic crops and could be applied for other genome walking applications.

  13. Reconstruction of the original mycoflora in pelleted feed by PCR-SSCP and qPCR.

    PubMed

    Dorn-In, Samart; Fahn, Carmen; Hölzel, Christina S; Wenz, Sebastian; Hartwig, Isabella; Schwaiger, Karin; Bauer, Johann

    2014-10-01

    Ground feeds for pigs were investigated for fungal contamination before and after pelleting (subsamples in total n = 24) by cultural and molecular biological methods. A fungal-specific primer pair ITS1/ITS5.8R was used to amplify fungal DNA; PCR products were processed for the PCR-SSCP method. In the resulting acrylamide gel, more than 85% of DNA bands of ground feeds were preserved after pelleting. Twenty-two DNA bands were sequenced; all represented fungal DNA. The level of fungal DNA in ground feed samples was equivalent to 4.77-5.69 log10  CFU g(-1) , calculated by qPCR using a standard curve of Aspergillus flavus. In pelleted feed, the level of fungal DNA was in average ± 0.07 log10 different from ground feed. Quantified by cultural methods, the fresh ground feeds contained up to 4.51 log10  CFU g(-1) culturable fungi, while there was < 2.83 log10  CFU g(-1) detected in pelleted feeds. This result shows that, while the process of pelleting reduced the amount of living fungi dramatically, it did not affect the total fungal DNA in feed. Thus, the described methodology was able to reconstruct the fungal microbiota in feeds and reflected a considerable fungal contamination of raw materials such as grains.

  14. The inhibitory effect of pentobarbitone on reverse transcription-PCR.

    PubMed

    Hyun, Changbaig; Filippich, Lucio John; Hughes, Ian

    2005-01-31

    Pentobarbitone sodium (Sodium 5-ethyl-5[1-methylbutyl]-pentobarbitone) is a short-acting barbiturate that is commonly used to euthanase animals. As part of our studies into the molecular genetics of copper toxicosis in Bedlington terrier dogs, reverse-transcription (RT)-PCR was noted to always fail on RNA samples collected from livers of dogs sacrificed by pentobarbitone injection. When samples were collected without pentobarbitone, however, RT-PCR was always successful. We suspected the possible inhibition by pentobarbitone sodium of either reverse transcriptase or Taq polymerase. In vitro studies showed that pentobarbitone interference of PCR occurred at >4 microg/microl. To identify if pentobarbitone produced competitive inhibition, each components (Taq polymerase, MgCl(2), dNTP, etc.) of the PCR was individually altered. However, inhibition still persisted, suggesting that multiple PCR components may be affected. Also it was shown that pentobarbitone interference was not dependent on the PCR product size. Simple dilution of pentobarbitone contaminated DNA solutions, and the addition of bovine serum albumin (BSA) to the PCR mix overcame pentobarbitone interference. In vivo, PCR by pentobarbitone was found to be compounded by high DNA concentration and pentobarbitone contamination. In addition, both high DNA concentration and pentobarbitone contamination could be overcome through dilution and the addition of BSA. Further work is required to quantify pentobarbitone concentration in the liver-extracted DNA and RNA samples before this inhibition effect on PCR can be fully elucidated.

  15. Buoyancy-Driven Polymerase Chain Reaction (PCR) Devices

    SciTech Connect

    Ness, K D; Wheeler, E K; Benett, W; Stratton, P; Christian, A; Chen, A; Ortega, J; Weisgraber, T H; Goodson, K E

    2004-09-28

    Polymerase chain reaction (PCR) facilitates DNA detection by significantly increasing the concentration of specific DNA segments. A new class of PCR instruments uses a buoyancy-driven re-circulating flow to thermally cycle the DNA sample and benefits from reduced cycle times, low sample volumes, a miniaturized format, and low power consumption. This paper analyzes a specific buoyancy PCR device in a micro-channel ''race-track'' geometry to determine key parameters about PCR cycle times and other figures of merit as functions of device dimensions. The 1-D model balances the buoyancy driving force with frictional losses. A hydrostatic pressure imbalance concept is used between the left and right sides of the fluid loop to calculate the buoyancy driving force. Velocity and temperature distributions within the channels are determined from two-dimensional analysis of the channel section, with developing region effects included empirically through scaled values of the local Nusselt number. Good agreement between four independent verification steps validate the 1-D simulation approach: (1) analytical expressions for the thermal entrance length are compared against, (2) comparison with a full 3-D finite element simulation, (3) comparison with an experimental flow field characterization, and (4) calculation of the minimum PCR runtime required to get a positive PCR signal from the buoyancy-driven PCR device. The 1-D approach closely models an actual buoyancy-driven PCR device and can further be used as a rapid design tool to simulate buoyancy PCR flows and perform detailed design optimizations studies.

  16. Quantifying RNA allelic ratios by microfluidic multiplex PCR and sequencing.

    PubMed

    Zhang, Rui; Li, Xin; Ramaswami, Gokul; Smith, Kevin S; Turecki, Gustavo; Montgomery, Stephen B; Li, Jin Billy

    2014-01-01

    We developed a targeted RNA sequencing method that couples microfluidics-based multiplex PCR and deep sequencing (mmPCR-seq) to uniformly and simultaneously amplify up to 960 loci in 48 samples independently of their gene expression levels and to accurately and cost-effectively measure allelic ratios even for low-quantity or low-quality RNA samples. We applied mmPCR-seq to RNA editing and allele-specific expression studies. mmPCR-seq complements RNA-seq for studying allelic variations in the transcriptome.

  17. 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.

  18. Comparison of droplet digital PCR with quantitative real-time PCR for determination of zygosity in transgenic maize.

    PubMed

    Xu, Xiaoli; Peng, Cheng; Wang, Xiaofu; Chen, Xiaoyun; Wang, Qiang; Xu, Junfeng

    2016-12-01

    This study evaluated the applicability of droplet digital PCR (ddPCR) as a tool for maize zygosity determination using quantitative real-time PCR (qPCR) as a reference technology. Quantitative real-time PCR is commonly used to determine transgene copy number or GMO zygosity characterization. However, its effectiveness is based on identical reaction efficiencies for the transgene and the endogenous reference gene. Additionally, a calibrator sample should be utilized for accuracy. Droplet digital PCR is a DNA molecule counting technique that directly counts the absolute number of target and reference DNA molecules in a sample, independent of assay efficiency or external calibrators. The zygosity of the transgene can be easily determined using the ratio of the quantity of the target gene to the reference single copy endogenous gene. In this study, both the qPCR and ddPCR methods were used to determine insect-resistant transgenic maize IE034 zygosity. Both methods performed well, but the ddPCR method was more convenient because of its absolute quantification property.

  19. Hypervariable spacer regions are good sites for developing specific PCR-RFLP markers and PCR primers for screening actinorhizal symbionts.

    PubMed

    Varehese, Rajani; Chauhan, Vineeta S; Misra, Arvind K

    2003-06-01

    While the ribosomal RNA like highly conserved genes are good molecular chronometers for establishing phylogenetic relationships, they can also be useful in securing the amplification of adjoining hyper-variable regions. These regions can then be used for developing specific PCR primers or PCR-RFL profiles to be used as molecular markers. We report here the use of ITS region of rrn operon of Frankia for developing PCR-RFL profiles capable of discriminating between closely related frankiae. We have also made use of the ITS1 region of the nuclear rrn operon of Alnus nepalensis (D Don) for designing a PCR primer for specific amplification of nuclear DNA of this tree.

  20. Real-time PCR for the detection of Salmonella spp. in food: An alternative approach to a conventional PCR system suggested by the FOOD-PCR project.

    PubMed

    Hein, Ingeborg; Flekna, Gabriele; Krassnig, Martina; Wagner, Martin

    2006-09-01

    A real-time PCR assay using non-patented primers and a TaqMan probe for the detection and quantification of Salmonella spp. is presented. The assay is based on an internationally validated conventional PCR system, which was suggested as a standard method for the detection of Salmonella spp. in the FOOD-PCR project. The assay was sensitive and specific. Consistent detection of 9.5 genome equivalents per PCR reaction was achieved, whereas samples containing an average of 0.95 genome equivalents per reaction were inconsistently positive. The assay performed equally well as a commercially available real-time PCR assay and allowed sensitive detection of Salmonella spp. in artificially contaminated food. After enrichment for 16 h in buffered peptone water (BPW) or universal pre-enrichment broth (UPB) 2.5 CFU/25 g salmon and minced meat, and 5 CFU/25 g chicken meat and 25 ml raw milk were detected. Enrichment in BPW yielded higher numbers of CFU/ml than UPB for all matrices tested. However, the productivity of UPB was sufficient, as all samples were positive with both real-time PCR methods, including those containing less than 300 CFU/ml enrichment broth (enrichment of 5 CFU/25 ml raw milk in UPB).

  1. Quantitative PCR Method for Diagnosis of Citrus Bacterial Canker†

    PubMed Central

    Cubero, J.; Graham, J. H.; Gottwald, T. R.

    2001-01-01

    For diagnosis of citrus bacterial canker by PCR, an internal standard is employed to ensure the quality of the DNA extraction and that proper requisites exist for the amplification reaction. The ratio of PCR products from the internal standard and bacterial target is used to estimate the initial bacterial concentration in citrus tissues with lesions. PMID:11375206

  2. Applications of competitor RNA in diagnostic reverse transcription-PCR.

    PubMed

    Kleiboeker, Steven B

    2003-05-01

    Detection of RNA viruses by reverse transcription (RT)-PCR has proven to be a useful approach for the diagnosis of infections caused by many viral pathogens. However, adequate controls are required for each step of the RT-PCR protocol to ensure the accuracies of diagnostic test results. Heterologous competitor RNA can be used as a control for a number of different aspects of diagnostic RT-PCR. Competitor RNA can be applied to assessments of the efficiency of RNA recovery during extraction procedures, detection of endogenous RT-PCR inhibitors that could lead to false-negative results, and quantification of viral template in samples used for diagnosis; competitor RNA can also be used as a positive control for the RT-PCR. In the present study, heterologous competitor RNA was synthesized by a method that uses two long oligonucleotide primers containing primer binding sites for RT-PCR amplification of porcine reproductive and respiratory syndrome virus or West Nile virus. Amplification of the competitor RNA by RT-PCR resulted in a product that was easily distinguished from the amplification product of viral RNA by agarose gel electrophoresis. Assessment of a variety of RNA samples prepared from routine submissions to a veterinary diagnostic laboratory found that either partial or complete inhibition of the RT-PCR could be demonstrated for approximately 20% of the samples. When inhibition was detected, either dilution of the sample or RNA extraction by an alternative protocol proved successful in eliminating the source of inhibition.

  3. ANIMAL DNA IN PCR REAGENTS PLAGUES ANCIENT DNA RESEARCH

    EPA Science Inventory

    Ancient DNA analysis is becoming widespread. These studies use polymerase chain reaction (PCR) to amplify minute quantities of heavily damaged template. Unusual steps are taken to achieve the sensitivity necessary to detect ancient DNA, including high-cycle PCR amplification targ...

  4. Detection of Salmonella spp. in oysters by PCR.

    PubMed Central

    Bej, A K; Mahbubani, M H; Boyce, M J; Atlas, R M

    1994-01-01

    PCR DNA amplification of a region of the himA gene of Salmonella typhimurium specifically detected Salmonella spp. In oysters, 1 to 10 cells of Salmonella spp. were rapidly detected by the PCR following a pre-enrichment step to increase sensitivity and to ensure that detection was based on the presence of viable Salmonella spp. Images PMID:8117091

  5. Interlaboratory Comparison of Quantitative PCR Test Results for Dehalococcoides

    EPA Science Inventory

    Quantitative PCR (qPCR) techniques have been widely used to measure Dehalococcoides (Dhc) DNA in the groundwater at field sites for several years. Interpretation of these data may be complicated when different laboratories using alternate methods conduct the analysis. An...

  6. DNA probes and PCR for diagnosis of parasitic infections.

    PubMed Central

    Weiss, J B

    1995-01-01

    DNA probe and PCR-based assays to identify and detect parasites are technically complex; however, they have high sensitivity, directly detect parasites independent of the immunocompetence or previous clinical history of the patient, and can distinguish between organisms that are morphologically similar. Diagnosis of parasites is often based on direct detection by microscopy, which is insensitive and laborious and can lack specificity. Most PCR-based assays were more sensitive than DNA probe assays. The development of PCR-based diagnostic assays requires multiple steps following the initial selection of oligonucleotide primers and reporter probe. Generally, the ability to detect the DNA of one parasite was attained by PCR; however, advances in the preparation of samples for PCR (extraction of DNA while removing PCR inhibitors) will be required to achieve that sensitivity with human specimens. Preliminary PCR systems have been developed for many different parasites, yet few have been evaluated with a large number of clinical specimens and/or under field conditions. Those evaluations are essential for determination of clinical and field utility and performance and of the most appropriate application of the assay. Several situations in which PCR-based diagnosis will result in epidemiologic, medical, or public health advances have been identified. PMID:7704890

  7. Evaluation of Aspergillus PCR protocols for testing serum specimens.

    PubMed

    White, P Lewis; Mengoli, Carlo; Bretagne, Stéphane; Cuenca-Estrella, Manuel; Finnstrom, Niklas; Klingspor, Lena; Melchers, Willem J G; McCulloch, Elaine; Barnes, Rosemary A; Donnelly, J Peter; Loeffler, Juergen

    2011-11-01

    A panel of human serum samples spiked with various amounts of Aspergillus fumigatus genomic DNA was distributed to 23 centers within the European Aspergillus PCR Initiative to determine analytical performance of PCR. Information regarding specific methodological components and PCR performance was requested. The information provided was made anonymous, and meta-regression analysis was performed to determine any procedural factors that significantly altered PCR performance. Ninety-seven percent of protocols were able to detect a threshold of 10 genomes/ml on at least one occasion, with 83% of protocols reproducibly detecting this concentration. Sensitivity and specificity were 86.1% and 93.6%, respectively. Positive associations between sensitivity and the use of larger sample volumes, an internal control PCR, and PCR targeting the internal transcribed spacer (ITS) region were shown. Negative associations between sensitivity and the use of larger elution volumes (≥100 μl) and PCR targeting the mitochondrial genes were demonstrated. Most Aspergillus PCR protocols used to test serum generate satisfactory analytical performance. Testing serum requires less standardization, and the specific recommendations shown in this article will only improve performance.

  8. Comparison between ICT and PCR for diagnosis of Chlamydia trachomatis.

    PubMed

    Khan, E R; Hossain, M A; Paul, S K; Mahmud, C; Hasan, M M; Rahman, M M; Nahar, K; Kubayashi, N

    2012-04-01

    Chlamydia trachomatis is an obligate intracellular gram-negative bacterium which is the most prevalent cause of bacterial sexually transmitted infections (STI). The present study was carried to diagnose genital Chlamydia trachomatis infection among women of reproductive age, attending Mymensingh Medical College Hospital, during July 2009 to June 2010 by Immunochromatographic test (ICT) and Polymerase chain reaction (PCR). A total of 70 females were included in this study. Out of 70 cases 56 were symptomatic and 14 asymptomatic. Endocervical swabs were collected from each of the cases and examined by Immunochromatographic test (ICT) for antigen detection and Polymerase chain reaction (PCR) for detection of endogenous plasmid-based nucleic acid. A total 29(41.4%) of the cases were found positive for C. trachomatis either by ICT or PCR. Of the 56 symptomatic cases, 19(33.9%) were found ICT positive and 17(30.4%) were PCR positive. Among 14 asymptomatic females, 2(14.3%) were ICT positive and none were PCR positive. Though PCR is highly sensitive but a total of twelve cases were found ICT positive but PCR negative. It may be due to presence of plasmid deficient strain of C trachomatis which could be amplified by ompA based (Chromosomal gene) multiplex PCR.

  9. Comparison of two DNA extractions and nested PCR, real-time PCR, a new commercial PCR assay, and bacterial culture for detection of Mycobacterium avium subsp. paratuberculosis in bovine feces.

    PubMed

    Christopher-Hennings, Jane; Dammen, Matthew A; Weeks, Shelleen R; Epperson, William B; Singh, Shri N; Steinlicht, Gina L; Fang, Ying; Skaare, Jessica L; Larsen, Jill L; Payeur, Janet B; Nelson, Eric A

    2003-03-01

    In this study, 5 combinations of 2 DNA extractions and 3 polymerase chain reaction (PCR) techniques were compared with culture for the detection of Mycobacterium paratuberculosis directly from bovine feces. These combinations included a new commercial extraction technique combined with a commercial PCR/Southern blot technique, nested PCR (nPCR), or real-time PCR, and a university-developed extraction combined with nPCR or real-time PCR. Four of the 5 combinations had statistically similar sensitivities between 93% and 100% and specificity between 95% and 100%, when compared with culture results from 63 bovine fecal samples. These results indicated that using a commercial extraction with a commercial PCR/Southern blot, nPCR, or real-time PCR, or a university-developed extraction with real-time PCR would result in similar sensitivities to culture for the identification of M. paratuberculosis from bovine feces and are valid alternatives to culture.

  10. Detection of episomal banana streak badnavirus by IC-PCR.

    PubMed

    Harper, G; Dahal, G; Thottappilly, G; Hull, R

    1999-04-01

    A polymerase chain reaction (PCR) based strategy to detect episomal banana streak badnavirus (BSV) in banana and plantain plants that carry integrated BSV sequences was developed. Antisera used in immuno-capture polymerase chain reaction (IC-PCR) are capable of binding a large number of BSV serotypes. The primers used for PCR are capable of annealing to and amplifying across the aspartic protease-reverse transcriptase domain boundaries of both episomal and integrated BSV sequences and result in similar or identical sequence size fragments from either template. However, we show that under the conditions selected for IC-PCR, nuclear, mitochondrial or chloroplast genomic sequences are not amplified and thus only captured episomal BSV is amplified. IC-PCR is suitable for the large-scale screening of Musa for episomal BSV which is necessary for germplasm movement.

  11. Engineered DNA polymerase improves PCR results for plastid DNA1

    PubMed Central

    Schori, Melanie; Appel, Maryke; Kitko, AlexaRae; Showalter, Allan M.

    2013-01-01

    • Premise of the study: Secondary metabolites often inhibit PCR and sequencing reactions in extractions from plant material, especially from silica-dried and herbarium material. A DNA polymerase that is tolerant to inhibitors improves PCR results. • Methods and Results: A novel DNA amplification system, including a DNA polymerase engineered via directed evolution for improved tolerance to common plant-derived PCR inhibitors, was evaluated and PCR parameters optimized for three species. An additional 31 species were then tested with the engineered enzyme and optimized protocol, as well as with regular Taq polymerase. • Conclusions: PCR products and high-quality sequence data were obtained for 96% of samples for rbcL and 79% for matK, compared to 29% and 21% with regular Taq polymerase. PMID:25202519

  12. Rapid diagnosis of poliovirus infection by PCR amplification.

    PubMed Central

    Chezzi, C

    1996-01-01

    A single-tube, single-primer-set reverse transcription-PCR assay was developed for the rapid detection of polioviruses in infected tissue culture fluids and clinical materials. The poliovirus-specific PCR primers are located in the VP1-2A region of the poliovirus genome. They generate a 290-bp product and can be used in duplex reactions with general enterovirus primers. The primers span the region used for genotype determination, so that genotype analysis of wild-type polioviruses can be performed by direct sequencing of the PCR products. Of 125 virus isolates typed as polioviruses by neutralization assays, 125 (100%) were also positive by PCR, and of 38 isolates typed as non-polio enteroviruses by conventional techniques, 38 (100%) were also negative by PCR. The assay described here is rapid, highly sensitive, and specific and has clinical applicability in the diagnosis of poliovirus infections. PMID:8784577

  13. Teaching PCR Through Inquiry in an Undergraduate Biology Laboratory Course

    NASA Astrophysics Data System (ADS)

    Dorighi, K. M.; Betancourt, J.; Sapp, J.; Quan, T. K.; Lee, J.

    2010-12-01

    In this paper, we describe the design and implementation of an inquiry-based laboratory unit on the Polymerase Chain Reaction (PCR). This unit was designed and taught for the undergraduate Eukaryotic Genetics Laboratory class (Bio105L) at the University of California, Santa Cruz. Our activity utilizes an authentic molecular biology research question to teach the underlying molecular mechanisms and experimental technique of PCR, as well as fundamental scientific process skills such as planning experiments, making predictions and interpreting data. In particular, the activity prompts students to use PCR to determine which gene has been deleted in a region of the Drosophila genome. During this activity, students also gained technical experience in common molecular biology techniques, learned about additional applications of PCR and used a hands-on approach to model each step of PCR.

  14. Transcript quantitation in total yeast cellular RNA using kinetic PCR

    PubMed Central

    Kang, John J.; Watson, Robert M.; Fisher, Mary E.; Higuchi, Russell; Gelfand, David H.; Holland, Michael J.

    2000-01-01

    Kinetically monitored, reverse transcriptase-initiated PCR (kinetic RT–PCR, kRT–PCR) is a novel application of kinetic PCR for high throughput transcript quantitation in total cellular RNA. The assay offers the simplicity and flexibility of an enzyme assay with distinct advantages over DNA microarray hybridization and SAGE technologies for certain applications. The reproducibility, sensitivity and accuracy of the kRT–PCR were assessed for yeast transcripts previously quantitated by a variety of methods including SAGE analysis. Changes in transcript levels between different genetic or physiological cell states were reproducibly quantitated with an accuracy of ±20%. The assay was sufficiently sensitive to quantitate yeast transcripts over a range of more than five orders of magnitude, including low abundance transcripts encoding cell cycle and transcriptional regulators. PMID:10606670

  15. Multiplex PCR identification of Taenia spp. in rodents and carnivores.

    PubMed

    Al-Sabi, Mohammad N S; Kapel, Christian M O

    2011-11-01

    The genus Taenia includes several species of veterinary and public health importance, but diagnosis of the etiological agent in definitive and intermediate hosts often relies on labor intensive and few specific morphometric criteria, especially in immature worms and underdeveloped metacestodes. In the present study, a multiplex PCR, based on five primers targeting the 18S rDNA and ITS2 sequences, produced a species-specific banding patterns for a range of Taenia spp. Species typing by the multiplex PCR was compared to morphological identification and sequencing of cox1 and/or 12S rDNA genes. As compared to sequencing, the multiplex PCR identified 31 of 32 Taenia metacestodes from rodents, whereas only 14 cysts were specifically identified by morphology. Likewise, the multiplex PCR identified 108 of 130 adult worms, while only 57 were identified to species by morphology. The tested multiplex PCR system may potentially be used for studies of Taenia spp. transmitted between rodents and carnivores.

  16. PCR thermal management in an integrated Lab on Chip

    NASA Astrophysics Data System (ADS)

    Singh, Janak; Ekaputri, Mayang

    2006-04-01

    Thermal management modelling and simulations of a polymerase chain reaction (PCR) device to be integrated on a lab on chip (LOC) have been carried out and presented. A typical MEMS PCR in symmetrical configuration is the base model for this study. When the PCR device is integrated on a fluidic chip with many other bio-analysis components such as DNA extraction, RNA extraction, electro-chemical sensor, flow through components and channels etc., thermal symmetry required for uniform temperature across the PCR chamber is normally lost. In this paper, ANSYS 8.0 simulations in varying conditions and corresponding physical basis have been investigated and presented. Model optimizations are carried out when PCR chamber is placed, one, in the centre (symmetry) and two, in the corner (asymmetry) of the integrated chip. In both cases, temperature uniformity within ±0.5 °C variation is obtained.

  17. [Detection of influenza virus (RT-PCR assay and others)].

    PubMed

    Matsuzaki, Yoko

    2003-11-01

    Viral isolation is the conventional method for influenza virus diagnosis but it is less useful for immediate patient management. RT-PCR is the sensitive and rapid assay for the detection of respiratory viruses. Single step and multiplex RT-PCR is able to detect several viruses simultaneously in a single reaction. Real time PCR(TaqMan method) is able to detect the amplicon directly by release of a fluorescent reporter of the probe during the amplification reactions. This procedure can save time since it eliminates post-PCR processing steps. These RT-PCR methods should be useful for the accurate and rapid diagnosis of influenza virus infection, especially severe cases such as pneumonia and encephalopathy.

  18. PCR detection of Babesia ovata from questing ticks in Japan.

    PubMed

    Sivakumar, Thillaiampalam; Tattiyapong, Muncharee; Okubo, Kazuhiro; Suganuma, Keisuke; Hayashida, Kyoko; Igarashi, Ikuo; Zakimi, Satoshi; Matsumoto, Kotaro; Inokuma, Hisashi; Yokoyama, Naoaki

    2014-04-01

    Babesia ovata is a tick-transmitted hemoprotozoan parasite of cattle. In the present study, we analyzed tick DNA samples (n=1459) prepared from questing ticks collected from various cattle pastures in Hokkaido (Shibecha, Taiki, Otofuke, Memuro, and Shin-Hidaka districts) and Okinawa (Yonaguni Island) prefectures of Japan for B. ovata. When all the tick DNA samples were screened by a previously described B. ovata-specific apical membrane antigen-1 (AMA-1) gene-based polymerase chain reaction (PCR) assay, none of the DNA samples was positive. Therefore, we developed a PCR assay based on the protozoan beta-tubulin (β-tubulin) gene to detect B. ovata from ticks in Japan. In the specificity test, the PCR assay amplified the expected 444-bp target gene fragment from B. ovata DNA. No PCR products were amplified from DNA samples from other blood pathogens, bovine blood, or ticks. In addition, the PCR assay detected 100 fg of B. ovata-genomic DNA extracted from an in vitro culture of the parasites. Subsequently, when all the tick DNA samples were screened by this new PCR assay, 18 were positive for B. ovata. Positive samples were found only in the Yonaguni and Memuro areas. In Okinawa, where all the ticks were identified as Haemaphysalis longicornis, 9.7% of the samples were PCR-positive, while a single tick (Ixodes ovatus) from Memuro was infected with B. ovata. When the nucleotide sequences of the PCR amplicons were phylogenetically analyzed, they formed a separate clade containing a previously described β-tubulin gene sequence from B. ovata (Miyake strain), confirming that the PCR assay had detected only B. ovata from the tick DNA samples. This is the first report that describes the PCR detection of B. ovata in ticks. The findings warrant transmission experiments to evaluate I. ovatus as a potential vector of B. ovata.

  19. RNase H-dependent PCR (rhPCR): improved specificity and single nucleotide polymorphism detection using blocked cleavable primers

    PubMed Central

    2011-01-01

    Background The polymerase chain reaction (PCR) is commonly used to detect the presence of nucleic acid sequences both in research and diagnostic settings. While high specificity is often achieved, biological requirements sometimes necessitate that primers are placed in suboptimal locations which lead to problems with the formation of primer dimers and/or misamplification of homologous sequences. Results Pyrococcus abyssi (P.a.) RNase H2 was used to enable PCR to be performed using blocked primers containing a single ribonucleotide residue which are activated via cleavage by the enzyme (rhPCR). Cleavage occurs 5'-to the RNA base following primer hybridization to the target DNA. The requirement of the primer to first hybridize with the target sequence to gain activity eliminates the formation of primer-dimers and greatly reduces misamplification of closely related sequences. Mismatches near the scissile linkage decrease the efficiency of cleavage by RNase H2, further increasing the specificity of the assay. When applied to the detection of single nucleotide polymorphisms (SNPs), rhPCR was found to be far more sensitive than standard allele-specific PCR. In general, the best discrimination occurs when the mismatch is placed at the RNA:DNA base pair. Conclusion rhPCR eliminates the formation of primer dimers and markedly improves the specificity of PCR with respect to off-target amplification. These advantages of the assay should find utility in challenging qPCR applications such as genotyping, high level multiplex assays and rare allele detection. PMID:21831278

  20. An investigation of PCR inhibition using Plexor(®) -based quantitative PCR and short tandem repeat amplification.

    PubMed

    Thompson, Robyn E; Duncan, George; McCord, Bruce R

    2014-11-01

    A common problem in forensic DNA typing is PCR inhibition resulting in allele dropout and peak imbalance. In this paper, we have utilized the Plexor(®) real-time PCR quantification kit to evaluate PCR inhibition. This is performed by adding increasing concentrations of various inhibitors and evaluating changes in melt curves and PCR amplification efficiencies. Inhibitors examined included calcium, humic acid, collagen, phenol, tannic acid, hematin, melanin, urea, bile salts, EDTA, and guanidinium thiocyanate. Results were plotted and modeled using mathematical simulations. In general, we found that PCR inhibitors that bind DNA affect melt curves and CT takeoff points while those that affect the Taq polymerase tend to affect the slope of the amplification curve. Mixed mode effects were also visible. Quantitative PCR results were then compared with subsequent STR amplification using the PowerPlex(®) 16 HS System. The overall results demonstrate that real-time PCR can be an effective method to evaluate PCR inhibition and predict its effects on subsequent STR amplifications.

  1. How good is a PCR efficiency estimate: Recommendations for precise and robust qPCR efficiency assessments.

    PubMed

    Svec, David; Tichopad, Ales; Novosadova, Vendula; Pfaffl, Michael W; Kubista, Mikael

    2015-03-01

    We have examined the imprecision in the estimation of PCR efficiency by means of standard curves based on strategic experimental design with large number of technical replicates. In particular, how robust this estimation is in terms of a commonly varying factors: the instrument used, the number of technical replicates performed and the effect of the volume transferred throughout the dilution series. We used six different qPCR instruments, we performed 1-16 qPCR replicates per concentration and we tested 2-10 μl volume of analyte transferred, respectively. We find that the estimated PCR efficiency varies significantly across different instruments. Using a Monte Carlo approach, we find the uncertainty in the PCR efficiency estimation may be as large as 42.5% (95% CI) if standard curve with only one qPCR replicate is used in 16 different plates. Based on our investigation we propose recommendations for the precise estimation of PCR efficiency: (1) one robust standard curve with at least 3-4 qPCR replicates at each concentration shall be generated, (2) the efficiency is instrument dependent, but reproducibly stable on one platform, and (3) using a larger volume when constructing serial dilution series reduces sampling error and enables calibration across a wider dynamic range.

  2. Field effect sensors for PCR applications

    NASA Astrophysics Data System (ADS)

    Taing, Meng-Houit; Sweatman, Denis R.

    2004-03-01

    The use of field effect sensors for biological and chemical sensing is widely employed due to its ability to make detections based on charge and surface potential. Because proteins and DNA almost always carry a charge [1], silicon can be used to micro fabricate such a sensor. The EIS structure (Electrolyte on Insulator on Silicon) provides a novel, label-free and simple to fabricate way to make a field effect DNA detection sensor. The sensor responds to fluctuating capacitance caused by a depletion layer thickness change at the surface of the silicon substrate through DNA adsorption onto the dielectric oxide/PLL (Poly-L-Lysine) surface. As DNA molecules diffuse to the sensor surface, they are bound to their complimentary capture probes deposited on the surface. The negative charge exhibited by the DNA forces negative charge carriers in the substrate to move away from the surface. This causes an n-type depletion layer substrate to thicken and a p-type to thin. The depletion layer thickness can be measured by its capacitance using an LCR meter. This experiment is conducted using the ConVolt (constant voltage) approach. Nucleic acids are amplified by an on chip PCR (Polymerase Chain Reaction) system and then fed into the sensor. The low ionic solution strength will ensure that counter-ions do not affect the sensor measurements. The sensor surface contains capture probes that bind to the pathogen. The types of pathogens we"ll be detecting include salmonella, campylobacter and E.Coli DNA. They are held onto the sensor surface by the positively charged Poly-L-Lysine layer. The electrolyte is biased through a pseudo-reference electrode. Pseudo reference electrodes are usually made from metals such as Platinum or Silver. The problem associated with "floating" biasing electrodes is they cannot provide stable biasing potentials [2]. They drift due to surface charging effects and trapped charges on the surface. To eliminate this, a differential system consisting of 2 sensors

  3. Real time PCR in childhood tuberculosis: a valuable diagnostic tool.

    PubMed

    Dayal, Rajeshwar; Kashyap, Haripal; Pounikar, Gajanand; Kamal, Raj; Yadav, Neeraj Kumar; Singh, Manoj Kumar; Chauhan, Devendra Singh; Goyal, Ankur

    2015-02-01

    The present study was conducted to detect and quantitate Mycobacterium tuberculosis from various body fluid specimens of cases of tuberculosis by real time PCR technique and compare results with conventional PCR technique and culture. One hundred fifteen children (<18 y) with tuberculosis (diagnosed as per IAP guidelines) and 32 disease matched controls from the Department of Pediatrics, S.N. Medical College, Agra, were included in the study. Different body fluids (CSF, gastric aspirate, pleural fluid, ascitic fluid and lymph node aspirate) were subjected to culture, conventional PCR targeting insertion sequence 1S6110 and Real time PCR targeting 16srRNA of Mycobacterium tuberculosis. Real time PCR showed significantly better results than culture in all body fluids (p < 0.05). It was superior to conventional PCR in CSF (p < 0.05) but showed comparable results in gastric aspirate, pleural fluid, ascitic fluid and lymph node aspirate (p > 0.05). Hence, real time PCR is a promising diagnostic tool for childhood tuberculosis, particularly tubercular meningitis.

  4. A comparison of four methods for PCR inhibitor removal.

    PubMed

    Hu, Qingqing; Liu, Yuxuan; Yi, Shaohua; Huang, Daixin

    2015-05-01

    Biological samples collected from the crime scenes often contain some compounds that can inhibit the polymerase chain reaction (PCR). The removal of PCR inhibitors from the extracts prior to the PCR amplification is vital for successful forensic DNA typing. This paper aimed to evaluate the ability of four different methods (PowerClean® DNA Clean-Up kit, DNA IQ™ System, Phenol-Chloroform extraction and Chelex®-100 methods) to remove eight commonly encountered PCR inhibitors including: melanin, humic acid, collagen, bile salt, hematin, calcium ions, indigo and urea. Each of these PCR inhibitors was effectively removed by the PowerClean® DNA Clean-Up kit and DNA IQ™ System as demonstrated by generating more complete short tandem repeat (STR) profiles from the cleaned up inhibitor samples than from the raw inhibitor samples. The Phenol-Chloroform extraction and Chelex®-100 methods, however, could only remove some of eight PCR inhibitors. Our results demonstrated that the PowerClean® DNA Clean-Up kit and DNA IQ™ System were very effective for the removal of known PCR inhibitors that are routinely found in DNA extracts from forensic samples.

  5. Multiplex PCR to detect four different tomato-infecting pathogens.

    PubMed

    Quintero-Vásquez, Gabriela Alejandra; Bazán-Tejeda, María Luisa; Martínez-Peñafiel, Eva; Kameyama-Kawabe, Luis; Bermúdez-Cruz, Rosa María

    2013-07-01

    This work was aimed to develop a multiplex PCR assay to detect infectious agents such as Clavibacter michiganensis subsp. michiganensis, Fusarium sp, Leveillula taurica, and begomoviruses in tomato (Solanum lycopersicum) plants. Specific primer sets of each pathogen were designed based on intergenic ribosomal RNA sequences for the first three, whereas for begomoviruses, primers were designed based on conserved regions. The design also considered that the length (200-800 bp) of the PCR products was resolvable by electrophoresis; thus 296, 380, 457, and 731 bp fragments for Clavibacter, Fusarium, Leveillula, and begomoviruses, respectively, were considered. PCR conditions were optimized to amplify all the products in a single tube from genomic DNA and circumvent PCR inhibitors from infected plants. Finally, when the multiplex PCR assay was tested with tomato plants infected with any of the four pathogens, specific PCR products confirmed the presence of the pathogens. Optimized PCR multiplex allowed for the accurate and simultaneous detection of Clavibacter, Fusarium, Leveillula, and begomoviruses in infected plants or seeds from tomato.

  6. Molecular characterization of Corynebacterium pseudotuberculosis isolates using ERIC-PCR.

    PubMed

    Guimarães, Alessandro de Sá; Dorneles, Elaine Maria Seles; Andrade, Giovanna Ivo; Lage, Andrey Pereira; Miyoshi, Anderson; Azevedo, Vasco; Gouveia, Aurora Maria Guimarães; Heinemann, Marcos Bryan

    2011-12-15

    Caseous lymphadenitis is an infectious sheep and goats disease caused by Corynebacterium pseudotuberculosis and characterized by abscesses in superficial and visceral lymph nodes. C. pseudotuberculosis strains isolated from these hosts have been shown to be very difficult to type by the existing methods. The aim of this study is evaluating the potential of the Enterobacterial Repetitive Intergenic Consensus (ERIC-PCR) as a tool for molecular typing of C. pseudotuberculosis strains isolated in sheep. One hundred and twenty seven isolates of C. pseudotuberculosis were isolated from lesions suspected to have had caseous lymphadenitis collected from sheep at the slaughterhouse. Animals were from 24 flocks in 13 municipalities of the Minas Gerais State, Brazil. Species identification of the isolates was performed by routine biochemical tests and mPCR. Fingerprint was performed by RAPD using ERIC-1R, ERIC-2 and ERIC-1R+ERIC-2 primers. Seventeen different genotypes were generated by ERIC 1-PCR, 21 genotypes by ERIC 2-PCR and 21 genotypes by ERIC 1+2-PCR. Hunter-Gaston Discrimination Index (HGDI) found for ERIC 1, ERIC 2, ERIC 1+2 PCR were 0.69, 0.87, and 0.84, respectively. For most herds evaluated observed at most three different genotypes among isolates from animals of these property, in all ERIC-PCR assays. However a few flocks observed between four and nine genotypes per flock. The W Kendall value found for correlation among the three techniques of ERIC-PCR was 0.91 (P<5.0 x 10(-6)). The results show that ERIC-PCR has good discriminatory power and advantages over other DNA-based typing methods, making it a useful tool to discriminate C. pseudotuberculosis isolates.

  7. Droplet digital polymerase chain reaction (PCR) outperforms real-time PCR in the detection of environmental DNA from an invasive fish species.

    PubMed

    Doi, Hideyuki; Takahara, Teruhiko; Minamoto, Toshifumi; Matsuhashi, Saeko; Uchii, Kimiko; Yamanaka, Hiroki

    2015-05-05

    Environmental DNA (eDNA) has been used to investigate species distributions in aquatic ecosystems. Most of these studies use real-time polymerase chain reaction (PCR) to detect eDNA in water; however, PCR amplification is often inhibited by the presence of organic and inorganic matter. In droplet digital PCR (ddPCR), the sample is partitioned into thousands of nanoliter droplets, and PCR inhibition may be reduced by the detection of the end-point of PCR amplification in each droplet, independent of the amplification efficiency. In addition, real-time PCR reagents can affect PCR amplification and consequently alter detection rates. We compared the effectiveness of ddPCR and real-time PCR using two different PCR reagents for the detection of the eDNA from invasive bluegill sunfish, Lepomis macrochirus, in ponds. We found that ddPCR had higher detection rates of bluegill eDNA in pond water than real-time PCR with either of the PCR reagents, especially at low DNA concentrations. Limits of DNA detection, which were tested by spiking the bluegill DNA to DNA extracts from the ponds containing natural inhibitors, found that ddPCR had higher detection rate than real-time PCR. Our results suggest that ddPCR is more resistant to the presence of PCR inhibitors in field samples than real-time PCR. Thus, ddPCR outperforms real-time PCR methods for detecting eDNA to document species distributions in natural habitats, especially in habitats with high concentrations of PCR inhibitors.

  8. PCR amplification on microarrays of gel immobilized oligonucleotides

    DOEpatents

    Strizhkov, Boris; Tillib, Sergei; Mikhailovich, Vladimir; Mirzabekov, Andrei

    2003-11-04

    The invention relates two general methods for performing PCR amplification, combined with the detection and analysis of the PCR products on a microchip. In the first method, the amplification occurs both outside and within a plurality of gel pads on a microchip, with at least one oligonucleotide primer immobilized in a gel pad. In the second method, PCR amplification also takes place within gel pads on a microchip, but the pads are surrounded by a hydrophobic liquid such as that which separates the individual gel pads into environments which resemble micro-miniaturized test tubes.

  9. PCR diagnosis of Pneumocystis pneumonia: a bivariate meta-analysis.

    PubMed

    Lu, Yuan; Ling, Guoya; Qiang, Chenyi; Ming, Qinshou; Wu, Cong; Wang, Ke; Ying, Zouxiao

    2011-12-01

    We undertook a bivariate meta-analysis to assess the overall accuracy of respiratory specimen PCR assays for diagnosing Pneumocystis pneumonia. The summary sensitivity and specificity were 0.99 (95% confidence interval, 0.96 to 1.00) and 0.90 (0.87 to 0.93). Subgroup analyses showed that quantitative PCR analysis and the major surface glycoprotein gene target had the highest specificity value (0.93). Respiratory specimen PCR results are sufficient to confirm or exclude the disease for at-risk patients suspected of having Pneumocystis pneumonia.

  10. Preparation of DNA-containing extract for PCR amplification

    DOEpatents

    Dunbar, John M.; Kuske, Cheryl R.

    2006-07-11

    Environmental samples typically include impurities that interfere with PCR amplification and DNA quantitation. Samples of soil, river water, and aerosol were taken from the environment and added to an aqueous buffer (with or without detergent). Cells from the sample are lysed, releasing their DNA into the buffer. After removing insoluble cell components, the remaining soluble DNA-containing extract is treated with N-phenacylthiazolium bromide, which causes rapid precipitation of impurities. Centrifugation provides a supernatant that can be used or diluted for PCR amplification of DNA, or further purified. The method may provide a DNA-containing extract sufficiently pure for PCR amplification within 5–10 minutes.

  11. Identification and quantification of ice nucleation active microorganisms by digital droplet PCR (ddPCR)

    NASA Astrophysics Data System (ADS)

    Linden, Martin; Pöschl, Ulrich; Fröhlich-Nowoisky, Janine

    2015-04-01

    Several bioaerosol types, including bacteria, fungi, pollen and lichen, have been identified as sources of biological ice nucleators (IN) which induce ice formation already at temperatures as high as -10 °C or above. Accordingly, they potentially contribute widely to environmental ice nucleation in the atmosphere and are of great interest in the study of natural heterogenous ice nucleation processes. Ice nucleation active microorganisms have been found and studied among bacteria (Proteobacteria) and fungi (phyla Basidiomycota and Ascomycota). The mechanisms enabling the microorganisms to ice nucleation are subject to ongoing research. While it has been demonstrated that whole cells can act as ice nucleators in the case of bacteria due to the presence of specific membrane proteins, cell-free ice nucleation active particles seem to be responsible for this phenomenon in fungi and lichen. The identification and quantification of these ice nucleation active microorganisms and their IN in atmospheric samples is crucial to understand their contribution to the pool of atmospheric IN. This is not a trivial task since the respective microorganisms are often prevalent in lowest concentrations and a variety of states, be it viable cells, spores or cell debris from dead cells. Molecular biology provides tools to identify and quantify ice nucleation active microorganisms independent of their state by detecting genetic markers specific for the organism of interest. Those methods are not without their drawbacks in terms of sample material concentration required or reliable standardization. Digital Droplet Polymerase Chain Reaction (ddPCR) was chosen for our demands as a more elegant, quick and specific method in the investigation of ice nucleation active microorganisms in atmospheric samples. The advantages of ddPCR lie in the simultaneous detection and quantification of genetic markers and their original copy numbers in a sample. This is facilitated by the fractionation of the

  12. Oligoribonucleotide (ORN) interference-PCR (ORNi-PCR): a simple method for suppressing PCR amplification of specific DNA sequences using ORNs.

    PubMed

    Tanigawa, Naoki; Fujita, Toshitsugu; Fujii, Hodaka

    2014-01-01

    Polymerase chain reaction (PCR) amplification of multiple templates using common primers is used in a wide variety of molecular biological techniques. However, abundant templates sometimes obscure the amplification of minor species containing the same primer sequences. To overcome this challenge, we used oligoribonucleotides (ORNs) to inhibit amplification of undesired template sequences without affecting amplification of control sequences lacking complementarity to the ORNs. ORNs were effective at very low concentrations, with IC50 values for ORN-mediated suppression on the order of 10 nM. DNA polymerases that retain 3'-5' exonuclease activity, such as KOD and Pfu polymerases, but not those that retain 5'-3' exonuclease activity, such as Taq polymerase, could be used for ORN-mediated suppression. ORN interference-PCR (ORNi-PCR) technology should be a useful tool for both molecular biology research and clinical diagnosis.

  13. COMplementary Primer ASymmetric PCR (COMPAS-PCR) Applied to the Identification of Salmo salar, Salmo trutta and Their Hybrids

    PubMed Central

    2016-01-01

    Avoiding complementarity between primers when designing a PCR assay constitutes a central rule strongly anchored in the mind of the molecular scientist. 3’-complementarity will extend the primers during PCR elongation using one another as template, consequently disabling further possible involvement in traditional target amplification. However, a 5’-complementarity will leave the primers unchanged during PCR cycles, albeit sequestered to one another, therefore also suppressing target amplification. We show that 5’-complementarity between primers may be exploited in a new PCR method called COMplementary-Primer-Asymmetric (COMPAS)-PCR, using asymmetric primer concentrations to achieve target PCR amplification. Moreover, such a design may paradoxically reduce spurious non-target amplification by actively sequestering the limiting primer. The general principles were demonstrated using 5S rDNA direct repeats as target sequences to design a species-specific assay for identifying Salmo salar and Salmo trutta using almost fully complementary primers overlapping the same target sequence. Specificity was enhanced by using 3’-penultimate point mutations and the assay was further developed to enable identification of S. salar x S. trutta hybrids by High Resolution Melt analysis in a 35 min one-tube assay. This small paradigm shift, using highly complementary primers for PCR, should help develop robust assays that previously would not be considered. PMID:27783658

  14. Whole blood Nested PCR and Real-time PCR amplification of Talaromyces marneffei specific DNA for diagnosis.

    PubMed

    Lu, Sha; Li, Xiqing; Calderone, Richard; Zhang, Jing; Ma, Jianchi; Cai, Wenying; Xi, Liyan

    2016-02-01

    Talaromyces marneffei is a dimorphic pathogenic fungus, which is a life-threatening invasive mycosis in the immunocompromised host. Prompt diagnosis of T. marneffei infection remains difficult although there has been progress in attempts to expedite the diagnosis of this infection. We previously demonstrated the value of nested polymerase chain reaction (PCR) to detect T. marneffei in paraffin embedded tissue samples with high sensitivity and specificity. In this study, this assay was used to detect the DNA of T. marneffei in whole blood samples. Real-time PCR assay was also evaluated to identify T. marneffei in the same samples. Twenty out of 30 whole blood samples (67%) collected from 23 patients were found positive by using the nested PCR assay, while 23/30 (77%) samples were found positive by using the real-time PCR assay. In order to express accurately the fungal loads, we used a normalized linearized plasmid as an internal control for real-time PCR. The assay results were correlated as the initial quantity (copies/μl) with fungal burden. These data indicate that combination of nested PCR and real-time PCR assay provides an attractive alternative for identification of T. marneffei DNA in whole blood samples of HIV-infected patients.

  15. COMplementary Primer ASymmetric PCR (COMPAS-PCR) Applied to the Identification of Salmo salar, Salmo trutta and Their Hybrids.

    PubMed

    Anglès d'Auriac, Marc B

    2016-01-01

    Avoiding complementarity between primers when designing a PCR assay constitutes a central rule strongly anchored in the mind of the molecular scientist. 3'-complementarity will extend the primers during PCR elongation using one another as template, consequently disabling further possible involvement in traditional target amplification. However, a 5'-complementarity will leave the primers unchanged during PCR cycles, albeit sequestered to one another, therefore also suppressing target amplification. We show that 5'-complementarity between primers may be exploited in a new PCR method called COMplementary-Primer-Asymmetric (COMPAS)-PCR, using asymmetric primer concentrations to achieve target PCR amplification. Moreover, such a design may paradoxically reduce spurious non-target amplification by actively sequestering the limiting primer. The general principles were demonstrated using 5S rDNA direct repeats as target sequences to design a species-specific assay for identifying Salmo salar and Salmo trutta using almost fully complementary primers overlapping the same target sequence. Specificity was enhanced by using 3'-penultimate point mutations and the assay was further developed to enable identification of S. salar x S. trutta hybrids by High Resolution Melt analysis in a 35 min one-tube assay. This small paradigm shift, using highly complementary primers for PCR, should help develop robust assays that previously would not be considered.

  16. Specific detection of viable Legionella cells by combined use of photoactivated ethidium monoazide and PCR/real-time PCR.

    PubMed

    Chang, Bin; Sugiyama, Kanji; Taguri, Toshitsugu; Amemura-Maekawa, Junko; Kura, Fumiaki; Watanabe, Haruo

    2009-01-01

    Legionella organisms are prevalent in manmade water systems and cause legionellosis in humans. A rapid detection method for viable Legionella cells combining ethidium monoazide (EMA) and PCR/real-time PCR was assessed. EMA could specifically intercalate and cleave the genomic DNA of heat- and chlorine-treated dead Legionella cells. The EMA-PCR assay clearly showed an amplified fragment specific for Legionella DNA from viable cells, but it could not do so for DNA from dead cells. The number of EMA-treated dead Legionella cells estimated by real-time PCR exhibited a 10(4)- to 10(5)-fold decrease compared to the number of dead Legionella cells without EMA treatment. Conversely, no significant difference in the numbers of EMA-treated and untreated viable Legionella cells was detected by the real-time PCR assay. The combined assay was also confirmed to be useful for specific detection of culturable Legionella cells from water samples obtained from spas. Therefore, the combined use of EMA and PCR/real-time PCR detects viable Legionella cells rapidly and specifically and may be useful in environmental surveillance for Legionella.

  17. Sensitive simultaneous detection of seven sexually transmitted agents in semen by multiplex-PCR and of HPV by single PCR.

    PubMed

    Gimenes, Fabrícia; Medina, Fabiana Soares; Abreu, André Luelsdorf Pimenta de; Irie, Mary Mayumi Taguti; Esquiçati, Isis Baroni; Malagutti, Natália; Vasconcellos, Vinícius Rodrigo Bulla; Discacciati, Michele Garcia; Bonini, Marcelo Gialluisi; Maria-Engler, Silvya Stuchi; Consolaro, Marcia Edilaine Lopes

    2014-01-01

    Sexually transmitted diseases (STDs) may impair sperm parameters and functions thereby promoting male infertility. To date limited molecular studies were conducted to evaluate the frequency and type of such infections in semen Thus, we aimed at conceiving and validating a multiplex PCR (M-PCR) assay for the simultaneous detection of the following STD pathogens in semen: Chlamydia trachomatis, Neisseria gonorrhoeae, Mycoplasma genitalium, Trichomonas vaginalis, Herpes virus simplex (HSV) -1 and -2, and Treponema pallidum; We also investigated the potential usefulness of this M-PCR assay in screening programs for semen pathogens. In addition, we aimed: to detect human Papillomavirus (HPV) and genotypes by single PCR (sPCR) in the same semen samples; to determine the prevalence of the seven STDs, HPV and co-infections; to assess the possibility that these infections affect semen parameters and thus fertility. The overall validation parameters of M-PCR were extremely high including agreement (99.2%), sensitivity (100.00%), specificity (99.70%), positive (96.40%) and negative predictive values (100.00%) and accuracy (99.80%). The prevalence of STDs was very high (55.3%). Furthermore, associations were observed between STDs and changes in semen parameters, highlighting the importance of STD detection in semen. Thus, this M-PCR assay has great potential for application in semen screening programs for pathogens in infertility and STD clinics and in sperm banks.

  18. Sensitive Simultaneous Detection of Seven Sexually Transmitted Agents in Semen by Multiplex-PCR and of HPV by Single PCR

    PubMed Central

    de Abreu, André Luelsdorf Pimenta; Irie, Mary Mayumi Taguti; Esquiçati, Isis Baroni; Malagutti, Natália; Vasconcellos, Vinícius Rodrigo Bulla; Discacciati, Michele Garcia; Bonini, Marcelo Gialluisi; Maria-Engler, Silvya Stuchi; Consolaro, Marcia Edilaine Lopes

    2014-01-01

    Sexually transmitted diseases (STDs) may impair sperm parameters and functions thereby promoting male infertility. To date limited molecular studies were conducted to evaluate the frequency and type of such infections in semen Thus, we aimed at conceiving and validating a multiplex PCR (M-PCR) assay for the simultaneous detection of the following STD pathogens in semen: Chlamydia trachomatis, Neisseria gonorrhoeae, Mycoplasma genitalium, Trichomonas vaginalis, Herpes virus simplex (HSV) −1 and −2, and Treponema pallidum; We also investigated the potential usefulness of this M-PCR assay in screening programs for semen pathogens. In addition, we aimed: to detect human Papillomavirus (HPV) and genotypes by single PCR (sPCR) in the same semen samples; to determine the prevalence of the seven STDs, HPV and co-infections; to assess the possibility that these infections affect semen parameters and thus fertility. The overall validation parameters of M-PCR were extremely high including agreement (99.2%), sensitivity (100.00%), specificity (99.70%), positive (96.40%) and negative predictive values (100.00%) and accuracy (99.80%). The prevalence of STDs was very high (55.3%). Furthermore, associations were observed between STDs and changes in semen parameters, highlighting the importance of STD detection in semen. Thus, this M-PCR assay has great potential for application in semen screening programs for pathogens in infertility and STD clinics and in sperm banks. PMID:24921247

  19. A PCR assay and PCR-restriction fragment length polymorphism combination identifying the 3 primary Mycoplasma species causing mastitis.

    PubMed

    Boonyayatra, S; Fox, L K; Besser, T E; Sawant, A; Gay, J M; Raviv, Z

    2012-01-01

    The focus of the current research was to develop real-time PCR assays with improved sensitivity and the capacity to simultaneously speciate the 3 most common mycoplasma mastitis agents: Mycoplasma bovis, Mycoplasma californicum, and Mycoplasma bovigenitalium. Real-time PCR was chosen because it provides rapid results. Partial 16S rRNA gene sequencing was used as the gold standard for evaluating candidate real-time PCR assays. To ascertain the real-time PCR assay specificity, reference strains of Mycoplasma species, Acholeplasma axanthum, and common gram-positive and gram-negative mastitis pathogens were tested. No cross-reactions were observed. Mycoplasma spp. isolated from bovine milk samples (n=228) and other organ sites (n=40) were tested by the real-time PCR assays and the partial 16S rRNA gene sequencing assay. Overall accuracy of this novel real-time PCR was 98.51%; 4 of 228 isolates identified as M. bovis by the partial 16S rRNA gene sequencing assay were identified as both M. bovis and M. californicum by real-time PCR. Subsequent amplicon sequencing suggested the presence of both M. bovis and M. californicum in these 4 samples. Using a cycle threshold of 37, the detection limits for real-time PCR were 10 copies of DNA template for both M. bovis and M. bovigenitalium, and 1 copy for M. californicum. This real-time PCR assay is a diagnostic technique that may be used as a screening tool or as a confirmation test for mycoplasma mastitis.

  20. Clinical Performance of Aspergillus PCR for Testing Serum and Plasma: a Study by the European Aspergillus PCR Initiative.

    PubMed

    White, P Lewis; Barnes, Rosemary A; Springer, Jan; Klingspor, Lena; Cuenca-Estrella, Manuel; Morton, C Oliver; Lagrou, Katrien; Bretagne, Stéphane; Melchers, Willem J G; Mengoli, Carlo; Donnelly, J Peter; Heinz, Werner J; Loeffler, Juergen

    2015-09-01

    Aspergillus PCR testing of serum provides technical simplicity but with potentially reduced sensitivity compared to whole-blood testing. With diseases for which screening to exclude disease represents an optimal strategy, sensitivity is paramount. The associated analytical study confirmed that DNA concentrations were greater in plasma than those in serum. The aim of the current investigation was to confirm analytical findings by comparing the performance of Aspergillus PCR testing of plasma and serum in the clinical setting. Standardized Aspergillus PCR was performed on plasma and serum samples concurrently obtained from hematology patients in a multicenter retrospective anonymous case-control study, with cases diagnosed according to European Organization for Research and Treatment of Cancer/Invasive Fungal Infections Cooperative Group and the National Institute of Allergy and Infectious Diseases Mycoses Study Group (EORTC/MSG) consensus definitions (19 proven/probable cases and 42 controls). Clinical performance and clinical utility (time to positivity) were calculated for both kinds of samples. The sensitivity and specificity for Aspergillus PCR when testing serum were 68.4% and 76.2%, respectively, and for plasma, they were 94.7% and 83.3%, respectively. Eighty-five percent of serum and plasma PCR results were concordant. On average, plasma PCR was positive 16.8 days before diagnosis and was the earliest indicator of infection in 13 cases, combined with other biomarkers in five cases. On average, serum PCR was positive 10.8 days before diagnosis and was the earliest indicator of infection in six cases, combined with other biomarkers in three cases. These results confirm the analytical finding that the sensitivity of Aspergillus PCR using plasma is superior to that using serum. PCR positivity occurs earlier when testing plasma and provides sufficient sensitivity for the screening of invasive aspergillosis while maintaining methodological simplicity.

  1. Quantitative PCR for genetic markers of human fecal pollution

    EPA Science Inventory

    Assessment of health risk and fecal bacteria loads associated with human fecal pollution requires reliable host-specific analytical methods and a rapid quantification approach. We report the development of quantitative PCR assays for enumeration of two recently described hum...

  2. Quantitative PCR for Genetic Markers of Human Fecal Pollution

    EPA Science Inventory

    Assessment of health risk and fecal bacteria loads associated with human fecal pollution requires reliable host-specific analytical methods and a rapid quantificationapproach. We report the development of quantitative PCR assays for quantification of two recently described human-...

  3. Identification of Alocasia odora (Kuwazuimo in Japanese) Using PCR Method.

    PubMed

    Hagino, Kayo; Nakano, Hisako; Shimizu, Motomu; Terai, Akiko; Ogai, Mami; Aragane, Masako; Abe, Tomohiro; Sasamoto, Takeo

    2017-01-01

    Kuwazuimo (Alocasia odora) and shimakuwazuimo (Alocasia cucullata) are evergreen perennial plants that originated in East Asia. Although inedible, they are occasionally eaten by mistake because they resemble satoimo (Colocasia esculenta), and this has caused food poisoning in Japan. It is not easy to determine the cause of a food poisoning outbreak from the shape or chemical composition when the available sample is small. Therefore, we developed a new primer pair for PCR to identify kuwazuimo and shimakuwazuimo in small samples, based on the internal transcribed spacer (ITS) region of ribosomal DNA. Using PCR with the developed primer pair, we detected all samples of kuwazuimo obtained from the market, while excluding 17 other kinds of crops. The samples were identified as shimakuwazuimo by DNA sequencing of the PCR products. The present PCR method showed high specificity and was confirmed to be applicable to the identification of kuwazuimo and shimakuwazuimo from various crops.

  4. Single-molecule emulsion PCR in microfluidic droplets.

    PubMed

    Zhu, Zhi; Jenkins, Gareth; Zhang, Wenhua; Zhang, Mingxia; Guan, Zhichao; Yang, Chaoyong James

    2012-06-01

    The application of microfluidic droplet PCR for single-molecule amplification and analysis has recently been extensively studied. Microfluidic droplet technology has the advantages of compartmentalizing reactions into discrete volumes, performing highly parallel reactions in monodisperse droplets, reducing cross-contamination between droplets, eliminating PCR bias and nonspecific amplification, as well as enabling fast amplification with rapid thermocycling. Here, we have reviewed the important technical breakthroughs of microfluidic droplet PCR in the past five years and their applications to single-molecule amplification and analysis, such as high-throughput screening, next generation DNA sequencing, and quantitative detection of rare mutations. Although the utilization of microfluidic droplet single-molecule PCR is still in the early stages, its great potential has already been demonstrated and will provide novel solutions to today's biomedical engineering challenges in single-molecule amplification and analysis.

  5. Rapid PCR amplification of DNA utilizing Coriolis effects.

    PubMed

    Mårtensson, Gustaf; Skote, Martin; Malmqvist, Mats; Falk, Mats; Asp, Allan; Svanvik, Nicke; Johansson, Arne

    2006-08-01

    A novel polymerase chain reaction (PCR) method is presented that utilizes Coriolis and centrifugal effects, produced by rotation of the sample disc, in order to increase internal circulatory rates, and with them temperature homogenization and mixing speeds. A proof of concept has been presented by testing a rapid 45-cycle PCR DNA amplification protocol. During the repeated heating and cooling that constitutes a PCR process, the 100 microL samples were rotated at a speed equivalent to an effective acceleration of gravity of 7,000 g. A cycle time of 20.5 s gave a total process time of 15 min to complete the 45 cycles. A theoretical and numerical analysis of the resulting flow, which describes the increased mixing and temperature homogenization, is presented. The device gives excellent reaction speed efficiency, which is beneficial for rapid PCR.

  6. DNA Microarray-Based PCR Ribotyping of Clostridium difficile

    PubMed Central

    Ehricht, Ralf; Slickers, Peter; Baier, Vico; Neubauer, Heinrich; Zimmermann, Stefan; Rabold, Denise; Lübke-Becker, Antina; Seyboldt, Christian

    2014-01-01

    This study presents a DNA microarray-based assay for fast and simple PCR ribotyping of Clostridium difficile strains. Hybridization probes were designed to query the modularly structured intergenic spacer region (ISR), which is also the template for conventional and PCR ribotyping with subsequent capillary gel electrophoresis (seq-PCR) ribotyping. The probes were derived from sequences available in GenBank as well as from theoretical ISR module combinations. A database of reference hybridization patterns was set up from a collection of 142 well-characterized C. difficile isolates representing 48 seq-PCR ribotypes. The reference hybridization patterns calculated by the arithmetic mean were compared using a similarity matrix analysis. The 48 investigated seq-PCR ribotypes revealed 27 array profiles that were clearly distinguishable. The most frequent human-pathogenic ribotypes 001, 014/020, 027, and 078/126 were discriminated by the microarray. C. difficile strains related to 078/126 (033, 045/FLI01, 078, 126, 126/FLI01, 413, 413/FLI01, 598, 620, 652, and 660) and 014/020 (014, 020, and 449) showed similar hybridization patterns, confirming their genetic relatedness, which was previously reported. A panel of 50 C. difficile field isolates was tested by seq-PCR ribotyping and the DNA microarray-based assay in parallel. Taking into account that the current version of the microarray does not discriminate some closely related seq-PCR ribotypes, all isolates were typed correctly. Moreover, seq-PCR ribotypes without reference profiles available in the database (ribotype 009 and 5 new types) were correctly recognized as new ribotypes, confirming the performance and expansion potential of the microarray. PMID:25411174

  7. DNA microarray-based PCR ribotyping of Clostridium difficile.

    PubMed

    Schneeberg, Alexander; Ehricht, Ralf; Slickers, Peter; Baier, Vico; Neubauer, Heinrich; Zimmermann, Stefan; Rabold, Denise; Lübke-Becker, Antina; Seyboldt, Christian

    2015-02-01

    This study presents a DNA microarray-based assay for fast and simple PCR ribotyping of Clostridium difficile strains. Hybridization probes were designed to query the modularly structured intergenic spacer region (ISR), which is also the template for conventional and PCR ribotyping with subsequent capillary gel electrophoresis (seq-PCR) ribotyping. The probes were derived from sequences available in GenBank as well as from theoretical ISR module combinations. A database of reference hybridization patterns was set up from a collection of 142 well-characterized C. difficile isolates representing 48 seq-PCR ribotypes. The reference hybridization patterns calculated by the arithmetic mean were compared using a similarity matrix analysis. The 48 investigated seq-PCR ribotypes revealed 27 array profiles that were clearly distinguishable. The most frequent human-pathogenic ribotypes 001, 014/020, 027, and 078/126 were discriminated by the microarray. C. difficile strains related to 078/126 (033, 045/FLI01, 078, 126, 126/FLI01, 413, 413/FLI01, 598, 620, 652, and 660) and 014/020 (014, 020, and 449) showed similar hybridization patterns, confirming their genetic relatedness, which was previously reported. A panel of 50 C. difficile field isolates was tested by seq-PCR ribotyping and the DNA microarray-based assay in parallel. Taking into account that the current version of the microarray does not discriminate some closely related seq-PCR ribotypes, all isolates were typed correctly. Moreover, seq-PCR ribotypes without reference profiles available in the database (ribotype 009 and 5 new types) were correctly recognized as new ribotypes, confirming the performance and expansion potential of the microarray.

  8. Quantification of HEV RNA by Droplet Digital PCR

    PubMed Central

    Nicot, Florence; Cazabat, Michelle; Lhomme, Sébastien; Marion, Olivier; Sauné, Karine; Chiabrando, Julie; Dubois, Martine; Kamar, Nassim; Abravanel, Florence; Izopet, Jacques

    2016-01-01

    The sensitivity of real-time PCR for hepatitis E virus (HEV) RNA quantification differs greatly among techniques. Standardized tools that measure the real quantity of virus are needed. We assessed the performance of a reverse transcription droplet digital PCR (RT-ddPCR) assay that gives absolute quantities of HEV RNA. Analytical and clinical validation was done on HEV genotypes 1, 3 and 4, and was based on open reading frame (ORF)3 amplification. The within-run and between-run reproducibilities were very good, the analytical sensitivity was 80 HEV RNA international units (IU)/mL and linearities of HEV genotype 1, 3 and 4 were very similar. Clinical validation based on 45 samples of genotype 1, 3 or 4 gave results that correlated well with a validated reverse transcription quantitative PCR (RT-qPCR) assay (Spearman rs = 0.89, p < 0.0001). The RT-ddPCR assay is a sensitive method and could be a promising tool for standardizing HEV RNA quantification in various sample types. PMID:27548205

  9. PCR test for detecting Taenia solium cysticercosis in pig carcasses.

    PubMed

    Sreedevi, Chennuru; Hafeez, Mohammad; Kumar, Putcha Anand; Rayulu, Vukka Chengalva; Subramanyam, Kothapalli Venkata; Sudhakar, Krovvidi

    2012-01-01

    Polymerase chain reaction (PCR) test was employed to detect Taenia solium DNA in muscle lesions for validation of the meat inspection results of slaughtered pigs. Two sets of oligonucleotide primers, one targeted against the large subunit rRNA gene (TBR primers) and the other targeted against cytochrome c oxidase subunit 1 gene (Cox1 primers) of T. solium were used in this study. On reactivity in PCR test, the TBR primers and the Cox1 primers yielded products of 286 and 984 bp, respectively, in cysticercosis positive cases. Both the sets of primers were found to be highly specific, since they did not yield any PCR product in negative controls. A total of 225 pig carcasses were screened for cysticercosis by meat inspection, out of which 25 carcasses with visible cysts (16 viable and 9 degenerated cysts) were also confirmed to be positive for cysticercosis in PCR test. However, out of the 35 carcasses with suspected lesions on meat inspection, only two were found to be positive for cysticercosis in PCR test. The detection limits for both the primer sets were analyzed. The TBR primer set could detect up to 10 pg of cysticercus DNA, whereas the Cox1 primer set could detect only up to 1 ng. It is evident from the study that PCR test is an efficient tool for validation of meat inspection results and also to rule out ambiguity in carcass judgment of suspected cases of porcine cysticercosis.

  10. Improved PCR Amplification of Broad Spectrum GC DNA Templates.

    PubMed

    Guido, Nicholas; Starostina, Elena; Leake, Devin; Saaem, Ishtiaq

    2016-01-01

    Many applications in molecular biology can benefit from improved PCR amplification of DNA segments containing a wide range of GC content. Conventional PCR amplification of DNA sequences with regions of GC less than 30%, or higher than 70%, is complex due to secondary structures that block the DNA polymerase as well as mispriming and mis-annealing of the DNA. This complexity will often generate incomplete or nonspecific products that hamper downstream applications. In this study, we address multiplexed PCR amplification of DNA segments containing a wide range of GC content. In order to mitigate amplification complications due to high or low GC regions, we tested a combination of different PCR cycling conditions and chemical additives. To assess the fate of specific oligonucleotide (oligo) species with varying GC content in a multiplexed PCR, we developed a novel method of sequence analysis. Here we show that subcycling during the amplification process significantly improved amplification of short template pools (~200 bp), particularly when the template contained a low percent of GC. Furthermore, the combination of subcycling and 7-deaza-dGTP achieved efficient amplification of short templates ranging from 10-90% GC composition. Moreover, we found that 7-deaza-dGTP improved the amplification of longer products (~1000 bp). These methods provide an updated approach for PCR amplification of DNA segments containing a broad range of GC content.

  11. PCR performance of a thermostable heterodimeric archaeal DNA polymerase.

    PubMed

    Killelea, Tom; Ralec, Céline; Bossé, Audrey; Henneke, Ghislaine

    2014-01-01

    DNA polymerases are versatile tools used in numerous important molecular biological core technologies like the ubiquitous polymerase chain reaction (PCR), cDNA cloning, genome sequencing, and nucleic acid based diagnostics. Taking into account the multiple DNA amplification techniques in use, different DNA polymerases must be optimized for each type of application. One of the current tendencies is to reengineer or to discover new DNA polymerases with increased performance and broadened substrate spectra. At present, there is a great demand for such enzymes in applications, e.g., forensics or paleogenomics. Current major limitations hinge on the inability of conventional PCR enzymes, such as Taq, to amplify degraded or low amounts of template DNA. Besides, a wide range of PCR inhibitors can also impede reactions of nucleic acid amplification. Here we looked at the PCR performances of the proof-reading D-type DNA polymerase from P. abyssi, Pab-polD. Fragments, 3 kilobases in length, were specifically PCR-amplified in its optimized reaction buffer. Pab-polD showed not only a greater resistance to high denaturation temperatures than Taq during cycling, but also a superior tolerance to the presence of potential inhibitors. Proficient proof-reading Pab-polD enzyme could also extend a primer containing up to two mismatches at the 3' primer termini. Overall, we found valuable biochemical properties in Pab-polD compared to the conventional Taq, which makes the enzyme ideally suited for cutting-edge PCR-applications.

  12. PCR performance of a thermostable heterodimeric archaeal DNA polymerase

    PubMed Central

    Killelea, Tom; Ralec, Céline; Bossé, Audrey; Henneke, Ghislaine

    2014-01-01

    DNA polymerases are versatile tools used in numerous important molecular biological core technologies like the ubiquitous polymerase chain reaction (PCR), cDNA cloning, genome sequencing, and nucleic acid based diagnostics. Taking into account the multiple DNA amplification techniques in use, different DNA polymerases must be optimized for each type of application. One of the current tendencies is to reengineer or to discover new DNA polymerases with increased performance and broadened substrate spectra. At present, there is a great demand for such enzymes in applications, e.g., forensics or paleogenomics. Current major limitations hinge on the inability of conventional PCR enzymes, such as Taq, to amplify degraded or low amounts of template DNA. Besides, a wide range of PCR inhibitors can also impede reactions of nucleic acid amplification. Here we looked at the PCR performances of the proof-reading D-type DNA polymerase from P. abyssi, Pab-polD. Fragments, 3 kilobases in length, were specifically PCR-amplified in its optimized reaction buffer. Pab-polD showed not only a greater resistance to high denaturation temperatures than Taq during cycling, but also a superior tolerance to the presence of potential inhibitors. Proficient proof-reading Pab-polD enzyme could also extend a primer containing up to two mismatches at the 3' primer termini. Overall, we found valuable biochemical properties in Pab-polD compared to the conventional Taq, which makes the enzyme ideally suited for cutting-edge PCR-applications. PMID:24847315

  13. Rapid diagnosis of goose viral infections by multiplex PCR.

    PubMed

    Chen, Zongyan; Li, Chuanfeng; Li, Guoxin; Yu, Hai; Jiang, Yifeng; Yan, Liping; Meng, Chunchun; Zhou, Yanjun; Tong, Guangzhi; Liu, Guangqing

    2013-08-01

    Goose parvovirus (GPV), newcastle disease virus (NDV), goose herpesvirus (GHV) and goose adenovirus (GAV) are considered collectively to be four of the most important and widespread viruses of geese. Because all of these viruses cause similar pathological changes, histological differentiation among these viruses is difficult. A reliable, specific and sensitive multiplex PCR (mPCR) assay was developed for the combined detection of GPV, NDV, GHV and GAV in clinical samples of geese. Using the mPCR technique, single infections with GPV (28/76; 36.8%), NDV (9/76; 11.8%), GHV (3/76; 3.9%) and GAV (12/76; 15.8%) were identified in the samples; co-infections with GAV and either GPV or NDV (31.6%; 24/76) were also identified with this approach. The results for all of the samples tested were the same in both the uPCR and mPCR systems. The mPCR approach is considered to be useful for routine molecular diagnosis and epidemiological applications in geese.

  14. Specific PCR product primer design using memetic algorithm.

    PubMed

    Yang, Cheng-Hong; Cheng, Yu-Huei; Chuang, Li-Yeh; Chang, Hsueh-Wei

    2009-01-01

    To provide feasible primer sets for performing a polymerase chain reaction (PCR) experiment, many primer design methods have been proposed. However, the majority of these methods require a relatively long time to obtain an optimal solution since large quantities of template DNA need to be analyzed. Furthermore, the designed primer sets usually do not provide a specific PCR product size. In recent years, evolutionary computation has been applied to PCR primer design and yielded promising results. In this article, a memetic algorithm (MA) is proposed to solve primer design problems associated with providing a specific product size for PCR experiments. The MA is compared with a genetic algorithm (GA) using an accuracy formula to estimate the quality of the primer design and test the running time. Overall, 50 accession nucleotide sequences were sampled for the comparison of the accuracy of the GA and MA for primer design. Five hundred runs of the GA and MA primer design were performed with PCR product lengths of 150-300 bps and 500-800 bps, and two different methods of calculating T(m) for each accession nucleotide sequence were tested. A comparison of the accuracy results for the GA and MA primer design showed that the MA primer design yielded better results than the GA primer design. The results further indicate that the proposed method finds optimal or near-optimal primer sets and effective PCR products in a dry dock experiment. Related materials are available online at http://bio.kuas.edu.tw/ma-pd/.

  15. Comparison of Droplet Digital PCR and qPCR for the Quantification of Shiga Toxin-Producing Escherichia coli in Bovine Feces.

    PubMed

    Verhaegen, Bavo; De Reu, Koen; De Zutter, Lieven; Verstraete, Karen; Heyndrickx, Marc; Van Coillie, Els

    2016-05-18

    Cattle are considered to be the main reservoir for Shiga toxin-producing Escherichia coli (STEC) and are often the direct or indirect source of STEC outbreaks in humans. Accurate measurement of the concentration of shed STEC in cattle feces could be a key answer to questions concerning transmission of STEC, contamination sources and efficiency of treatments at farm level. Infected animals can be identified and the contamination level quantified by real-time quantitative PCR (qPCR), which has its specific limitations. Droplet digital PCR (ddPCR) has been proposed as a method to overcome many of the drawbacks of qPCR. This end-point amplification PCR is capable of absolute quantification independent from any reference material and is less prone to PCR inhibition than qPCR. In this study, the qPCR-based protocol described by Verstraete et al. (2014) for Shiga toxin genes stx1 and stx2 and the intimin gene eae quantification was optimized for ddPCR analysis. The properties of ddPCR and qPCR using two different mastermixes (EMM: TaqMan(®) Environmental Master Mix 2.0; UMM: TaqMan(®) Universal PCR Master Mix) were evaluated, using standard curves and both artificial and natural contaminated cattle fecal samples. In addition, the susceptibility of these assays to PCR-inhibitors was investigated. Evaluation of the standard curves and both artificial and natural contaminated cattle fecal samples suggested a very good agreement between qPCR using EMM and ddPCR. Furthermore, similar sensitivities and no PCR inhibition were recorded for both assays. On the other hand, qPCR using UMM was clearly prone to PCR inhibition. In conclusion, the ddPCR technique shows potential for the accurate absolute quantification of STEC on the farms, without relying on standardized reference material.

  16. Comparison of Droplet Digital PCR and qPCR for the Quantification of Shiga Toxin-Producing Escherichia coli in Bovine Feces

    PubMed Central

    Verhaegen, Bavo; De Reu, Koen; De Zutter, Lieven; Verstraete, Karen; Heyndrickx, Marc; Van Coillie, Els

    2016-01-01

    Cattle are considered to be the main reservoir for Shiga toxin-producing Escherichia coli (STEC) and are often the direct or indirect source of STEC outbreaks in humans. Accurate measurement of the concentration of shed STEC in cattle feces could be a key answer to questions concerning transmission of STEC, contamination sources and efficiency of treatments at farm level. Infected animals can be identified and the contamination level quantified by real-time quantitative PCR (qPCR), which has its specific limitations. Droplet digital PCR (ddPCR) has been proposed as a method to overcome many of the drawbacks of qPCR. This end-point amplification PCR is capable of absolute quantification independent from any reference material and is less prone to PCR inhibition than qPCR. In this study, the qPCR-based protocol described by Verstraete et al. (2014) for Shiga toxin genes stx1 and stx2 and the intimin gene eae quantification was optimized for ddPCR analysis. The properties of ddPCR and qPCR using two different mastermixes (EMM: TaqMan® Environmental Master Mix 2.0; UMM: TaqMan® Universal PCR Master Mix) were evaluated, using standard curves and both artificial and natural contaminated cattle fecal samples. In addition, the susceptibility of these assays to PCR-inhibitors was investigated. Evaluation of the standard curves and both artificial and natural contaminated cattle fecal samples suggested a very good agreement between qPCR using EMM and ddPCR. Furthermore, similar sensitivities and no PCR inhibition were recorded for both assays. On the other hand, qPCR using UMM was clearly prone to PCR inhibition. In conclusion, the ddPCR technique shows potential for the accurate absolute quantification of STEC on the farms, without relying on standardized reference material. PMID:27213452

  17. Absolute quantification of olive oil DNA by droplet digital-PCR (ddPCR): Comparison of isolation and amplification methodologies.

    PubMed

    Scollo, Francesco; Egea, Leticia A; Gentile, Alessandra; La Malfa, Stefano; Dorado, Gabriel; Hernandez, Pilar

    2016-12-15

    Olive oil is considered a premium product for its nutritional value and health benefits, and the ability to define its origin and varietal composition is a key step towards ensuring the traceability of the product. However, isolating the DNA from such a matrix is a difficult task. In this study, the quality and quantity of olive oil DNA, isolated using four different DNA isolation protocols, was evaluated using the qRT-PCR and ddPCR techniques. The results indicate that CTAB-based extraction methods were the best for unfiltered oil, while Nucleo Spin-based extraction protocols showed greater overall reproducibility. The use of both qRT-PCR and ddPCR led to the absolute quantification of the DNA copy number. The results clearly demonstrate the importance of the choice of DNA-isolation protocol, which should take into consideration the qualitative aspects of DNA and the evaluation of the amplified DNA copy number.

  18. Detection of Leishmania infantum in 4 different dog samples by real-time PCR and ITS-1 nested PCR.

    PubMed

    Carvalho Ferreira, Aline Leandra; Carregal, Virgínia Mendes; de Almeida Ferreira, Sidney; Leite, Rodrigo Souza; de Andrade, Antero Silva Ribeiro

    2014-04-01

    The canine visceral leishmaniasis (CVL) diagnosis is an important step of visceral leishmaniasis control program in Brazil, which involves the elimination of infected dogs, the main animal reservoir host of the disease. The aim of the present study was to evaluate a sensitive real-time PCR method for Leishmania infantum detection in 4 different clinical samples of dogs, including the noninvasive conjunctival swab (CS) sample. The results of real-time PCR were compared with those obtained using internal transcribed spacer 1 nested PCR. Animals were divided into 2 groups based on the absence or presence of CVL clinical sings. The CS associated with real-time PCR, using primers addressed to kinetoplast DNA minicircles, was able to detect L. infantum infection in 96.7% of dogs without clinical signs and in 100% of the symptomatic animals, demonstrating the importance of these procedures for diagnosing CVL.

  19. Assessing UV Inactivation of Adenovirus 41 Using Integrated Cell Culture Real-Time qPCR/RT-qPCR.

    PubMed

    Ding, Ning; Craik, Stephen A; Pang, Xiaoli; Lee, Bonita; Neumann, Norman F

    2017-04-01

      Enteric adenoviruses are among most UV-resistant viruses in water. Cytopathic effects (CPE)-based cell culture TCID50 assay as a conventional virus assessment approach has major drawbacks for enteric adenovirus since it is selective on cell lines and takes longer time to show CPE. Integrated cell culture real-time quantitative PCR (ICC-qPCR) and reverse transcriptase (RT)-qPCR were applied in this study, in comparison with TCID50, to assess UV inactivation of adenovirus type 41 (Ad41) in water. Adenovirus type 41 was exposed to UV doses of 40, 80, 160, and 320 mJ/cm2 using a collimated beam apparatus. There was no significant difference of inactivation at conducted UV doses between measurements using TCID50 assay and ICC-RT-qPCR. Both assays fitted the Chick-Watson model at 95% confidence level. The inactivation measured by ICC-qPCR did not fit the Chick-Watson model. In summary, ICC-RT-qPCR is the most appropriate alternate to CPE-based assay for assessing UV inactivation of enteric adenoviruses.

  20. Inverse PCR for subtyping of Acinetobacter baumannii carrying ISAba1.

    PubMed

    Kim, Shukho; Park, Yun-Ju; Kim, Jungmin

    2016-05-01

    Acinetobacter baumannii has been prevalent in nosocomial infections, often causing outbreaks in intensive care units. ISAba1 is an insertion sequence that has been identified only in A. baumannii and its copy number varies among strains. It has been reported that ISAba1 provides a promoter for bla(OXA-51-like), bla(OXA-23-like), and bla(ampC), which are associated with the resistance of A. baumannii to carbapenems and cephalosporins. The main purpose of this study was to develop a novel inverse PCR method capable of typing A. baumannii strains. The method involves three major steps: cutting of genomic DNA with a restriction enzyme, ligation, and PCR. In the first step, bacterial genomic DNA was digested with DpnI. In the second step, the digested genomic DNAs were ligated to form intramolecular circular DNAs. In the last step, the ligated circular DNAs were amplified by PCR with primers specific for ISAba1 and the amplified PCR products were electrophoresed. Twenty-two clinical isolates of A. baumannii were used for the evaluation of the inverse PCR (iPCR) typing method. Dendrogram analysis revealed two major clusters, similar to pulsed-field gel electrophoresis (PFGE) results. Three ISAba1-associated genes--bla(ampC), bla(OXA-66-like), and csuD--were amplified and detected in the clinical isolates. This novel iPCR typing method is comparable to PFGE in its ability to discriminate A. baumannii strains, and is a promising molecular epidemiological tool for investigating A. baumannii carrying ISAba1.

  1. Early diagnosis of Lassa fever by reverse transcription-PCR.

    PubMed

    Demby, A H; Chamberlain, J; Brown, D W; Clegg, C S

    1994-12-01

    We developed a method based on a coupled reverse transcription-PCR (RT-PCR) for the detection of Lassa virus using primers specific for regions of the S RNA segment which are well conserved between isolates from Sierra Leone, Liberia, and Nigeria. The specificity of the assay was confirmed by Southern blotting with a chemiluminescent probe. The assay was able to detect 1 to 10 copies of a plasmid or an RNA transcript containing the target sequence. There was complete concordance between RT-PCR and virus culture for the detection of Lassa virus in a set of 29 positive and 32 negative serum samples obtained on admission to the hospital from patients suspected of having Lassa fever in Sierra Leone. Specificity was confirmed by the failure of amplification of specific products from serum samples collected from 129 healthy blood donors in Sierra Leone or from tissue culture supernatants from cells infected with related arenaviruses (Mopeia, lymphocytic choriomeningitis, Tacaribe, and Pichinde viruses). Sequential serum samples from 29 hospitalized patients confirmed to have Lassa fever were tested by RT-PCR and for Lassa virus-specific antibodies by indirect immunofluorescence (IF). RT-PCR detected virus RNA in 79% of the patients at the time of admission, comparing favorably with IF, which detected antibodies in only 21% of the patients. Lassa virus RNA was detected by RT-PCR in all 29 patients by the third day of admission, whereas antibody was detectable by IF in only 52% of the patients. These results point to an important role for RT-PCR in the management of suspected cases of Lassa fever.

  2. Droplet-based micro oscillating-flow PCR chip

    NASA Astrophysics Data System (ADS)

    Wang, Wei; Li, Zhi-Xin; Luo, Rong; Lü, Shu-Hai; Xu, Ai-Dong; Yang, Yong-Jun

    2005-08-01

    Polymerase chain reactions (PCR), thermally activated chemical reactions which are widely used for nucleic acid amplification, have recently received much attention in microelectromechanical systems and micro total analysis systems because a wide variety of DNA/RNA molecules can be enriched by PCR for further analyses. In the present work, a droplet-based micro oscillating-flow PCR chip was designed and fabricated by the silicon microfabrication technique. Three different temperature zones, which were stable at denaturation, extension and annealing temperatures and isolated from each other by a thin-wall linkage, were integrated with a single, simple and straight microchannel to form the chip's basic functional structure. The PCR mixture was injected into the chip as a single droplet and flowed through the three temperature zones in the main microchannel in an oscillating manner to achieve the temperature maintenance and transitions. The chip's thermal performance was theoretically analyzed and numerically simulated. The results indicated that the time needed for the temperature of the droplet to change to the target value is less than 1 s, and the root mean square error of temperature is less than 0.2 °C. A droplet of 1 µl PCR mixture with standard HPV (Human Papilloma Virus)-DNA sample inside was amplified by the present chip and the results were analyzed by slab gel electrophoresis with separation of DNA markers in parallel. The electrophoresis results demonstrated that the micro oscillating-flow PCR chip successfully amplified the HPV-DNA, with a processing time of about 15 min which is significantly reduced compared to that for the conventional PCR instrument.

  3. Pneumocystis sp. in bats evaluated by qPCR.

    PubMed

    Cavallini Sanches, E M; Ferreiro, L; Andrade, C P; Pacheco, S M; Almeida, L L; Spanamberg, A; Wissmann, G

    2013-03-01

    Molecular techniques have revealed a high prevalence of Pneumocystis colonization in wild mammals. Accurate quantification of Pneumocystis sp. is essential for the correct interpretation of many research experiments investigating this organism. The objectives of this study were to detect the presence of Pneumocystis sp. in bats by qPCR, and to distinguish colonization from infection. Probes and primers for real time PCR (qPCR) were designed based on the gene of major surface glycoprotein (MSG) of Pneumocystis sp., in order to analyze 195 lung tissue samples from bats captured (2007-2009). All samples were also analyzed by nested PCR, using oligonucleotide primers designed for the gene encoding the mitochondrial small subunit rRNA (mtSSU rRNA) to confirm the results. The qPCR assay was standardized using a standard curve made with the DNA extracted from bronchoalveolar lavage positive for Pneumocystis jirovecii. The average Ct was found to be between 13 and 14 (calibration curve) for the detection of infection with Pneumocystis sp. and above these values for colonization. It was considered as negative samples the ones that had Ct values equal to 50. Out of the total 195 samples, 47 (24.1%) bat lung DNA samples were positive for Pneumocystis sp. by qPCR. The most common bat species found were: Tadarida brasiliensis (23.4%), Histiotus velatus (17.0%), Desmodus rotundus (14.9%) and Molossus molossus (8.5%). The average cycle threshold of the positive samples (bats) was 25.8 and standard deviation was 1.7. The DNA samples with Ct values greater than 14 suggest that these animals might be colonized by Pneumocystis sp. Results obtained in this study demonstrated the usefulness of the qPCR procedure for identification of Pneumocystis sp. and for distinction between its colonizing or infectious status in bats.

  4. Development of internal controls for PCR detection of Bacillus anthracis.

    PubMed

    Brightwell, G; Pearce, M; Leslie, D

    1998-12-01

    This work describes the development and evaluation of a multiplex polymerase chain reaction (PCR) for the detection of Bacillus anthracis strains harbouring plasmid pX02. The multiplex also incorporated an internal control (IC) to avoid false negative reactions. Internal controls consisted of plasmids containing modified PCR target sequences, corresponding to the capC and BA813 genes of B. anthracis, which were then co-amplified with the original target sequences using the same set of amplimers. The initial IC construct comprised of an internally deleted form of the genomic target sequence cloned into pUC19. A series of nested DNA fragments corresponding to the 23S rRNA sequences of Bacillus cereus were then subcloned into the point of deletion, producing a number of IC constructs with similar sequences but increasing product size on PCR amplification. Neither the presence of IC DNA template or IC PCR product size affected the specificity or non-specific cross-reactivity of the original PCR assay. The concentration of IC was critical, too much IC DNA template would out compete the genomic DNA template, thus giving a false negative result. However, when the concentration of IC was optimal assay sensitivity was not compromised.

  5. Optimizing methods for PCR-based analysis of predation

    PubMed Central

    Sint, Daniela; Raso, Lorna; Kaufmann, Rüdiger; Traugott, Michael

    2011-01-01

    Molecular methods have become an important tool for studying feeding interactions under natural conditions. Despite their growing importance, many methodological aspects have not yet been evaluated but need to be considered to fully exploit the potential of this approach. Using feeding experiments with high alpine carabid beetles and lycosid spiders, we investigated how PCR annealing temperature affects prey DNA detection success and how post-PCR visualization methods differ in their sensitivity. Moreover, the replicability of prey DNA detection among individual PCR assays was tested using beetles and spiders that had digested their prey for extended times postfeeding. By screening all predators for three differently sized prey DNA fragments (range 116–612 bp), we found that only in the longest PCR product, a marked decrease in prey detection success occurred. Lowering maximum annealing temperatures by 4 °C resulted in significantly increased prey DNA detection rates in both predator taxa. Among the three post-PCR visualization methods, an eightfold difference in sensitivity was observed. Repeated screening of predators increased the total number of samples scoring positive, although the proportion of samples testing positive did not vary significantly between different PCRs. The present findings demonstrate that assay sensitivity, in combination with other methodological factors, plays a crucial role to obtain robust trophic interaction data. Future work employing molecular prey detection should thus consider and minimize the methodologically induced variation that would also allow for better cross-study comparisons. PMID:21507208

  6. High-throughput PCR in silicon based microchamber array.

    PubMed

    Nagai, H; Murakami, Y; Yokoyama, K; Tamiya, E

    2001-12-01

    Highly integrated hybridization assay and capillary electrophoresis have improved the throughput of DNA analysis. The shift to high throughput analysis requires a high speed DNA amplification system, and several rapid PCR systems have been developed. In these thermal cyclers, the temperature was controlled by effective methodology instead of a large heating/cooling block preventing rapid thermal cycling. In our research, high speed PCR was performed using a silicon-based microchamber array and three heat blocks. The highly integrated microchamber array was fabricated by semiconductor microfabrication techniques. The temperature of the PCR microchamber was controlled by alternating between three heat blocks of different temperature. In general, silicon has excellent thermal conductivity, and the heat capacity is small in the miniaturized sample volume. Hence, the heating/cooling rate was rapid, approximately 16 degrees C/s. The rapid PCR was therefore completed in 18 min for 40 cycles. The thermal cycle time was reduced to 1/10 of a commercial PCR instrument (Model 9600, PE Applied Biosystems-3 h).

  7. Digital PCR using micropatterned superporous absorbent array chips.

    PubMed

    Wang, Yazhen; Southard, Kristopher M; Zeng, Yong

    2016-06-21

    Digital PCR (dPCR) is an emerging technology for genetic analysis and clinical diagnostics. To facilitate the widespread application of dPCR, here we developed a new micropatterned superporous absorbent array chip (μSAAC) which consists of an array of microwells packed with highly porous agarose microbeads. The packed beads construct a hierarchically porous microgel which confers superior water adsorption capacity to enable spontaneous filling of PDMS microwells for fluid compartmentalization without the need of sophisticated microfluidic equipment and operation expertise. Using large λ-DNA as the model template, we validated the μSAAC for stochastic partitioning and quantitative digital detection of DNA molecules. Furthermore, as a proof-of-concept, we conducted dPCR detection and single-molecule sequencing of a mutation prevalent in blood cancer, the chromosomal translocation t(14;18), demonstrating the feasibility of the μSAAC for analysis of disease-associated mutations. These experiments were carried out using the standard molecular biology techniques and instruments. Because of its low cost, ease of fabrication, and equipment-free liquid partitioning, the μSAAC is readily adaptable to general lab settings, which could significantly facilitate the widespread application of dPCR technology in basic research and clinical practice.

  8. Multiplex PCR Tests for Detection of Pathogens Associated with Gastroenteritis

    PubMed Central

    Zhang, Hongwei; Morrison, Scott; Tang, Yi-Wei

    2016-01-01

    Synopsis A wide range of enteric pathogens can cause infectious gastroenteritis. Conventional diagnostic algorithms including culture, biochemical identification, immunoassay and microscopic examination are time consuming and often lack sensitivity and specificity. Advances in molecular technology have as allowed its use as clinical diagnostic tools. Multiplex PCR based testing has made its way to gastroenterology diagnostic arena in recent years. In this article we present a review of recent laboratory developed multiplex PCR tests and current commercial multiplex gastrointestinal pathogen tests. We will focus on two FDA cleared commercial syndromic multiplex tests: Luminex xTAG GPP and Biofire FimArray GI test. These multiplex tests can detect and identify multiple enteric pathogens in one test and provide results within hours. Multiplex PCR tests have shown superior sensitivity to conventional methods for detection of most pathogens. The high negative predictive value of these multiplex tests has led to the suggestion that they be used as screening tools especially in outbreaks. Although the clinical utility and benefit of multiplex PCR test are to be further investigated, implementing these multiplex PCR tests in gastroenterology diagnostic algorithm has the potential to improve diagnosis of infectious gastroenteritis. PMID:26004652

  9. Optimizing methods for PCR-based analysis of predation.

    PubMed

    Sint, Daniela; Raso, Lorna; Kaufmann, Rüdiger; Traugott, Michael

    2011-09-01

    Molecular methods have become an important tool for studying feeding interactions under natural conditions. Despite their growing importance, many methodological aspects have not yet been evaluated but need to be considered to fully exploit the potential of this approach. Using feeding experiments with high alpine carabid beetles and lycosid spiders, we investigated how PCR annealing temperature affects prey DNA detection success and how post-PCR visualization methods differ in their sensitivity. Moreover, the replicability of prey DNA detection among individual PCR assays was tested using beetles and spiders that had digested their prey for extended times postfeeding. By screening all predators for three differently sized prey DNA fragments (range 116-612 bp), we found that only in the longest PCR product, a marked decrease in prey detection success occurred. Lowering maximum annealing temperatures by 4 °C resulted in significantly increased prey DNA detection rates in both predator taxa. Among the three post-PCR visualization methods, an eightfold difference in sensitivity was observed. Repeated screening of predators increased the total number of samples scoring positive, although the proportion of samples testing positive did not vary significantly between different PCRs. The present findings demonstrate that assay sensitivity, in combination with other methodological factors, plays a crucial role to obtain robust trophic interaction data. Future work employing molecular prey detection should thus consider and minimize the methodologically induced variation that would also allow for better cross-study comparisons.

  10. Electrochemistry-based real-time PCR on a microchip.

    PubMed

    Yeung, Stephen S W; Lee, Thomas M H; Hsing, I-Ming

    2008-01-15

    The development of handheld instruments for point-of-care DNA analysis can potentially contribute to the medical diagnostics and environmental monitoring for decentralized applications. In this work, we demonstrate the implementation of a recently developed electrochemical real-time polymerase chain reaction (ERT-PCR) technique on a silicon-glass microchip for simultaneous DNA amplification and detection. This on-chip ERT-PCR process requires the extension of an oligonucleotide in both solution and at solid phases and intermittent electrochemical signal measurement in the presence of all the PCR reagents. Several important parameters, related to the surface passivation and electrochemical scanning of working electrodes, were investigated. It was found that the ERT-PCR's onset thermal cycle ( approximately 3-5), where the analytical signal begins to be distinguishable from the background, is much lower than that of the fluorescence-based counterparts for high template DNA situations (3 x 10(6) copies/microL). By carefully controlling the concentrations of the immobilized probe and the enzyme polymerase, improvements have been made in obtaining a meaningful electrochemical signal using a lower initial template concentration. This ERT-PCR technique on a microchip platform holds significant promise for rapid DNA detection for point-of-care testing applications.

  11. Application of PCR and real-time PCR for monitoring cyanobacteria, Microcystis spp. and Cylindrospermopsis raciborskii in Macau freshwater reservoir

    NASA Astrophysics Data System (ADS)

    Zhang, Weiying; Lou, Inchio; Ung, Wai Kin; Kong, Yijun; Mok, Kai Meng

    2014-06-01

    Freshwater algal blooms have become a growing concern world-wide. They are caused by a high level of cyanobacteria, predominantly Microcystis spp. and Cylindrospermopsis raciborskii, which can produce microcystin and cylindrospermopsin, respectively. Longtime exposure to these cyanotoxins may affect public health, thus reliable detection, quantification, and enumeration of these harmful algae species has become a priority in water quality management. Traditional manual enumeration of algal bloom cells primarily involves microscopic identification which limited by inaccuracy and time-consumption.With the development of molecular techniques and an increasing number of microbial sequences available in the Genbank database, the use of molecular methods can be used for more rapid, reliable, and accurate detection and quantification. In this study, multiplex polymerase chain reaction (PCR) and real-time quantitative PCR (qPCR) techniques were developed and applied for monitoring cyanobacteria Microcystis spp. and C. raciborskii in the Macau Storage Reservoir (MSR). The results showed that the techniques were successful for identifying and quantifying the species in pure cultures and mixed cultures, and proved to be a potential application for water sampling in MSR. When the target species were above 1 million cells/L, similar cell numbers estimated by microscopic enumeration and qPCR were obtained. Further quantification in water samples indicated that the ratio of the estimated number of cell by microscopy and qPCR was 0.4-12.9 for cyanobacteria and 0.2-3.9 for C. raciborskii. However, Microcystis spp. was not observed by manual enumeration, while it was detected at low levels by qPCR, suggesting that qPCR is more sensitive and accurate. Thus the molecular approaches provide an additional reliable monitoring option to traditional microscopic enumeration for the ecosystems monitoring program.

  12. Quantification of viable bacteria in wastewater treatment plants by using propidium monoazide combined with quantitative PCR (PMA-qPCR).

    PubMed

    Li, Dan; Tong, Tiezheng; Zeng, Siyu; Lin, Yiwen; Wu, Shuxu; He, Miao

    2014-02-01

    The detection of viable bacteria in wastewater treatment plants (WWTPs) is very important for public health, as WWTPs are a medium with a high potential for waterborne disease transmission. The aim of this study was to use propidium monoazide (PMA) combined with the quantitative polymerase chain reaction (PMA-qPCR) to selectively detect and quantify viable bacteria cells in full-scale WWTPs in China. PMA was added to the concentrated WWTP samples at a final concentration of 100 micromol/L and the samples were incubated in the dark for 5 min, and then lighted for 4 min prior to DNA extraction and qPCR with specific primers for Escherichia coli and Enterococci, respectively. The results showed that PMA treatment removed more than 99% of DNA from non-viable cells in all the WWTP samples, while matrices in sludge samples markedly reduced the effectiveness of PMA treatment. Compared to qPCR, PMA-qPCR results were similar and highly linearly correlated to those obtained by culture assay, indicating that DNA from non-viable cells present in WWTP samples can be eliminated by PMA treatment, and that PMA-qPCR is a reliable method for detection of viable bacteria in environmental samples. This study demonstrated that PMA-qPCR is a rapid and selective detection method for viable bacteria in WWTP samples, and that WWTPs have an obvious function in removing both viable and non-viable bacteria. The results proved that PMA-qPCR is a promising detection method that has a high potential for application as a complementary method to the standard culture-based method in the future.

  13. [Use of nested PCR in detection of the plague pathogen].

    PubMed

    Glukhov, A I; Gordeev, S A; Al'tshuler, M L; Zykova, I E; Severin, S E

    2003-07-01

    Causative agents of plague, i.e. bacterium Yersina pestis (in the subcutaneous tissues of rodents) and their cutaneous parasites need to be isolated to enable plague prevention. A comparatively new method of polymerase chain reaction (PCR) opens up new possibilities of determining Y. pestis just within several hours and without any cultivation. The article contains a description of the PCR-method, which makes it possible to distinguish the culture of Y. pestis from cultures of other microorganism, including speci of Yersina. The method is of the cluster-type, i.e. it is made up of subsequent PC reactions with the substrate for the second reaction being the product of the first one. The cluster nature of the method preconditions a higher sensitivity and specificity versus the ordinary PCR.

  14. Heat properties of an integrated micro PCR vessel

    NASA Astrophysics Data System (ADS)

    Zhao, Zhan; Cui, Dafu; Yu, Zhongyao; Wang, Li; Xia, Shanhong; Cui, Zheng

    2001-09-01

    The PCR amplification is based on multiple temperature cycles of DNA synthesis; each includes denaturation of the template, annealing of the primers to complementary sites in the template and primer extension. The key technique of PCR amplification is the heating control in design and fabrication of its chip form. The specifications of the chip are heat properties. In this paper the heat properties of a micro PCR vessel integration heater and temperature sensor was introduced. The temperature distribution of the vessel was simulated with software tool IntelliSuite. The temperatures cycles were measured and the time response of the chip was discussed. It is found that the integrate micro vessel is a very useful tool not only for DNA synthesis but also as a biochemical reactor for many other biological and chemical analyses.

  15. Noninvasive genotyping of common marmoset (Callithrix jacchus) by fingernail PCR.

    PubMed

    Takabayashi, Shuji; Katoh, Hideki

    2015-07-01

    The common marmoset (Callithrix jacchus) is a New World primate that is a useful model for medical studies. In this study, we report a convenient, reliable, and noninvasive procedure to genotype a living common marmoset by using fingernails. This method was used to successfully genotype DNA by restriction fragment length polymorphism (RFLP) PCR without prior purification, by using the KOD FX PCR enzyme kit. Additionally, there is no sample contamination from hematopoietic chimera derived from fused placenta in utero. We compared chimeric levels between various tissues in females with male littermates using quantitative fluorescent (QF)-PCR to prepare a reliable DNA source for genetic analyses, such as genotyping, gene mapping, or genomic sequencing. The chimerism detected appeared to be restricted to lymphatic tissues, such as bone marrow, thymus, spleen, lymph nodes and blood cells. As a result, DNA from fingernails with the quick is the best DNA source for genetic research in living marmosets.

  16. Detection of alcohol-tolerant hiochi bacteria by PCR.

    PubMed Central

    Nakagawa, T; Shimada, M; Mukai, H; Asada, K; Kato, I; Fujino, K; Sato, T

    1994-01-01

    We report a sensitive and rapid method for detection of hiochi bacteria by PCR. This method involves the electrophoresis of amplified DNA. Nucleotide sequences of the spacer region between 16S and 23S rRNA genes of 11 Lactobacillus strains were identified by analysis of PCR products. Five primers were designed by analysis of similarities among these sequences. A single cell of Lactobacillus casei subsp. casei could be detected when purified genomic DNA was used as the template. When various cell concentrations of L. casei subsp. casei were added to 50 ml of pasteurized sake and the cells were recovered, the detection limit was about one cell. No discrete band was observed in electrophoresis after PCR when human, Escherichia coli, mycoplasma, Acholeplasma, yeast, or mold DNA was used as the template. Images PMID:7510942

  17. Multiplex PCR for identification of herpes virus infections in adolescents.

    PubMed

    Durzyńska, Julia; Pacholska-Bogalska, Joanna; Kaczmarek, Maria; Hanć, Tomasz; Durda, Magdalena; Skrzypczak, Magdalena; Goździcka-Józefiak, Anna

    2011-02-01

    The aim of the study was to develop a multiplex PCR (mPCR) for a rapid and simultaneous detection of herpes simplex 1 (HSV-1), herpes simplex 2 (HSV-2), and human cytomegalovirus (HCMV) DNA in squamous oral cells obtained from adolescents. Accuracy of the method was tested in a group of 513 adolescents, almost 11% of subjects were positive for infection with herpes viruses. Correlations with gender, age, and place of residence were sought. A similar incidence of HSV-2 and HCMV was found (4.3% and 5.4%, respectively) and the incidence of HSV-1 was the lowest (1%) in the study group. Conversely to HSV-2, HCMV was detected mostly in the youngest individuals. The same occurrence of all viruses was observed in boys and girls. The mPCR method described is suggested as a useful tool for epidemiologic studies of active herpes infections.

  18. Identification of neotropical felid faeces using RCP-PCR.

    PubMed

    Roques, S; Adrados, B; Chavez, C; Keller, C; Magnusson, W E; Palomares, F; Godoy, J A

    2011-01-01

    Faeces similarity among sympatric felid species has generally hampered their use in distributional, demographic and dietary studies. Here, we present a new and simple approach based on a set of species-specific primers, for the unambiguous identification of faeces from sympatric neotropical felids (i.e. puma, jaguar, jaguarundi and ocelot/ margay). This method, referred to as rapid classificatory protocol-PCR (RCP-PCR), consists of a single-tube multiplex PCR yielding species-specific banding patterns on agarose gel. The method was optimized with samples of known origin (14 blood and 15 fresh faeces) and validated in faecal samples of unknown origin (n = 138), for some of which (n = 40) we also obtained species identification based on mtDNA sequencing. This approach proved reliable and provides high identification success rates from faeces. Its simplicity and cost effectiveness should facilitate its application for routine surveys of presence and abundance of these species.

  19. Design and evaluation of consensus PCR assays for henipaviruses.

    PubMed

    Feldman, K S; Foord, A; Heine, H G; Smith, I L; Boyd, V; Marsh, G A; Wood, J L N; Cunningham, A A; Wang, L-F

    2009-10-01

    Henipaviruses were first discovered in the 1990s, and their potential threat to public health is of increasing concern with increasing knowledge. Old-world fruit bats are the reservoir hosts for these viruses, and spill-over events cause lethal infections in a wide range of mammalian species, including humans. In anticipation of these spill-over events, and to investigate further the geographical range of these genetically diverse viruses, assays for detection of known and potentially novel strains of henipaviruses are required. The development of multiple consensus PCR assays for the detection of henipaviruses, including both SYBR Green and TaqMan real-time PCRs and a conventional heminested PCR is described. The assays are highly sensitive and have defined specificity. In addition to being useful tools for detection of known and novel henipaviruses, evaluation of assay efficiency and sensitivity across both biological and synthetic templates has provided valuable insight into consensus PCR design and use.

  20. [Application of rapid PCR to authenticate medicinal snakes].

    PubMed

    Chen, Kang; Jiang, Chao; Yuan, Yuan; Huang, Lu-Qi; Li, Man

    2014-10-01

    To obtained an accurate, rapid and efficient method for authenticate medicinal snakes listed in Chinese Pharmacopoeia (Zaocysd humnades, Bungarus multicinctus, Agkistrodon acutus), a rapid PCR method for authenticate snakes and its adulterants was established based on the classic molecular authentication methods. DNA was extracted by alkaline lysis and the specific primers were amplified by two-steps PCR amplification method. The denatured and annealing temperature and cycle numbers were optimized. When 100 x SYBR Green I was added in the PCR product, strong green fluorescence was visualized under 365 nm UV whereas adulterants without. The whole process can complete in 30-45 minutes. The established method provides the technical support for authentication of the snakes on field.

  1. Authentication of medicinal plants by SNP-based multiplex PCR.

    PubMed

    Lee, Ok Ran; Kim, Min-Kyeoung; Yang, Deok-Chun

    2012-01-01

    Highly variable intergenic spacer and intron regions from nuclear and cytoplasmic DNA have been used for species identification. Noncoding internal transcribed spacers (ITSs) located in 18S-5.8S-26S, and 5S ribosomal RNA genes (rDNAs) represent suitable region for medicinal plant authentication. Noncoding regions from two cytoplasmic DNA, chloroplast DNA (trnT-F intergenic spacer region), and mitochondrial DNA (fourth intron region of nad7 gene) are also successfully applied for the proper identification of medicinal plants. Single-nucleotide polymorphism (SNP) sites obtained from the amplification of intergenic spacer and intron regions are properly utilized for the verification of medicinal plants in species level using multiplex PCR. Multiplex PCR as a variant of PCR technique used to amplify more than two loci simultaneously.

  2. Using PCR to Target Misconceptions about Gene Expression †

    PubMed Central

    Wright, Leslie K.; Newman, Dina L.

    2013-01-01

    We present a PCR-based laboratory exercise that can be used with first- or second-year biology students to help overcome common misconceptions about gene expression. Biology students typically do not have a clear understanding of the difference between genes (DNA) and gene expression (mRNA/protein) and often believe that genes exist in an organism or cell only when they are expressed. This laboratory exercise allows students to carry out a PCR-based experiment designed to challenge their misunderstanding of the difference between genes and gene expression. Students first transform E. coli with an inducible GFP gene containing plasmid and observe induced and un-induced colonies. The following exercise creates cognitive dissonance when actual PCR results contradict their initial (incorrect) predictions of the presence of the GFP gene in transformed cells. Field testing of this laboratory exercise resulted in learning gains on both knowledge and application questions on concepts related to genes and gene expression. PMID:23858358

  3. Rapid PCR of STR markers: Applications to human identification.

    PubMed

    Romsos, Erica L; Vallone, Peter M

    2015-09-01

    Multiplex PCR with fluorescently labeled primers has been an essential method for the amplification of short tandem repeats used in human identify testing. Within the STR workflow of extraction, quantitation, amplification, separation, and detection, multiplex PCR is commonly identified as the bottleneck in the process. The time requirement of up to three hours to complete 28-30 cycles of multiplex PCR for STR genotyping is the greatest amount of time required for a single step within the process. The historical use of commercially available thermal cyclers and heat stable polymerases may have given the impression that large multiplex will always require long PCR cycling times to ensure that all of the varying sized targets (typically 100-400bp) can be amplified in a balanced manner throughout the multiplex. However, with the advent of improved polymerases and faster thermal cyclers the time required for the amplification of large STR multiplexes is no longer on the order of three hours, but as little as 14min. Faster amplification times can be performed while retaining the balance and integrity of large multiplex PCRs by implementation of alternate polymerases and thermal cyclers. With the reduction in PCR cycling times there has also been an impact on the development of integrated and microfluidics devices which employ the use of reduced or rapid thermal cycling protocols as part of their integration. Similarly, PCR inhibitor resistant polymerases can also reduce overall STR processing times for reference samples by eliminating the need for DNA extraction and purification that is additionally implemented within the development of integrated DNA typing devices.

  4. Monitoring of geosmin producing Anabaena circinalis using quantitative PCR.

    PubMed

    Tsao, Hsiang-Wei; Michinaka, Atsuko; Yen, Hung-Kai; Giglio, Steven; Hobson, Peter; Monis, Paul; Lin, Tsair-Fuh

    2014-02-01

    Geosmin is one of the most commonly detected off-flavor chemicals present in reservoirs and drinking water systems. Quantitative real-time PCR (qPCR) is useful for quantifying geosmin-producers by focusing on the gene encoding geosmin synthase, which is responsible for geosmin synthesis. In this study, several primers and probes were designed and evaluated to detect the geosmin synthase gene in cyanobacteria. The specificity of primer and probe sets was tested using 21 strains of laboratory cultured cyanobacteria isolated from surface waters in Australia (18) and Taiwan (2), including 6 strains with geosmin producing ability. The results showed that the primers designed in this study could successfully detect all geosmin producing strains tested. The selected primers were used in a qPCR assay, and the calibration curves were linear from 5 × 10(1) to 5 × 10(5) copies mL(-1), with a high correlation coefficient (R(2) = 0.999). This method was then applied to analyze samples taken from Myponga Reservoir, South Australia, during a cyanobacterial bloom event. The results showed good correlations between qPCR techniques and traditional methods, including cell counts determined by microscopy and geosmin concentration measured using gas chromatography (GC) coupled with a mass selective detector (MSD). Results demonstrate that qPCR could be used for tracking geosmin-producing cyanobacteria in drinking water reservoirs. The qPCR assay may provide water utilities with the ability to properly characterize a taste and odor episode and choose appropriate management and treatment options.

  5. Revealing the Diversity and Quantity of Peritrich Ciliates in Environmental Samples Using Specific Primer-based PCR and Quantitative PCR

    PubMed Central

    Liu, Xihan; Gong, Jun

    2012-01-01

    Peritrichs are a diverse, ecologically important ciliate group usually with a complex life cycle. To date, the community of the peritrichs has been investigated by using morphology-based methods such as living observation and silver staining. Here we show a molecular approach for characterizing the diversity and quantity of free-living peritrichs in environmental samples. We newly designed four peritrich-specific primers targeting 18S rRNA genes that allow clone library construction, screening and analysis. A quantitative real-time PCR (qPCR) assay was developed to quantify peritrichs in environmental samples by using rDNA copy number as an indicator. DNA extracted from four water samples of contrasting environmental gradients was analysed. The results showed that the peritrich community was differentiated among these samples, and that the diversity decreased with the increase of water salinity. The qPCR results are consistent with the library sequence analysis in terms of quantity variations from sample to sample. The development of peritrich-specific primers, for the first time, for conventional PCR and qPCR assays, provides useful molecular tools for revealing the diversity and quantity of peritrich ciliates in environmental samples. Also, our study illustrates the potential of these molecular tools to ecological studies of other ciliate groups in diverse environments. PMID:23100023

  6. Detection and Quantification of Wallemia sebi in Aerosols by Real-Time PCR, Conventional PCR, and Cultivation

    PubMed Central

    Zeng, Qing-Yin; Westermark, Sven-Olof; Rasmuson-Lestander, Åsa; Wang, Xiao-Ru

    2004-01-01

    Wallemia sebi is a deuteromycete fungus commonly found in agricultural environments in many parts of the world and is suspected to be a causative agent of farmer's lung disease. The fungus grows slowly on commonly used culture media and is often obscured by the fast-growing fungi. Thus, its occurrence in different environments has often been underestimated. In this study, we developed two sets of PCR primers specific to W. sebi that can be applied in either conventional PCR or real-time PCR for rapid detection and quantification of the fungus in environmental samples. Both PCR systems proved to be highly specific and sensitive for W. sebi detection even in a high background of other fungal DNAs. These methods were employed to investigate the presence of W. sebi in the aerosols of a farm. The results revealed a high concentration of W. sebi spores, 107 m−3 by real-time PCR and 106 m−3 by cultivation, which indicates the prevalence of W. sebi in farms handling hay and grain and in cow barns. The methods developed in this study could serve as rapid, specific, and sensitive means of detecting W. sebi in aerosol and surface samples and could thus facilitate investigations of its distribution, ecology, clinical diagnosis, and exposure risk assessment. PMID:15574929

  7. [Do Multiplex PCR techniques displace classical cultures in microbiology?].

    PubMed

    Auckenthaler, Raymond; Risch, Martin

    2015-02-01

    Multiplex PCR technologies progressively find their way in clinical microbiology. This technique allows the simultaneous amplification of multiple DNA targets in a single test run for the identification of pathogens up to the species level. Various pathogens of infectious diseases can be detected by a symptom-oriented approach clearly and quickly with high reliability. Essentially multiplex PCR panels are available for clarification of gastrointestinal, respiratory, sexually transmitted infections and meningitis. Today's offer from industry, university hospitals and large private laboratories of Switzerland is tabulated and commented.

  8. Novel PCR assay for determining the genetic sex of mice.

    PubMed

    McFarlane, L; Truong, V; Palmer, J S; Wilhelm, D

    2013-01-01

    A number of studies require the determination of the genetic sex of mouse embryos before sexual differentiation and/or of mutant mice that display partial or complete sex reversal. The majority of current methods for sexing by PCR involve multiplexing of 2 primer pairs. We have developed a novel sexing PCR using a single primer pair that amplifies fragments from the X and the Y chromosome with a clear size difference between the respective amplicons. This assay provides a rapid and reliable method to identify the genetic sex of mice across different mouse strains.

  9. Sheep poxvirus identification by PCR in cell cultures.

    PubMed

    Mangana-Vougiouka, O; Markoulatos, P; Koptopoulos, G; Nomikou, K; Bakandritsos, N; Papadopoulos, O

    1999-01-01

    A simple, rapid and specific diagnostic polymerase chain reaction (PCR) method was developed for sheep poxvirus identification. The primers used were from the sequenced genomes of the capripox viruses KS-1 and InS-1. Six different sheep pox isolates were tested against two orf (parapox) and three animal herpesviruses as controls. Material from uninfected cell cultures was also used as control. The sensitivity of the PCR was approximately equivalent with each of the two primers and for the six sheep pox isolates. All the negative control virus DNAs were negative and differed clearly from those of the sheep pox strains.

  10. PCR Amplicon Prediction from Multiplex Degenerate Primer and Probe Sets

    SciTech Connect

    Gardner, S. N.

    2013-08-08

    Assessing primer specificity and predicting both desired and off-target amplification products is an essential step for robust PCR assay design. Code is described to predict potential polymerase chain reaction (PCR) amplicons in a large sequence database such as NCBI nt from either singleplex or a large multiplexed set of primers, allowing degenerate primer and probe bases, with target mismatch annotates amplicons with gene information automatically downloaded from NCBI, and optionally it can predict whether there are also TaqMan/Luminex probe matches within predicted amplicons.

  11. Real-time PCR in Food Science: Introduction.

    PubMed

    Rodriguez-Lazaro, David; Hernandez, Marta

    2013-01-01

    Food safety and quality control programmes are increasingly applied throughout the production food chain in order to guarantee added value products as well as to minimize the risk of infection for the consumer. The development of real-time PCR has represented one of the most significant advances in food diagnostics as it provides rapid, reliable and quantitative results. These aspects become increasingly important for the agricultural and food industry. Different strategies for real-time PCR diagnostics have been developed including unspecific detection independent of the target sequence using fluorescent dyes such as SYBR Green, or by sequence-specific fluorescent oligonucleotide probes such as TaqMan probes or molecular beacons.

  12. [Basics of PCR and related techniques applied in veterinary parasitology].

    PubMed

    Ben Abderrazak, S

    2004-01-01

    We attempte through the following overall review pertaining to the basics of PCR techniques (Polymerase Chain Reaction), to introduce the main applications used in veterinary parasitology. A major problem restricting the application possibilities of molecular biology techniques is of quantitative nature. Amplification techniques represent a real revolution, for it makes possible the production of tens, even hundreds of nanogrammes of sequences when starting from very small quantities. The PCR technique has dramatically transformed the strategies used so far in molecular biology and subsequently research and medical diagnosis.

  13. Microsatellite amplification in plants: optimization procedure of major PCR components.

    PubMed

    Ghaffari, Sana; Hasnaoui, Nejib

    2013-01-01

    Microsatellites (SSRs) are the most informative and popular class of molecular markers used for diverse purposes, particularly in plants: genetic diversity study, marker assisted selection, breeding, mapping, phylogenetics and phylogeography, systematics, etc. They have become a routine technique practically in each laboratory for studying molecular plant genetics. Despite their wide utilization, however, setup and optimization of various conditions involved in PCR amplification is a prerequisite for reliable inference of results. In this chapter, we describe optimization of SSR-PCR conditions and give ranges of concentrations for different parameters. The protocol provided here is inspired from bench work on the use of microsatellite to study diversity of Vitis vinifera germplasm.

  14. Multiplex PCR for Detection and Typing of Porcine Circoviruses

    PubMed Central

    Ouardani, M.; Wilson, L.; Jetté, R.; Montpetit, C.; Dea, S.

    1999-01-01

    Sets of oligonucleotide primers were designed according to the sequences of the open reading frames (ORFs) ORF1 and ORF2 of the prototype nonpathogenic PK-15 strain of porcine circovirus (PCV) type 1 (PCV-1). By the PCR performed with the various primer sets, genomic DNA or RNA from other bacterial or viral pathogens of the respiratory tracts of pigs could not be amplified. A positive amplification reaction could be visualized with DNA extracted from a viral suspension containing as few as 10 viral particles per ml. No DNA fragment could be amplified from lysates of continuous porcine cell lines (PT, ST, and PFT cells) known to be negative for PCV. When tested with clinical samples from pigs, the results of the single PCR method showed nearly 93% (13 of 14 samples) correlation with histopathological and immunohistochemical findings. Interestingly, subclinical PCV infections could be detected by single PCR with clinical samples that have been submitted from animals with irrelevant cases of respiratory and/or enteric problems. On the basis of the nucleotide sequences of PCV strains (PCV-2) recently associated with outbreaks of postweaning multisystemic wasting syndrome (PWMS) in Quebec, Canada, pig farms, other primers were designed from the PCV-1 genome, and these primers failed to amplify genomic fragments specific to the ORF1 or ORF2 genes of clinical isolates associated with PWMS but amplified DNA from the PCV-1 strain. Two rapid multiplex PCR (mPCR) methods have been developed to distinguish between both genotypes of PCV. By those two mPCR methods, (i) species-specific primer pairs were used to amplify a DNA fragment of 488 bp specific for the ORF2 genes of both genotypes, whereas a 375-bp fragment was amplified from the ORF1 gene of the PCV-1 strain only, or (ii) species-specific primer pairs were used to amplify a DNA fragment of 646 bp specific for the ORF1 genes of both genotypes, whereas a 425-bp fragment was amplified from the ORF2 gene of the PCV-1 strain

  15. High-throughput quantitative real-time PCR.

    PubMed

    Arany, Zoltan P

    2008-07-01

    Recent technical advances in quantitative real-time PCR (qRT-PCR) have allowed for extensive miniaturization, thereby rendering the technique amenable to high-throughput assays. Large numbers of different nucleic acids can now rapidly be measured quantitatively. Many investigations can benefit from this approach, including determination of gene expression in hundreds of samples, determination of hundreds of genes in a few samples, or even quantification of nucleic acids other than mRNA. A simple technique is described here to quantify 1880 transcripts of choice from any number of starting RNA samples.

  16. Gold nanoparticle-based RT-PCR and real-time quantitative RT-PCR assays for detection of Japanese encephalitis virus

    NASA Astrophysics Data System (ADS)

    Huang, Su-Hua; Yang, Tsuey-Ching; Tsai, Ming-Hong; Tsai, I.-Shou; Lu, Huang-Chih; Chuang, Pei-Hsin; Wan, Lei; Lin, Ying-Ju; Lai, Chih-Ho; Lin, Cheng-Wen

    2008-10-01

    Virus isolation and antibody detection are routinely used for diagnosis of Japanese encephalitis virus (JEV) infection, but the low level of transient viremia in some JE patients makes JEV isolation from clinical and surveillance samples very difficult. We describe the use of gold nanoparticle-based RT-PCR and real-time quantitative RT-PCR assays for detection of JEV from its RNA genome. We tested the effect of gold nanoparticles on four different PCR systems, including conventional PCR, reverse-transcription PCR (RT-PCR), and SYBR green real-time PCR and RT-PCR assays for diagnosis in the acute phase of JEV infection. Gold nanoparticles increased the amplification yield of the PCR product and shortened the PCR time compared to the conventional reaction. In addition, nanogold-based real-time RT-PCR showed a linear relationship between Ct and template amount using ten-fold dilutions of JEV. The nanogold-based RT-PCR and real-time quantitative RT-PCR assays were able to detect low levels (1-10 000 copies) of the JEV RNA genomes extracted from culture medium or whole blood, providing early diagnostic tools for the detection of low-level viremia in the acute-phase infection. The assays described here were simple, sensitive, and rapid approaches for detection and quantitation of JEV in tissue cultured samples as well as clinical samples.

  17. Critical appraisal of quantitative PCR results in colorectal cancer research: can we rely on published qPCR results?

    PubMed

    Dijkstra, J R; van Kempen, L C; Nagtegaal, I D; Bustin, S A

    2014-06-01

    The use of real-time quantitative polymerase chain reaction (qPCR) in cancer research has become ubiquitous. The relative simplicity of qPCR experiments, which deliver fast and cost-effective results, means that each year an increasing number of papers utilizing this technique are being published. But how reliable are the published results? Since the validity of gene expression data is greatly dependent on appropriate normalisation to compensate for sample-to-sample and run-to-run variation, we have evaluated the adequacy of normalisation procedures in qPCR-based experiments. Consequently, we assessed all colorectal cancer publications that made use of qPCR from 2006 until August 2013 for the number of reference genes used and whether they had been validated. Using even these minimal evaluation criteria, the validity of only three percent (6/179) of the publications can be adequately assessed. We describe common errors, and conclude that the current state of reporting on qPCR in colorectal cancer research is disquieting. Extrapolated to the study of cancer in general, it is clear that the majority of studies using qPCR cannot be reliably assessed and that at best, the results of these studies may or may not be valid and at worst, pervasive incorrect normalisation is resulting in the wholesale publication of incorrect conclusions. This survey demonstrates that the existence of guidelines, such as MIQE, is necessary but not sufficient to address this problem and suggests that the scientific community should examine its responsibility and be aware of the implications of these findings for current and future research.

  18. Designing multiplex PCR system of Campylobacter jejuni for efficient typing by improving monoplex PCR binary typing method.

    PubMed

    Yamada, Kazuhiro; Ibata, Ami; Suzuki, Masahiro; Matsumoto, Masakado; Yamashita, Teruo; Minagawa, Hiroko; Kurane, Ryuichiro

    2015-01-01

    Campylobacter jejuni is responsible for the majority of Campylobacter infections. As the molecular epidemiological study of outbreaks, pulsed-field gel electrophoresis (PFGE) is performed in general. But PFGE has several problems. PCR binary typing (P-BIT) method is a typing method for Campylobacter spp. that was recently developed, and was reported to have a similar discriminatory power and stability to those of PFGE. We modified the P-BIT method from 18 monoplex PCRs to two multiplex PCR systems (mP-BIT). The same results were obtained from monoplex PCRs using original primers and multiplex PCR in the representative isolates. The mP-BIT can analyze 48 strains at a time by using 96-well PCR systems and can identify C. jejuni because mP-BIT includes C. jejuni marker. The typing of the isolates by the mP-BIT and PFGE demonstrated generally concordant results and the mP-BIT method (D = 0.980) has a similar discriminatory power to that of PFGE with SmaI digest (D = 0.975) or KpnI digest (D = 0.987) as with original article. The mP-BIT method is quick, simple and easy, and comes to be able to perform it at low cost by having become a multiplex PCR system. Therefore, the mP-BIT method with two multiplex PCR systems has high potential for a rapid first-line surveillance typing assay of C. jejuni and can be used for routine surveillance and outbreak investigations of C. jejuni in the future.

  19. qPCR assays to quantify genes and gene expression associated with microbial perchlorate reduction.

    PubMed

    De Long, Susan K; Kinney, Kerry A; Kirisits, Mary Jo

    2010-11-01

    Quantitative PCR (qPCR) assays targeting cld (developed in this work) and pcrA (previously described) were used to quantify these perchlorate-related genes in a perchlorate-reducing enrichment culture. Transcript copies were quantified in perchlorate-reducing Rhodocyclaceae strain JDS4. Oxygen and nitrate inhibited expression of cld and pcrA.

  20. Use of Droplet Digital PCR for Estimation of Fish Abundance and Biomass in Environmental DNA Surveys

    PubMed Central

    Doi, Hideyuki; Uchii, Kimiko; Takahara, Teruhiko; Matsuhashi, Saeko; Yamanaka, Hiroki; Minamoto, Toshifumi

    2015-01-01

    An environmental DNA (eDNA) analysis method has been recently developed to estimate the distribution of aquatic animals by quantifying the number of target DNA copies with quantitative real-time PCR (qPCR). A new quantitative PCR technology, droplet digital PCR (ddPCR), partitions PCR reactions into thousands of droplets and detects the amplification in each droplet, thereby allowing direct quantification of target DNA. We evaluated the quantification accuracy of qPCR and ddPCR to estimate species abundance and biomass by using eDNA in mesocosm experiments involving different numbers of common carp. We found that ddPCR quantified the concentration of carp eDNA along with carp abundance and biomass more accurately than qPCR, especially at low eDNA concentrations. In addition, errors in the analysis were smaller in ddPCR than in qPCR. Thus, ddPCR is better suited to measure eDNA concentration in water, and it provides more accurate results for the abundance and biomass of the target species than qPCR. We also found that the relationship between carp abundance and eDNA concentration was stronger than that between biomass and eDNA by using both ddPCR and qPCR; this suggests that abundance can be better estimated by the analysis of eDNA for species with fewer variations in body mass. PMID:25799582

  1. Electrochemical genosensor based on peptide nucleic acid-mediated PCR and asymmetric PCR techniques: Electrostatic interactions with a metal cation.

    PubMed

    Kerman, Kagan; Vestergaard, Mun'delanji; Nagatani, Naoki; Takamura, Yuzuru; Tamiya, Eiichi

    2006-04-01

    The unique structure of peptide nucleic acids (PNAs), linking the N-(2-aminoethyl)glycine units that create a neutral backbone, and prevent it from acting as a primer for DNA polymerase, has been utilized in an electrochemical biosensor scheme for simple and sensitive detection of hybridization. When the PNA is targeted against a single-nucleotide polymorphism (SNP) or wild-type site on the gene, PNA-mediated polymerase chain reaction (PCR) clamping method effectively blocks the formation of a PCR product. In our report, PNA probe for PCR clamping was targeted against the wild-type site of alcohol dehydrogenase. The electrostatic interactions between the negatively charged DNA and neutral PNA molecules with redox-active metal cation cobalt(III)hexamine ([Co(NH3)6]3+) were monitored using differential pulse voltammetry. The electrostatic binding of [Co(NH3)6]3+ to DNA provided the basis for the discrimination against PNA/PNA, PNA/DNA, and DNA/DNA hybrid molecules. We have optimized the experimental conditions, such as probe concentration, [Co(NH3)6]3+ concentration, accumulation time for [Co(NH3)6]3+, and target concentration. A new pretreatment method has also been employed to allow fast and simple detection of hybridization reaction between the PCR amplicon and the probe on glassy carbon electrode (GCE) surface. This method was based on the application of a high-temperature treatment (95 degrees C, 5 min), followed by a 1-min incubation in the presence of DNA primers. The excess concentration of DNA primers prevented the rehybridization of the denatured strands, while enabling the target gene sequence to bind with the immobilized probe. Additionally, asymmetric PCR was employed to detect the presence of genetically modified organism in standard Roundup Ready soybean samples. The amplicons of asymmetric PCR, which were predominantly single-stranded DNA as a result of unequal primer concentration, hybridized with the DNA probe on the sensor surface efficiently. The

  2. Deletion-targeted multiplex PCR (DTM-PCR) for identification of Beijing/W genotypes of Mycobacterium tuberculosis.

    PubMed

    Chen, Jing; Tsolaki, Anthony G; Shen, Xin; Jiang, Xi; Mei, Jian; Gao, Qian

    2007-09-01

    Beijing/W strains of Mycobacterium tuberculosis cause the vast majority of tuberculosis cases in Shanghai, China. Such highly prevalent strains are considered as hypervirulent and are often associated with multi-drug resistance, treatment failure and HIV status. We present a reliable and fast detection method to identify these Beijing/W strains, which can be applied to screening large numbers of samples at low cost. Using this Deletion-Targeted Multiplex PCR (DTM-PCR) method for detecting these strains, we obtained 100% sensitivity and specificity.

  3. A novel multiplex quantitative DNA array based PCR (MQDA-PCR) for quantification of transgenic maize in food and feed.

    PubMed

    Rudi, Knut; Rud, Ida; Holck, Askild

    2003-06-01

    We have developed a novel multiplex quantitative DNA array based PCR method (MQDA-PCR). The MQDA-PCR is general and may be used in all areas of biological science where simultaneous quantification of multiple gene targets is desired. We used quantification of transgenic maize in food and feed as a model system to show the applicability of the method. The method is based on a two-step PCR. In the first few cycles bipartite primers containing a universal 5' 'HEAD' region and a 3' region specific to each genetically modified (GM) construct are employed. The unused primers are then degraded with a single-strand DNA-specific exonuclease. The second step of the PCR is run containing only primers consisting of the universal HEAD region. The removal of the primers is essential to create a competitive, and thus quantitative PCR. Oligo nucleotides hybridising to internal segments of the PCR products are then sequence specifically labelled in a cyclic linear signal amplification reaction. This is done both to increase the sensitivity and the specificity of the assay. Hybridisation of the labelled oligonucleotides to their complementary sequences in a DNA array enables multiplex detection. Quantitative information was obtained in the range 0.1-2% for the different GM constructs tested. Seventeen different food and feed samples were screened using a twelve-plex system for simultaneous detection of seven different GM maize events (Bt176, Bt11, Mon810, T25, GA21, CBH351 and DBT418). Ten samples were GM positive containing mainly mixtures of Mon810, Bt11 and Bt176 DNA. One sample contained appreciable amounts of GA21. An eight-plex MQDA-PCR system for detection of Mon810, Bt11 and Bt176 was evaluated by comparison with simplex 5' nuclease PCRs. There were no significant differences in the quantifications using the two approaches. The samples could, by both methods, be quantified as containing >2%, between 1 and 2%, between 0.1 and 1%, or <0.1% in 43 out of 47 determinations. The

  4. A comparative study of digital RT-PCR and RT-qPCR for quantification of Hepatitis A virus and Norovirus in lettuce and water samples.

    PubMed

    Coudray-Meunier, Coralie; Fraisse, Audrey; Martin-Latil, Sandra; Guillier, Laurent; Delannoy, Sabine; Fach, Patrick; Perelle, Sylvie

    2015-05-18

    Sensitive and quantitative detection of foodborne enteric viruses is classically achieved by quantitative RT-PCR (RT-qPCR). Recently, digital PCR (dPCR) was described as a novel approach to genome quantification without need for a standard curve. The performance of microfluidic digital RT-PCR (RT-dPCR) was compared to RT-qPCR for detecting the main viruses responsible for foodborne outbreaks (human Noroviruses (NoV) and Hepatitis A virus (HAV)) in spiked lettuce and bottled water. Two process controls (Mengovirus and Murine Norovirus) were used and external amplification controls (EAC) were added to examine inhibition of RT-qPCR and RT-dPCR. For detecting viral RNA and cDNA, the sensitivity of the RT-dPCR assays was either comparable to that of RT-qPCR (RNA of HAV, NoV GI, Mengovirus) or slightly (around 1 log10) decreased (NoV GII and MNV-1 RNA and of HAV, NoV GI, NoV GII cDNA). The number of genomic copies determined by dPCR was always from 0.4 to 1.7 log10 lower than the expected numbers of copies calculated by using the standard qPCR curve. Viral recoveries calculated by RT-dPCR were found to be significantly higher than by RT-qPCR for NoV GI, HAV and Mengovirus in water, and for NoV GII and HAV in lettuce samples. The RT-dPCR assay proved to be more tolerant to inhibitory substances present in lettuce samples. This absolute quantitation approach may be useful to standardize quantification of enteric viruses in bottled water and lettuce samples and may be extended to quantifying other human pathogens in food samples.

  5. Review of the taxonomy of the genus Arthrobacter, emendation of the genus Arthrobacter sensu lato, proposal to reclassify selected species of the genus Arthrobacter in the novel genera Glutamicibacter gen. nov., Paeniglutamicibacter gen. nov., Pseudoglutamicibacter gen. nov., Paenarthrobacter gen. nov. and Pseudarthrobacter gen. nov., and emended description of Arthrobacter roseus.

    PubMed

    Busse, Hans-Jürgen

    2016-01-01

    In this paper, the taxonomy of the genus Arthrobacter is discussed, from its first description in 1947 to the present state. Emphasis is given to intrageneric phylogeny and chemotaxonomic characteristics, concentrating on quinone systems, peptidoglycan compositions and polar lipid profiles. Internal groups within the genus Arthrobacter indicated from homogeneous chemotaxonomic traits and corresponding to phylogenetic grouping and/or high 16S rRNA gene sequence similarities are highlighted. Furthermore, polar lipid profiles and quinone systems of selected species are shown, filling some gaps concerning these chemotaxonomic traits. Based on phylogenetic groupings, 16S rRNA gene sequence similarities and homogeneity in peptidoglycan types, quinone systems and polar lipid profiles, a description of the genus Arthrobacter sensu lato and an emended description of Arthrobacter roseus are provided. Furthermore, reclassifications of selected species of the genus Arthrobacter into novel genera are proposed, namely Glutamicibacter gen. nov. (nine species), Paeniglutamicibacter gen. nov. (six species), Pseudoglutamicibacter gen. nov. (two species), Paenarthrobacter gen. nov. (six species) and Pseudarthrobacter gen. nov. (ten species).

  6. Using qPCR for Water Microbial Risk Assessments

    EPA Science Inventory

    Microbial risk assessment (MRA) has traditionally utilized microbiological data that was obtained by culture-based techniques that are expensive and time consuming. With the advent of PCR methods there is a realistic opportunity to conduct MRA studies economically, in less time,...

  7. Enumeration of Mycobacterium leprae Using Real-Time PCR

    PubMed Central

    Truman, Richard W.; Andrews, P. Kyle; Robbins, Naoko Y.; Adams, Linda B.; Krahenbuhl, James L.; Gillis, Thomas P.

    2008-01-01

    Mycobacterium leprae is not cultivable in axenic media, and direct microscopic enumeration of the bacilli is complex, labor intensive, and suffers from limited sensitivity and specificity. We have developed a real-time PCR assay for quantifying M. leprae DNA in biological samples. Primers were identified to amplify a shared region of the multicopy repeat sequence (RLEP) specific to M. leprae and tested for sensitivity and specificity in the TaqMan format. The assay was specific for M. leprae and able to detect 10 fg of purified M. leprae DNA, or approximately 300 bacteria in infected tissues. We used the RLEP TaqMan PCR to assess the short and long-term growth results of M. leprae in foot pad tissues obtained from conventional mice, a gene knock-out mouse strain, athymic nude mice, as well as from reticuloendothelial tissues of M. leprae–infected nine-banded armadillos. We found excellent correlative results between estimates from RLEP TaqMan PCR and direct microscopic counting (combined r = 0.98). The RLEP TaqMan PCR permitted rapid analysis of batch samples with high reproducibility and is especially valuable for detection of low numbers of bacilli. Molecular enumeration is a rapid, objective and highly reproducible means to estimate the numbers of M. leprae in tissues, and application of the technique can facilitate work with this agent in many laboratories. PMID:18982056

  8. Qualitative PCR method for Roundup Ready soybean: interlaboratory study.

    PubMed

    Kodama, Takashi; Kasahara, Masaki; Minegishi, Yasutaka; Futo, Satoshi; Sawada, Chihiro; Watai, Masatoshi; Akiyama, Hiroshi; Teshima, Reiko; Kurosawa, Yasunori; Furui, Satoshi; Hino, Akihiro; Kitta, Kazumi

    2011-01-01

    Quantitative and qualitative methods based on PCR have been developed for genetically modified organisms (GMO). Interlaboratory studies were previously conducted for GMO quantitative methods; in this study, an interlaboratory study was conducted for a qualitative method for a GM soybean, Roundup Ready soy (RR soy), with primer pairs designed for the quantitative method of RR soy studied previously. Fourteen laboratories in Japan participated. Each participant extracted DNA from 1.0 g each of the soy samples containing 0, 0.05, and 0.10% of RR soy, and performed PCR with primer pairs for an internal control gene (Le1) and RR soy followed by agarose gel electrophoresis. The PCR product amplified in this PCR system for Le1 was detected from all samples. The sensitivity, specificity, and false-negative and false-positive rates of the method were obtained from the results of RR soy detection. False-negative rates at the level of 0.05 and 0.10% of the RR soy samples were 6.0 and 2.3%, respectively, revealing that the LOD of the method was somewhat below 0.10%. The current study demonstrated that the qualitative method would be practical for monitoring the labeling system of GM soy in kernel lots.

  9. Citrus stubborn disease incidence determined by quantitative real time PCR

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Quantitative real-time (q) PCR was developed for detection of Spiroplasma citri, the causal agent of citrus stubborn disease (CSD), using the DNA binding fluorophore SYBR Green I. The primer pair, P58-3f/4r, developed based on sequences from the P58 putative adhesin multigene of the pathogen result...

  10. QUANTITATIVE PCR OF SELECTED ASPERGILLUS, PENICILLIUM AND PAECILOMYCES SPECIES

    EPA Science Inventory

    A total of 65 quantitative PCR (QPCR) assays, incorporating fluorigenic 5' nuclease (TaqMan®) chemistry and directed at the nuclear ribosomal RNA operon, internal transcribed spacer regions (ITS1 or ITS2) was developed and tested for the detection of Aspergillus, Penicillium and ...

  11. Real-Time PCR Quantification of Methanobrevibacter oralis in Periodontitis

    PubMed Central

    Bringuier, Amélie; Khelaifia, Saber; Richet, Hervé; Aboudharam, Gérard

    2013-01-01

    A real-time PCR assay developed to quantify Methanobrevibacter oralis indicated that its inoculum significantly correlated with periodontitis severity (P = 0.003), despite a nonsignificant difference in prevalence between controls (3/10) and patients (12/22) (P = 0.2, Fisher test). The M. oralis load can be used as a biomarker for periodontitis. PMID:23254133

  12. Fuel utilization in a progressive conversion reactor (PCR)

    SciTech Connect

    Leyse, C.F.; Judd, J.L.

    1981-05-01

    Preliminary studies indicate that for once-through fuel cycles, the PCR offers potential improvements over current LWRs in the following major areas: improved uranium utilization (reduced uranium demand), degraded plutonium product in spent fuel, reduced plutonium content of spent fuel, reduced amount of spent fuel, reduced fissile content of spent fuel, and reduced separative work.

  13. Identification of Staphylococcus spp. using (GTG)₅-PCR fingerprinting.

    PubMed

    Svec, Pavel; Pantůček, Roman; Petráš, Petr; Sedláček, Ivo; Nováková, Dana

    2010-12-01

    A group of 212 type and reference strains deposited in the Czech Collection of Microorganisms (Brno, Czech Republic) and covering 41 Staphylococcus species comprising 21 subspecies was characterised using rep-PCR fingerprinting with the (GTG)₅ primer in order to evaluate this method for identification of staphylococci. All strains were typeable using the (GTG)₅ primer and generated PCR products ranging from 200 to 4500 bp. Numerical analysis of the obtained fingerprints revealed (sub)species-specific clustering corresponding with the taxonomic position of analysed strains. Taxonomic position of selected strains representing the (sub)species that were distributed over multiple rep-PCR clusters was verified and confirmed by the partial rpoB gene sequencing. Staphylococcus caprae, Staphylococcus equorum, Staphylococcus sciuri, Staphylococcus piscifermentans, Staphylococcus xylosus, and Staphylococcus saprophyticus revealed heterogeneous fingerprints and each (sub)species was distributed over several clusters. However, representatives of the remaining Staphylococcus spp. were clearly separated in single (sub)species-specific clusters. These results showed rep-PCR with the (GTG)₅ primer as a fast and reliable method applicable for differentiation and straightforward identification of majority of Staphylococcus spp.

  14. Real-time PCR for Strongyloides stercoralis-associated meningitis.

    PubMed

    Nadir, Eyal; Grossman, Tamar; Ciobotaro, Pnina; Attali, Malka; Barkan, Daniel; Bardenstein, Rita; Zimhony, Oren

    2016-03-01

    Four immunocompromised patients, immigrants from Ethiopia, presented with diverse clinical manifestations of meningitis associated with Strongyloides stercoralis dissemination as determined by identification of intestinal larvae. The cerebrospinal fluid of 3 patients was tested by a validated (for stool) real-time PCR for S. stercoralis and was found positive, establishing this association.

  15. PCR Detection of Helicobacter pylori in Clinical Samples

    PubMed Central

    Rimbara, Emiko; Sasatsu, Masanori; Graham, David Y.

    2014-01-01

    Helicobacter pylori is an important pathogen whose primary niche is the human stomach. H. pylori is etio-logically associated with gastric inflammation (gastritis), peptic ulcer disease, and gastric cancer. Both noninvasive (e.g., urea breath and stool antigen tests) and invasive (gastric biopsy for histology, culture, or PCR) tests are used for diagnosis. PCR detection of H. pylori has been reported using a variety of clinical samples including gastric biopsy, gastric juice, saliva, dental plaque, and stools as well as environmental samples. Whenever possibly, noninvasive tests are preferred over invasive tests. H. pylori are excreted in the stool. Culture from stool is variable whereas stool antigen testing is widely used. Stool consists of a complicated mixture of commensal bacteria and chemicals and often includes inhibitors of PCR. Nevertheless, simple extraction methods are available to efficiently extract DNA from human stools and nested-PCR targeting the 23S rRNA gene have proven to be highly sensitive for the detection of H. pylori. Detection of clarithromycin susceptibility/resistance is important clinically and the mutation of the 23S rRNA gene responsible for resistance can also be detected using stool. This described method can be modified for other clinical samples such as gastric juice or biopsy material. PMID:23104297

  16. Halal authenticity of gelatin using species-specific PCR.

    PubMed

    Shabani, Hessam; Mehdizadeh, Mehrangiz; Mousavi, Seyed Mohammad; Dezfouli, Ehsan Ansari; Solgi, Tara; Khodaverdi, Mahdi; Rabiei, Maryam; Rastegar, Hossein; Alebouyeh, Mahmoud

    2015-10-01

    Consumption of food products derived from porcine sources is strictly prohibited in Islam. Gelatin, mostly derived from bovine and porcine sources, has many applications in the food and pharmaceutical industries. To ensure that food products comply with halal regulations, development of valid and reliable analytical methods is very much required. In this study, a species-specific polymerase chain reaction (PCR) assay using conserved regions of mitochondrial DNA (cytochrome b gene) was performed to evaluate the halal authenticity of gelatin. After isolation of DNA from gelatin powders with known origin, conventional PCR using species-specific primers was carried out on the extracted DNA. The amplified expected PCR products of 212 and 271 bp were observed for porcine and bovine gelatin, respectively. The sensitivity of the method was tested on binary gelatin mixtures containing 0.1%, 1%, 10%, and 100% (w/w) of porcine gelatin within bovine gelatin and vice versa. Although most of the DNA is degraded due to the severe processing steps of gelatin production, the minimum level of 0.1% w/w of both porcine and bovine gelatin was detected. Moreover, eight food products labeled as containing bovine gelatin and eight capsule shells were subjected to PCR examination. The results showed that all samples contained bovine gelatin, and the absence of porcine gelatin was verified. This method of species authenticity is very useful to verify whether gelatin and gelatin-containing food products are derived from halal ingredients.

  17. Introducing Undergraduate Students to Real-Time PCR

    ERIC Educational Resources Information Center

    Hancock, Dale; Funnell, Alister; Jack, Briony; Johnston, Jill

    2010-01-01

    An experiment is conducted, which in four 3 h laboratory sessions, introduces third year undergraduate Biochemistry students to the technique of real-time PCR in a biological context. The model used is a murine erythroleukemia cell line (MEL cells). These continuously cycling, immature red blood cells, arrested at an early stage in erythropoiesis,…

  18. A Trio of Human Molecular Genetics PCR Assays

    ERIC Educational Resources Information Center

    Reinking, Jeffrey L.; Waldo, Jennifer T.; Dinsmore, Jannett

    2013-01-01

    This laboratory exercise demonstrates three different analytical forms of the polymerase chain reaction (PCR) that allow students to genotype themselves at four different loci. Here, we present protocols to allow students to a) genotype a non-coding polymorphic Variable Number of Tandem Repeat (VNTR) locus on human chromosome 5 using conventional…

  19. Novel real-time PCR detection assay for Brucella suis

    PubMed Central

    Hänsel, C.; Mertens, K.; Elschner, M. C.; Melzer, F.

    2015-01-01

    Introduction Brucella suis is the causative agent of brucellosis in suidae and is differentiated into five biovars (bv). Biovars 1 and 3 possess zoonotic potential and can infect humans, whereas biovar 2 represents the main source of brucellosis in feral and domestic pigs in Europe. Both aspects, the zoonotic threat and the economic loss, emphasize the necessity to monitor feral and domestic pig populations. Available serological or PCR based methods lack sensitivity and specificity. Results Here a bioinformatics approach was used to identify a B. suis specific 17 bp repeat on chromosome II (BS1330_II0657 locus). This repeat is common for B. suis bv 1 to 4 and was used to develop a TaqMan probe assay. The average PCR efficiency was determined as 95% and the limit of detection as 12,5 fg/µl of DNA, equally to 3.7 bacterial genomes. This assay has the highest sensitivity of all previously described B. suis specific PCR assays, making it possible to detect 3-4 bacterial genomes per 1 µl of sample. The assay was tested 100% specific for B. suis and negative for other Brucella spp. and closely related non-Brucella species. Conclusions This novel qPCR assay could become a rapid, inexpensive and reliable screening method for large sample pools of B. suis 1 to 4. This method will be applicable for field samples after validation. PMID:26392898

  20. Application of nested PCR for diagnosis of histoplasmosis.

    PubMed

    Ohno, Hideaki; Tanabe, Koichi; Umeyama, Takashi; Kaneko, Yukihiro; Yamagoe, Satoshi; Miyazaki, Yoshitsugu

    2013-10-01

    Histoplasmosis is a fungal infection that, although not endemic in Japan, has seen a rise in the number of Japanese cases since the mid-1980s. Diagnosis of the disease is not straightforward, and the main method of detection, fungal culture (which has biosafety-related issues), is of low sensitivity in general. Alternative methods that depend on antibody or antigen detection have had limited use. We have developed a histoplasmosis detection method based on PCR amplification of the Histoplasma capsulatum M antigen gene. We compared this method with fungal culture and serological diagnostic techniques. Among five cases that were finally diagnosed as histoplasmosis, the fungal culture method was only successful in identifying one such case. Although the presence of anti-H. capsulatum antibodies was confirmed in three cases, our PCR method identified four of five cases of histoplasmosis. The performance of our PCR method could not be compared with the antigen detection method, which is used in the United States but is not routinely used in Japan. However, the PCR method was shown to have high sensitivity and specificity for H. capsulatum. Although the number of histoplasmosis cases examined in this study was small, our data suggest that the molecular diagnosis technique has potential for increasing the reliability of histoplasmosis diagnosis when used in combination with established methods.

  1. Screening ancient tuberculosis with qPCR: challenges and opportunities

    PubMed Central

    Harkins, Kelly M.; Buikstra, Jane E.; Campbell, Tessa; Bos, Kirsten I.; Johnson, Eric D.; Krause, Johannes; Stone, Anne C.

    2015-01-01

    The field of ancient DNA (aDNA) has rapidly accelerated in recent years as a result of new methods in next-generation sequencing, library preparation and targeted enrichment. Such research is restricted, however, by the highly variable DNA preservation within different tissues, especially when isolating ancient pathogens from human remains. Identifying positive candidate samples via quantitative PCR (qPCR) for downstream procedures can reduce reagent costs, increase capture efficiency and maximize the number of sequencing reads of the target. This study uses four qPCR assays designed to target regions within the Mycobacterium tuberculosis complex (MTBC) to examine 133 human skeletal samples from a wide geographical and temporal range, identified by the presence of skeletal lesions typical of chronic disseminated tuberculosis. Given the inherent challenges working with ancient mycobacteria, strict criteria must be used and primer/probe design continually re-evaluated as new data from bacteria become available. Seven samples tested positive for multiple MTBC loci, supporting them as strong candidates for downstream analyses. Using strict and conservative criteria, qPCR remains a fast and effective screening tool when compared with screening by more expensive sequencing and enrichment technologies. PMID:25487341

  2. DNA extraction protocol for rapid PCR detection of pathogenic bacteria

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Virtually all current assays for foodborne pathogens, including PCR assays, are conducted after lengthy cultural enrichment of the sample to increase the concentration of the target organism. This delays detection by many hours, prevents quantitation, and limits the ability to detect multiple organ...

  3. MOLD SPECIFIC QUANTITATIVE PCR: THE EMERGING STANDARD IN MOLD ANALYSIS

    EPA Science Inventory

    Today I will talk about the use of quantitative or Real time PCR for the standardized identification and quantification of molds. There are probably at least 100,000 species of molds or fungi. But there are actually about 100 typically found indoors. Some pose a threat to human...

  4. DETECTION OF FECAL ENTEROCOCCI USING A REAL TIME PCR METHOD

    EPA Science Inventory

    In spite of their importance in public health, the detection of fecal enterococci is performed via culturing methods that are time consuming and that are subject to inaccuracies that relate to their culturable status. In order to address these problems, a real time PCR (TaqMan) ...

  5. Genus identification of toxic plant by real-time PCR.

    PubMed

    Matsuyama, Shuji; Nishi, Katsuji

    2011-03-01

    Some plants have toxicities that are dangerous for humans. In the case of poisoning by toxic plants, a rapid and easy screening test is required for accurate medical treatment or forensic investigation. In this study, we designed specific primer pairs for identification of toxic plants, such as subgenus Aconitum, genus Ricinus, genus Illicium, and genus Scopolia, by internal transcribed spacer sequences of nuclear ribosomal DNA. Allied species of target plants, foods, and human DNA were not detected, but each primer pair provided a specific PCR product from the target plant using real-time PCR. This method can detect the subgenus Aconitum, genus Ricinus, and genus Scopolia with template DNA of 10 pg, respectively, and genus Illicium with 1 pg. Furthermore, each primer pair provided the exact PCR product from digested target plants in artificial gastric fluid. When a trace unknown plant sample in forensic investigation is collected from stomach contents, this PCR assay may be useful for screening toxic plants.

  6. Primer design for PCR reactions in forensic biology.

    PubMed

    Elkins, Kelly M

    2015-01-01

    The polymerase chain reaction (PCR) is a popular method to copy DNA in vitro. Its invention revolutionized fields ranging from clinical medicine to anthropology, molecular biology, and forensic biology. The method employs one of many available heat-stable DNA polymerases in a reaction that is repeated many times in situ. The DNA polymerase reads a template DNA strand and using the components of the reaction mix, catalyzes the addition of free 2'-deoxynucleotide triphosphate nitrogenous bases to short segment of DNA that forms a complement with the template via Watson-Crick base pairing. This short segment of DNA is referred to as a PCR primer and it is essential to the success of the reaction. The most widely used application of PCR in forensic labs is the amplification of short tandem repeat (STR) loci used in DNA typing. The STRs are routinely evaluated in concert with 16 or more reactions, a multiplex, run in one test tube simultaneously. In a multiplex, it is essential that the primers work specifically and accurately on the intended reactions without hindering the other reactions. The primers, which are very specific, also can be used to probe single nucleotide polymorphisms (SNPs) in a DNA sequence of interest by single base extension. Primers are often designed using one of many available automated software packages. Here the process of manually designing PCR primers for forensic biology using no-cost software is described.

  7. A PCR test for avian malaria in Hawaiian birds.

    PubMed

    Feldman, R A; Freed, L A; Cann, R L

    1995-12-01

    The decline of native Hawaiian forest birds since European contact is attributed to factors ranging from habitat destruction to interactions with introduced species. Remaining populations of Hawaiian honeycreepers (Fringillidae: Drepanidinae) are most abundant and diverse in high elevation refuges above the normal range of disease-carrying mosquitoes. Challenge experiments suggest that honeycreepers are highly susceptible to avian malaria (Plasmodium sp.) but resistance exists in some species. In order to detect low levels of malarial infection and quantify prevalence of Plasmodium in high elevation natural populations of Hawaiian birds, a polymerase chain reaction (PCR) based diagnostic test was developed that identifies rRNA genes of Plasmodium in avian blood samples. Quantitative competitive PCR (QC-PCR) experiments indicate that the detection limit of our test is an order of magnitude greater than that reported for human malaria DNA blot tests. Compared with standard histological methods, the PCR test detected a higher prevalence of diseased birds at mid-elevations. Malaria was detected in three species of native birds living in a high elevation wildlife refuge on the island of Hawaii and in four species from Maui. Our results show that avian malaria is more widespread in Hawaiian forests than previously thought, a finding that has important conservation implications for these threatened species.

  8. A novel nested PCR for the diagnosis of calicivirus infections in the cat.

    PubMed

    Marsilio, Fulvio; Di Martino, Barbara; Decaro, Nicola; Buonavoglia, Canio

    2005-01-05

    A novel nested PCR (nPCR) assay is reported on the diagnosis of the feline calicivirus (FCV) infection. The test was performed on 47 ocular and 40 pharyngeal swabs collected from 47 cats with respiratory syndrome; among the 87 samples examined, 18 ocular and 23 pharyngeal swabs were positive in nPCR. The nPCR sensitivity was compared to other diagnostic techniques such as virus isolation on cell culture and reverse transcriptase-polymerase chain reaction (RT-PCR). The nPCR was more sensitive than the virus isolation and RT-PCR; therefore, it can be used for calicivirosis diagnosis in cats.

  9. Two-temperature LATE-PCR endpoint genotyping

    PubMed Central

    Sanchez, J Aquiles; Abramowitz, Jessica D; Salk, Jesse J; Reis, Arthur H; Rice, John E; Pierce, Kenneth E; Wangh, Lawrence J

    2006-01-01

    Background In conventional PCR, total amplicon yield becomes independent of starting template number as amplification reaches plateau and varies significantly among replicate reactions. This paper describes a strategy for reconfiguring PCR so that the signal intensity of a single fluorescent detection probe after PCR thermal cycling reflects genomic composition. The resulting method corrects for product yield variations among replicate amplification reactions, permits resolution of homozygous and heterozygous genotypes based on endpoint fluorescence signal intensities, and readily identifies imbalanced allele ratios equivalent to those arising from gene/chromosomal duplications. Furthermore, the use of only a single colored probe for genotyping enhances the multiplex detection capacity of the assay. Results Two-Temperature LATE-PCR endpoint genotyping combines Linear-After-The-Exponential (LATE)-PCR (an advanced form of asymmetric PCR that efficiently generates single-stranded DNA) and mismatch-tolerant probes capable of detecting allele-specific targets at high temperature and total single-stranded amplicons at a lower temperature in the same reaction. The method is demonstrated here for genotyping single-nucleotide alleles of the human HEXA gene responsible for Tay-Sachs disease and for genotyping SNP alleles near the human p53 tumor suppressor gene. In each case, the final probe signals were normalized against total single-stranded DNA generated in the same reaction. Normalization reduces the coefficient of variation among replicates from 17.22% to as little as 2.78% and permits endpoint genotyping with >99.7% accuracy. These assays are robust because they are consistent over a wide range of input DNA concentrations and give the same results regardless of how many cycles of linear amplification have elapsed. The method is also sufficiently powerful to distinguish between samples with a 1:1 ratio of two alleles from samples comprised of 2:1 and 1:2 ratios of the

  10. Advances in digital polymerase chain reaction (dPCR) and its emerging biomedical applications.

    PubMed

    Cao, Lei; Cui, Xingye; Hu, Jie; Li, Zedong; Choi, Jane Ru; Yang, Qingzhen; Lin, Min; Ying Hui, Li; Xu, Feng

    2017-04-15

    Since the invention of polymerase chain reaction (PCR) in 1985, PCR has played a significant role in molecular diagnostics for genetic diseases, pathogens, oncogenes and forensic identification. In the past three decades, PCR has evolved from end-point PCR, through real-time PCR, to its current version, which is the absolute quantitive digital PCR (dPCR). In this review, we first discuss the principles of all key steps of dPCR, i.e., sample dispersion, amplification, and quantification, covering commercialized apparatuses and other devices still under lab development. We highlight the advantages and disadvantages of different technologies based on these steps, and discuss the emerging biomedical applications of dPCR. Finally, we provide a glimpse of the existing challenges and future perspectives for dPCR.

  11. A Basic Guide to Real Time PCR in Microbial Diagnostics: Definitions, Parameters, and Everything

    PubMed Central

    Kralik, Petr; Ricchi, Matteo

    2017-01-01

    Real time PCR (quantitative PCR, qPCR) is now a well-established method for the detection, quantification, and typing of different microbial agents in the areas of clinical and veterinary diagnostics and food safety. Although the concept of PCR is relatively simple, there are specific issues in qPCR that developers and users of this technology must bear in mind. These include the use of correct terminology and definitions, understanding of the principle of PCR, difficulties with interpretation and presentation of data, the limitations of qPCR in different areas of microbial diagnostics and parameters important for the description of qPCR performance. It is not our intention in this review to describe every single aspect of qPCR design, optimization, and validation; however, it is our hope that this basic guide will help to orient beginners and users of qPCR in the use of this powerful technique. PMID:28210243

  12. Methods for producing partially digested restriction DNA fragments and for producing a partially modified PCR product

    DOEpatents

    Wong, Kwong-Kwok

    2000-01-01

    The present invention is an improved method of making a partially modified PCR product from a DNA fragment with a polymerase chain reaction (PCR). In a standard PCR process, the DNA fragment is combined with starting deoxynucleoside triphosphates, a primer, a buffer and a DNA polymerase in a PCR mixture. The PCR mixture is then reacted in the PCR producing copies of the DNA fragment. The improvement of the present invention is adding an amount of a modifier at any step prior to completion of the PCR process thereby randomly and partially modifying the copies of the DNA fragment as a partially modified PCR product. The partially modified PCR product may then be digested with an enzyme that cuts the partially modified PCR product at unmodified sites thereby producing an array of DNA restriction fragments.

  13. STITCHER: A web resource for high-throughput design of primers for overlapping PCR applications.

    PubMed

    O'Halloran, Damien M

    2015-06-01

    Overlapping PCR is routinely used in a wide number of molecular applications. These include stitching PCR fragments together, generating fluorescent transcriptional and translational fusions, inserting mutations, making deletions, and PCR cloning. Overlapping PCR is also used for genotyping by traditional PCR techniques and in detection experiments using techniques such as loop-mediated isothermal amplification (LAMP). STITCHER is a web tool providing a central resource for researchers conducting all types of overlapping PCR experiments with an intuitive interface for automated primer design that's fast, easy to use, and freely available online (http://ohalloranlab.net/STITCHER.html). STITCHER can handle both single sequence and multi-sequence input, and specific features facilitate numerous other PCR applications, including assembly PCR, adapter PCR, and primer walking. Field PCR, and in particular, LAMP, offers promise as an on site tool for pathogen detection in underdeveloped areas, and STITCHER includes off-target detection features for pathogens commonly targeted using LAMP technology.

  14. Use of multiplex PCR and PCR restriction enzyme analysis for detection and exploration of the variability in the free-living amoeba Naegleria in the environment.

    PubMed

    Pélandakis, Michel; Pernin, Pierre

    2002-04-01

    A multiplex PCR was developed to simultaneously detect Naegleria fowleri and other Naegleria species in the environment. Multiplex PCR was also capable of identifying N. fowleri isolates with internal transcribed spacers of different sizes. In addition, restriction fragment length polymorphism analysis of the PCR product distinguished the main thermophilic Naegleria species from the sampling sites.

  15. Use of Multiplex PCR and PCR Restriction Enzyme Analysis for Detection and Exploration of the Variability in the Free-Living Amoeba Naegleria in the Environment

    PubMed Central

    Pélandakis, Michel; Pernin, Pierre

    2002-01-01

    A multiplex PCR was developed to simultaneously detect Naegleria fowleri and other Naegleria species in the environment. Multiplex PCR was also capable of identifying N. fowleri isolates with internal transcribed spacers of different sizes. In addition, restriction fragment length polymorphism analysis of the PCR product distinguished the main thermophilic Naegleria species from the sampling sites. PMID:11916734

  16. Detection of glycopeptide resistance genes in enterococci by multiplex PCR

    PubMed Central

    Bhatt, Puneet; Sahni, A.K.; Praharaj, A.K.; Grover, Naveen; Kumar, Mahadevan; Chaudhari, C.N.; Khajuria, Atul

    2014-01-01

    Background Vancomycin Resistant Enterococci (VRE) are a major cause of nosocomial infections. There are various phenotypic and genotypic methods of detection of glycopeptide resistance in enterococci. This study utilizes multiplex PCR for reliable detection of various glycopeptides resistance genes in VRE. Method This study was conducted to detect and to assess the prevalence of vancomycin resistance among enterococci isolates. From October 2011 to June 2013, a total of 96 non-repetitive isolates of enterococci from various clinical samples were analyzed. VRE were identified by Kirby Bauer disc diffusion method with Clinical and Laboratory Standards Institute (CLSI) guidelines. Minimum inhibitory concentration (MIC) of all isolates for vancomycin and teicoplanin was determined by E-test. Multiplex PCR was carried out for all enterococci isolates using six sets of primers. Results Out of 96 isolates, 14 (14.6%) were found to be resistant to vancomycin by vancomycin E-test method (MIC ≥32 μg/ml). Out of these 14 isolates, 13 were also resistant to teicoplanin (MIC ≥16 μg/ml). VanA gene was detected in all the 14 isolates by Multiplex PCR. One of the PCR amplicons was sent for sequencing and the sequence received was submitted in the GenBank (GenBank accession no. KF181100). Conclusion Prevalence of VRE in this study was 14.6%. Multiplex PCR is a robust, sensitive and specific technique, which can be used for rapid detection of various glycopeptide resistance genes. Rapid identification of patients infected or colonized with VRE is essential for implementation of appropriate control measures to prevent their spread. PMID:25609863

  17. A Ribeiroia spp. (Class: Trematoda) - Specific PCR-based diagnostic

    USGS Publications Warehouse

    Reinitz, D.M.; Yoshino, T.P.; Cole, R.A.

    2007-01-01

    Increased reporting of amphibian malformations in North America has been noted with concern in light of reports that amphibian numbers and species are declining worldwide. Ribeiroia ondatrae has been shown to cause a variety of types of malformations in amphibians. However, little is known about the prevalence of R. ondatrae in North America. To aid in conducting field studies of Ribeiroia spp., we have developed a polymerase chain reaction (PCR)-based diagnostic. Herein, we describe the development of an accurate, rapid, simple, and cost-effective diagnostic for detection of Ribeiroia spp. infection in snails (Planorbella trivolvis). Candidate oligonucleotide primers for PCR were designed via DNA sequence analyses of multiple ribosomal internal transcribed spacer-2 regions from Ribeiroia spp. and Echinostoma spp. Comparison of consensus sequences determined from both genera identified areas of sequence potentially unique to Ribeiroia spp. The PCR reliably produced a diagnostic 290-base pair (bp) product in the presence of a wide concentration range of snail or frog DNA. Sensitivity was examined with DNA extracted from single R. ondatrae cercaria. The single-tube PCR could routinely detect less than 1 cercariae equivalent, because DNA isolated from a single cercaria could be diluted at least 1:50 and still yield a positive result via gel electrophoresis. An even more sensitive nested PCR also was developed that routinely detected 100 fg of the 290-bp fragment. The assay did not detect furcocercous cercariae of certain Schistosomatidae, Echinostoma sp., or Sphaeridiotrema globulus nor adults of Clinostomum sp. or Cyathocotyle bushiensis. Field testing of 137 P. trivolvis identified 3 positives with no overt environmental cross-reactivity, and results concurred with microscopic examinations in all cases. ?? American Society of Parasitologists 2007.

  18. Discrimination between Mycoplasma mycoides subsp. capri and Mycoplasma capricolum subsp. capricolum using PCR-RFLP and PCR.

    PubMed

    Cillara, Grazia; Manca, Maria Giovanna; Longheu, Carla; Tola, Sebastiana

    2015-09-01

    In this study, the dihydrolipoyl dehydrogenase (lpdA) gene was used to distinguish Mycoplasma mycoides subsp. capri (Mmc) from Mycoplasma capricolum subsp. capricolum (Mcc), two of four Mycoplasma species that cause contagious agalactia in sheep and goats. After alignment of nucleotide sequences of both species, specific primer sets were designed from unchanging and variable gene segments. The first primer set LPD-C1-F/LPD-C1-R was used to amplify a 911 bp fragment that was subsequently co-digested with FastDigest PstI, SspI, EcoRI and ClaI enzymes. The PCR-RFLP profiles differentiated the two mycoplasma species. The second primer set was used to distinguish Mmc from Mcc by single tube PCR. Both methods were further applied to identify 54 isolates collected from dairy herds from different provinces in Sardinia. The results of this study showed that PCR-RFLP and PCR could be used in routine diagnosis for rapid and specific simultaneous discrimination of Mmc and Mcc.

  19. Nested-PCR and TaqMan real-time quantitative PCR assays for human adenoviruses in environmental waters.

    PubMed

    Huang, Wen-Chien; Chou, Yi-Pen; Kao, Po-Min; Hsu, Tsui-Kang; Su, Hung-Chang; Ho, Ying-Ning; Yang, Yi-Chun; Hsu, Bing-Mu

    2016-01-01

    Human adenovirus (HAdV) infections can occur throughout the year. Cases of HAdV-associated respiratory disease have been more common in the late winter, spring, and early summer. In this study, to provide viral pollution data for further epidemiological studies and governmental actions, the presence of HAdV in the aquatic environment was quantitatively surveyed in the summer. This study was conducted to compare the efficiencies of nested-PCR (polymerase chain reaction) and qPCR (quantitative PCR) for detecting HAdV in environmental waters. A total of 73 water samples were collected from Puzi River in Taiwan and subjected to virus concentration methods. In the results, qPCR had much better efficiency for specifying the pathogen in river sample. HAdV41 was detected most frequently in the river water sample (10.9%). The estimated HAdV concentrations ranged between 6.75 × 10(2) and 2.04 × 10(9) genome copies/L. Significant difference was also found in heterotrophic plate counts, conductivity, water temperature, and water turbidity between presence/absence of HAdV. HAdV in the Puzi River may pose a significant health risk.

  20. [Primers design and optimization of PCR and nested-PCR assays for the specific detection of Tritrichomonas foetus].

    PubMed

    Fernandes, Paula Rogério; Da Silva, Andréa Caetano; Gambarini, Maria Lúcia; Linhares, Guido Fontgalland C

    2008-01-01

    Tritrichomonas foetus is a pathogenic protozoan that causes a venereal disease in cattle known as bovine genital tricomonosis. In spite of the efficacy to recognize the target genomic DNA, the protocols so far developed for the diagnosis of this organism by PCR promote some inespecific amplifications or they are unable to discriminate T. foetus against other species within the genus. The objective of this study was to assess and optimize PCR and nested-PCR assays for the specific diagnosis of T. foetus, using novel primers selected from the alignment of sequences of the genes 18S rRNA, 5.8S rRNA, 28S rRNA and of the internal transcribed spacers of the rDNA (ITS1 and ITS2). A pair of primers was constructed for the genus-specific amplification of a 648 bp fragment and two others to amplify T. foetus species-specific fragments of 343 and 429 bp. No cross amplification was observed against Bos taurus genomic DNA neither against the DNA of usual bovine genital pathogens. Both, single and nested-PCR assays, presented analytical sensitivity to detect at least two T. foetus organisms.

  1. Application of multiplex PCR, pulsed-field gel electrophoresis (PFGE), and BOX-PCR for molecular analysis of enterococci

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The objective of the study was to use band-based molecular methods including BOX-PCR (Polymerase Chain Reaction) and Pulsed-Field Gel Electrophoresis (PFGE) to determine if genetically related enterococci were found among different stores, food types, or years. Enterococci were also characterized f...

  2. Quantitative real-time PCR (qPCR) for Eimeria tenella replication--Implications for experimental refinement and animal welfare.

    PubMed

    Nolan, Matthew J; Tomley, Fiona M; Kaiser, Pete; Blake, Damer P

    2015-10-01

    The Eimeria species are highly pathogenic parasites of chickens. Research aimed at reducing their impact is hindered by a lack of non-subjective, quantitative, tools to measure parasite replication in the host. The time-consuming, and often time-sensitive, nature of existing approaches precludes their use in large-scale genetic, epidemiological, and evolutionary analyses. We have used quantitative real-time PCR (qPCR) to accurately quantify Eimeria tenella in chicken tissue and shown this to be more efficient and sensitive than traditional methodologies. We tested four chicken-specific reference qPCR assays and found beta-actin (actb) to be optimal for sample normalisation. In an experimental setting, chickens were inoculated with 500, 1500, or 4500 E. tenella oocysts and parasite replication and the impact of infection measured by i) qPCR analysis of DNA extracted from caecal tissues collected at five and eight days post-infection (dpi), ii) faecal oocyst counts (FOCs) on samples taken from six to eight dpi, and iii) lesion scoring on caeca collected post-mortem at five and eight dpi. Quantitative real-time PCR test results indicated a significant dose-dependent increase in parasite numbers among study groups for samples collected five dpi (i.e., prior to gametogony) (R(2)=0.994) (p<0.002) but not in those from day eight (after most oocyst shedding) (R(2)=0.006) (p>0.379). A strong dose-dependent increase in parasite replication and severity of infection was also revealed by FOC (R(2)=0.997) and lesion scoring. Importantly, qPCR offers substantial improvements for animal welfare via improved statistical power and reduced group sizes in experimental studies. The described qPCR method overcomes subjective limitations of coproscopic quantification, allows reproducible medium- to high-throughput examination of tissues, faeces, and oocysts, and is a valuable tool for determining the impact of Eimeria infections in both experimental and field settings.

  3. Quantitative real-time PCR (qPCR) for Eimeria tenella replication — Implications for experimental refinement and animal welfare

    PubMed Central

    Nolan, Matthew J.; Tomley, Fiona M.; Kaiser, Pete; Blake, Damer P.

    2015-01-01

    The Eimeria species are highly pathogenic parasites of chickens. Research aimed at reducing their impact is hindered by a lack of non-subjective, quantitative, tools to measure parasite replication in the host. The time-consuming, and often time-sensitive, nature of existing approaches precludes their use in large-scale genetic, epidemiological, and evolutionary analyses. We have used quantitative real-time PCR (qPCR) to accurately quantify Eimeria tenella in chicken tissue and shown this to be more efficient and sensitive than traditional methodologies. We tested four chicken-specific reference qPCR assays and found beta-actin (actb) to be optimal for sample normalisation. In an experimental setting, chickens were inoculated with 500, 1500, or 4500 E. tenella oocysts and parasite replication and the impact of infection measured by i) qPCR analysis of DNA extracted from caecal tissues collected at five and eight days post-infection (dpi), ii) faecal oocyst counts (FOCs) on samples taken from six to eight dpi, and iii) lesion scoring on caeca collected post-mortem at five and eight dpi. Quantitative real-time PCR test results indicated a significant dose-dependent increase in parasite numbers among study groups for samples collected five dpi (i.e., prior to gametogony) (R2 = 0.994) (p < 0.002) but not in those from day eight (after most oocyst shedding) (R2 = 0.006) (p > 0.379). A strong dose-dependent increase in parasite replication and severity of infection was also revealed by FOC (R2 = 0.997) and lesion scoring. Importantly, qPCR offers substantial improvements for animal welfare via improved statistical power and reduced group sizes in experimental studies. The described qPCR method overcomes subjective limitations of coproscopic quantification, allows reproducible medium- to high-throughput examination of tissues, faeces, and oocysts, and is a valuable tool for determining the impact of Eimeria infections in both experimental and field settings

  4. Computational tradeoffs in multiplex PCR assay design for SNP genotyping

    PubMed Central

    Rachlin, John; Ding, Chunming; Cantor, Charles; Kasif, Simon

    2005-01-01

    Background Multiplex PCR is a key technology for detecting infectious microorganisms, whole-genome sequencing, forensic analysis, and for enabling flexible yet low-cost genotyping. However, the design of a multiplex PCR assays requires the consideration of multiple competing objectives and physical constraints, and extensive computational analysis must be performed in order to identify the possible formation of primer-dimers that can negatively impact product yield. Results This paper examines the computational design limits of multiplex PCR in the context of SNP genotyping and examines tradeoffs associated with several key design factors including multiplexing level (the number of primer pairs per tube), coverage (the % of SNP whose associated primers are actually assigned to one of several available tube), and tube-size uniformity. We also examine how design performance depends on the total number of available SNPs from which to choose, and primer stringency criterial. We show that finding high-multiplexing/high-coverage designs is subject to a computational phase transition, becoming dramatically more difficult when the probability of primer pair interaction exceeds a critical threshold. The precise location of this critical transition point depends on the number of available SNPs and the level of multiplexing required. We also demonstrate how coverage performance is impacted by the number of available snps, primer selection criteria, and target multiplexing levels. Conclusion The presence of a phase transition suggests limits to scaling Multiplex PCR performance for high-throughput genomics applications. Achieving broad SNP coverage rapidly transitions from being very easy to very hard as the target multiplexing level (# of primer pairs per tube) increases. The onset of a phase transition can be "delayed" by having a larger pool of SNPs, or loosening primer selection constraints so as to increase the number of candidate primer pairs per SNP, though the latter

  5. PCR detection and characterization of type-2 porcine circovirus.

    PubMed Central

    Hamel, A L; Lin, L L; Sachvie, C; Grudeski, E; Nayar, G P

    2000-01-01

    A polymerase chain reaction (PCR) assay was developed for detecting porcine circovirus (PCV). The assay readily detected type-2 PCV (PCV-2) and type-1 PCV (PCV-1). The PCR primers were designed based on DNA sequences conserved in all reported PCV genomes. Type 1 PCV and type 2 PCV both produced 438 bp amplification products, which were easily identified and differentiated from one another by restriction fragment length polymorphism (RFLP) analysis. Porcine circovirus was detected in 55% (931/1693) of randomly tested pigs with various clinical signs and lesions, most of which were difficult to differentiate from those associated with porcine reproductive and respiratory syndrome (PRRS). The PCR products from all positive clinical samples were identified by RFLP to be only PCV-2; DNA tested by PCR was extracted directly from one or more of lung, mesenteric or mediastinal lymph nodes, and tonsil. Type 2 PCV was also detected in 6% (2/34) of DNA extracted directly from semen of randomly chosen healthy boars. Positive PCR reactions from 554 diseased pigs were characterized by RFLP and categorized into 5 different profiles (A-E), of which 82.8% were PCV-2A (456/554), 3.0% were PCV-2B (17/554), 9.9% were PCV-2C (55/554), 1.1% were PCV-2D (6/554), and 3.2% were PCV-2E (18/554). The complete genomic nucleotide sequences of PCV-2A, B, C, D, and E were determined and found to have at least 95% homology compared with one another and with all other PCV-2 found in the GenBank database. All PCV-2 had less than 76% homology with PCV-1. This PCR assay will hopefully be useful to veterinary diagnostic laboratories for routine testing and surveillance of infection with PCV-2. The RFLP profiling system might be useful for preliminary characterization and identification of PCV isolates and might also benefit studies on the molecular epidemiology of PCV. Images Figure 1. PMID:10680656

  6. Comparison of Quantitative PCR and Droplet Digital PCR Multiplex Assays for Two Genera of Bloom-Forming Cyanobacteria, Cylindrospermopsis and Microcystis

    PubMed Central

    Te, Shu Harn; Chen, Enid Yingru

    2015-01-01

    The increasing occurrence of harmful cyanobacterial blooms, often linked to deteriorated water quality and adverse public health effects, has become a worldwide concern in recent decades. The use of molecular techniques such as real-time quantitative PCR (qPCR) has become increasingly popular in the detection and monitoring of harmful cyanobacterial species. Multiplex qPCR assays that quantify several toxigenic cyanobacterial species have been established previously; however, there is no molecular assay that detects several bloom-forming species simultaneously. Microcystis and Cylindrospermopsis are the two most commonly found genera and are known to be able to produce microcystin and cylindrospermopsin hepatotoxins. In this study, we designed primers and probes which enable quantification of these genera based on the RNA polymerase C1 gene for Cylindrospermopsis species and the c-phycocyanin beta subunit-like gene for Microcystis species. Duplex assays were developed for two molecular techniques—qPCR and droplet digital PCR (ddPCR). After optimization, both qPCR and ddPCR assays have high linearity and quantitative correlations for standards. Comparisons of the two techniques showed that qPCR has higher sensitivity, a wider linear dynamic range, and shorter analysis time and that it was more cost-effective, making it a suitable method for initial screening. However, the ddPCR approach has lower variability and was able to handle the PCR inhibition and competitive effects found in duplex assays, thus providing more precise and accurate analysis for bloom samples. PMID:26025892

  7. Comparison of Quantitative PCR and Droplet Digital PCR Multiplex Assays for Two Genera of Bloom-Forming Cyanobacteria, Cylindrospermopsis and Microcystis.

    PubMed

    Te, Shu Harn; Chen, Enid Yingru; Gin, Karina Yew-Hoong

    2015-08-01

    The increasing occurrence of harmful cyanobacterial blooms, often linked to deteriorated water quality and adverse public health effects, has become a worldwide concern in recent decades. The use of molecular techniques such as real-time quantitative PCR (qPCR) has become increasingly popular in the detection and monitoring of harmful cyanobacterial species. Multiplex qPCR assays that quantify several toxigenic cyanobacterial species have been established previously; however, there is no molecular assay that detects several bloom-forming species simultaneously. Microcystis and Cylindrospermopsis are the two most commonly found genera and are known to be able to produce microcystin and cylindrospermopsin hepatotoxins. In this study, we designed primers and probes which enable quantification of these genera based on the RNA polymerase C1 gene for Cylindrospermopsis species and the c-phycocyanin beta subunit-like gene for Microcystis species. Duplex assays were developed for two molecular techniques-qPCR and droplet digital PCR (ddPCR). After optimization, both qPCR and ddPCR assays have high linearity and quantitative correlations for standards. Comparisons of the two techniques showed that qPCR has higher sensitivity, a wider linear dynamic range, and shorter analysis time and that it was more cost-effective, making it a suitable method for initial screening. However, the ddPCR approach has lower variability and was able to handle the PCR inhibition and competitive effects found in duplex assays, thus providing more precise and accurate analysis for bloom samples.

  8. A prospective study on the incidence of Borrelia burgdorferi sensu lato infection after a tick bite in Sweden and on the Åland Islands, Finland (2008-2009).

    PubMed

    Wilhelmsson, Peter; Fryland, Linda; Lindblom, Pontus; Sjöwall, Johanna; Ahlm, Clas; Berglund, Johan; Haglund, Mats; Henningsson, Anna J; Nolskog, Peter; Nordberg, Marika; Nyberg, Clara; Ornstein, Katharina; Nyman, Dag; Ekerfelt, Christina; Forsberg, Pia; Lindgren, Per-Eric

    2016-02-01

    Lyme borreliosis (LB) is a common and increasing tick-borne disease in Europe. The risk of acquiring a Borrelia infection after a tick bite is not fully known. Therefore, we investigated the incidence of Borrelia infection after a bite by a Borrelia-infected tick and if the Borrelia load and/or the duration of tick-feeding influenced the risk of infection. During 2008-2009, ticks and blood samples were collected from 1546 tick-bitten persons from Sweden and the Åland Islands, Finland. Follow-up blood samples were taken 3 months after the tick bite. The duration of tick feeding was microscopically estimated and Borrelia was detected and quantified in ticks by real-time PCR. Anti-Borrelia antibodies were detected in sera using ELISA tests and immunoblot. Five percent (78/1546) of the study participants developed Borrelia infection (LB diagnosis and/or seroconversion) after a tick bite (45% bitten by Borrelia-infected ticks and 55% bitten by uninfected ticks). Of these, 33 developed LB (whereof 9 also seroconverted) while 45 participants seroconverted only. Experience of non-specific symptoms was more frequently reported by Borrelia-infected participants compared to uninfected participants. All who seroconverted removed "their" ticks significantly later than those who did not. The Borrelia load in the ticks did not explain the risk of seroconversion. Regional and sex differences in the Borrelia seroprevalence were found. The risk of developing a Borrelia infection after a bite by a Borrelia-infected tick is small but increases with the duration of tick feeding.

  9. Sporulation properties and antimicrobial susceptibility in endemic and rare Clostridium difficile PCR ribotypes.

    PubMed

    Zidaric, Valerija; Rupnik, Maja

    2016-06-01

    Increased sporulation and antibiotic resistance have been proposed to be associated with certain Clostridium difficile epidemic strains such as PCR ribotype 027. In this study we examined these properties in another widespread PCR ribotype, 014/020, in comparison to prevalent PCR ribotype 002 and a group of rarely represented PCR ribotypes. Highest sporulation was observed in 014/020 strains at 24 h, while after 72 h PCR ribotype 002 and rare PCR ribotypes formed higher total number of spores. PCR ribotype 014/020 strains exhibited slightly higher resistance to tested antimicrobials, followed by group of rare PCR ribotypes and less common PCR ribotype 002. Neither sporulation properties nor antibiotic resistance clearly differed in endemic and rare strains.

  10. False-Positive Viability PCR Results: An Association with Microtubes.

    PubMed

    Agustí, Gemma; Fittipaldi, Mariana; Codony, Francesc

    2017-03-01

    Currently, one of the most challenged points to expand the use of viability PCR technique is achieving the complete exclusion of dead cells amplification signals, thus avoiding the overestimation of live cells population. Considering that, and based on the hypothesis that DNA may be retained by microtube walls, the impact of the microtube was addressed on signals from live and heat-killed cells. A double-dye reagent, PEMAX™, which comprises a mix of photo-reactive azide forms of phenanthridium, was used in this work. We found that if both the incubation and the photoactivation steps are carried out in different microtubes, the dead cell signal is greatly reduced than when those steps are done in the same tube. Therefore, the strategy depicted in this study presents a simple and efficient step in minimizing false-positive signal when employing viability PCR.

  11. Tuberculosis diagnosed by PCR analysis of synovial fluid.

    PubMed

    Fujimoto, Nobukazu; Gemba, Kenichi; Yao, Atsushi; Ozaki, Shinji; Ono, Katsuichiro; Wada, Sae; Fujii, Yasuhiro; Namba, Yoshifumi; Kishimoto, Takumi

    2010-02-01

    Tuberculosis is a leading cause of mortality due to an infectious agent worldwide. It often affects multiple organs by hematogenous spread of Mycobacterium tuberculosis, but knee-joint involvement is extremely rare, comprising approximately 0.1% of all forms of tuberculosis. We present a case of tuberculous pleuritis with knee-joint involvement. Cytological and biochemical analysis of the pleural fluid and a biopsy specimen of the cervical lymph node indicated tuberculosis, but a definitive diagnosis was not given. A confirmed diagnosis was finally obtained through PCR analysis of the synovial fluid. Tuberculosis should be included in the differential diagnosis in patients with persistent pain and swelling of the knee. PCR analysis of the synovial fluid is a quick and useful method for the diagnosis.

  12. Development of a PCR for identification of Bordetella hinzii.

    PubMed

    Register, Karen B

    2013-06-01

    Bordetella hinzii infects primarily poultry and immunocompromised humans. It is closely related to the etiologic agent of turkey coryza, Bordetella avium. Distinguishing between B. avium and B. hinzii is difficult, and there is no method for identification of B. hinzii suitable for use by diagnostic laboratories. This report details the development of a B. hinzii-specific PCR targeting the ompA gene. Assay sensitivity is 100% based on analysis of 48 B. hinzii isolates from diverse geographic locations representing all known ribotypes. Evaluation of 71 isolates of B. avium and 20 other bacterial isolates from poultry, comprising gram-negative and gram-positive commensals and pathogens of nine genera, demonstrated an assay specificity of 100%. The ompA PCR is a rapid, reliable, and accurate method for identification of B. hinzii and provides a valuable new tool for veterinary diagnostic laboratories investigating poultry respiratory disease outbreaks.

  13. DPPrimer – A Degenerate PCR Primer Design Tool

    PubMed Central

    Gahoi, Shachi; Arya, L; Anil, Rai; Marla, ss

    2013-01-01

    Designed degenerate primers unlike conventional primers are superior in matching and amplification of large number of genes, from related gene families. DPPrimer tool was designed to predict primers for PCR amplification of homologous gene from related or diverse plant species. The key features of this tool include platform independence and user friendliness in primer design. Embedded features such as search for functional domains, similarity score selection and phylogebetic tree further enhance the user friendliness of DPPrimer tool. Performance of DPPrimer tool was evaluated by successful PCR amplification of ADP-glucose phosphorylase genes from wheat, barley and rice. Availability DPPrimer is freely accessible at http://202.141.12.147/DGEN_tool/index.html PMID:24307773

  14. Zip nucleic acids are potent hydrolysis probes for quantitative PCR

    PubMed Central

    Paris, Clément; Moreau, Valérie; Deglane, Gaëlle; Voirin, Emilie; Erbacher, Patrick; Lenne-Samuel, Nathalie

    2010-01-01

    Zip nucleic acids (ZNAs) are oligonucleotides conjugated with cationic spermine units that increase affinity for their target. ZNAs were recently shown to enable specific and sensitive reactions when used as primers for polymerase chain reaction (PCR) and reverse-transcription. Here, we report their use as quantitative PCR hydrolysis probes. Ultraviolet duplex melting data demonstrate that attachment of cationic residues to the 3′ end of an oligonucleotide does not alter its ability to discriminate nucleotides nor the destabilization pattern relative to mismatch location in the oligonucleotide sequence. The stability increase provided by the cationic charges allows the use of short dual-labeled probes that significantly improve single-nucleotide polymorphism genotyping. Longer ZNA probes were shown to display reduced background fluorescence, therefore, generating greater sensitivity and signal level as compared to standard probes. ZNA probes thus provide broad flexibility in assay design and also represent an effective alternative to minor groove binder- and locked nucleic-acid-containing probes. PMID:20071749

  15. An improved PCR method for gender identification of eagles.

    PubMed

    Chang, Hsueh-Wei; Chou, Ta-Ching; Gu, De-Leung; Cheng, Chun-An; Chang, Chia-Che; Yao, Cheng-Te; Chuang, Li-Yeh; Wen, Cheng-Hao; Chou, Yii-Cheng; Tan, Kock-Yee; Cheng, Chien-Chung

    2008-06-01

    Eagles are sexually monomorphic and therefore it is difficult to determine their gender, which is a crucial need for management purposes. In this study, we have developed an improved gender identification method by exploiting length differences between the Chromo-Helicase-DNA binding protein (CHD)-Z and CHD-W genes of Spilornis cheela hoya. By comparing DNA sequences for CHD-W and CHD-Z from 10 species of Falconiformes eagles we designed universal gender identification PCR primers that exploit differences in product size. Standard agarose gels were shown to easily distinguish between the 148-bp CHD-ZW and the 258-bp CHD-W PCR products. When used with 28 samples of S. cheela hoya, our improved universal primers provided a fast and precise gender identification assay.

  16. Chemical surface management for micro PCR in silicon chip thermocyclers

    NASA Astrophysics Data System (ADS)

    Felbel, Jana; Bieber, Ivonne; Koehler, Johann M.

    2002-11-01

    Silicon, silicon dioxide, glass and other key materials of micro system technology show an inhibiting effect on PCR. This negative influence becomes seriously, if devices are miniaturized, particularly in case of flow-through devices due to their high surface to volume ratio. In contrast, alkyl-substituted surfaces do not inhibit the reaction. Although the silanization improves the compatibility, the suppression of inhibition by wall surface treatment was not stable over longer time intervals. Therefore, the stability of chemical surface modifications was studied in dependence of silanization, material, pH, temperature and buffer composition. The efficiency of surface covering by molecular substitution was characterized by wetting experiments as well as by PCR test runs. The results show that the surface treatment can be optimized by the choice of silanization agents and the concentration of surface active additives.

  17. DNA probe and PCR-specific reaction for Lactobacillus plantarum.

    PubMed

    Quere, F; Deschamps, A; Urdaci, M C

    1997-06-01

    A 300 bp DNA fragment of Lactobacillus plantarum isolated by randomly amplified polymorphic DNA (RAPD) analysis was cloned and sequenced. This fragment was tested using a dot-blot DNA hybridization to technique for its ability to identify Lact. plantarum strains. This probe hybridized with all Lact. plantarum strains tested and with some strains of Lact. pentosus, albeit more weakly. Two internal primers of this probe were selected (LbP11 and LbP12) and polymerase chain reaction (PCR) was carried out. All Lact. plantarum strains tested amplified a 250 bp fragment contrary to the other LAB species tested. This specific PCR for Lact. plantarum was also performed from colonies grown on MRS medium with similar results. These methods enabled the rapid and specific detection and identification of Lact. plantarum.

  18. Targeted resequencing and variant validation using pxlence PCR assays.

    PubMed

    Coppieters, Frauke; Verniers, Kimberly; De Leeneer, Kim; Vandesompele, Jo; Lefever, Steve

    2016-01-01

    The advent of next-generation sequencing technologies had a profound impact on molecular diagnostics. PCR is a popular method for target enrichment of disease gene panels. Using our proprietary primer-design pipeline, primerXL, we have created almost one million assays covering over 98% of the human exome. Here we describe the assay specification and both in silico and wet-lab validation of a selected set of 2294 assays using both next-generation sequencing and Sanger sequencing. Using a universal PCR protocol without optimization, these assays result in high coverage uniformity and limited non-specific coverage. In addition, data indicates a positive correlation between the predictive in silico specificity score and the amount of assay non-specific coverage.

  19. Targeted resequencing and variant validation using pxlence PCR assays

    PubMed Central

    Coppieters, Frauke; Verniers, Kimberly; De Leeneer, Kim; Vandesompele, Jo; Lefever, Steve

    2015-01-01

    The advent of next-generation sequencing technologies had a profound impact on molecular diagnostics. PCR is a popular method for target enrichment of disease gene panels. Using our proprietary primer-design pipeline, primerXL, we have created almost one million assays covering over 98% of the human exome. Here we describe the assay specification and both in silico and wet-lab validation of a selected set of 2294 assays using both next-generation sequencing and Sanger sequencing. Using a universal PCR protocol without optimization, these assays result in high coverage uniformity and limited non-specific coverage. In addition, data indicates a positive correlation between the predictive in silico specificity score and the amount of assay non-specific coverage. PMID:27077044

  20. SNP genotyping using single-tube fluorescent bidirectional PCR.

    PubMed

    Waterfall, Christy M; Cobb, Benjamin D

    2002-07-01

    SNP genotyping is a well-populatedfield with a large number of assay formats offering accurate allelic discrimination. However, there remains a discord between the ultimate goal of rapid, inexpensive assays that do not require complex design considerations and involved optimization strategies. We describe the first integration of bidirectional allele-specific amplification, SYBR Green I, and rapid-cycle PCR to provide a homogeneous SNP-typing assay. Wild-type, mutant, and heterozygous alleles were easily discriminated in a single tube using melt curve profiling of PCR products alone. We demonstrate the effectiveness and reliability of this assay with a blinded trial using clinical samples from individuals with sickle cell anemia, sickle cell trait, or unaffected individuals. The tests were completed in less than 30 min without expensive fluorogenic probes, prohibiting design rules, or lengthy downstream processing for product analysis.

  1. Measuring Digital PCR Quality: Performance Parameters and Their Optimization

    PubMed Central

    Lievens, A.; Jacchia, S.; Kagkli, D.; Savini, C.; Querci, M.

    2016-01-01

    Digital PCR is rapidly being adopted in the field of DNA-based food analysis. The direct, absolute quantification it offers makes it an attractive technology for routine analysis of food and feed samples for their composition, possible GMO content, and compliance with labelling requirements. However, assessing the performance of dPCR assays is not yet well established. This article introduces three straightforward parameters based on statistical principles that allow users to evaluate if their assays are robust. In addition, we present post-run evaluation criteria to check if quantification was accurate. Finally, we evaluate the usefulness of Poisson confidence intervals and present an alternative strategy to better capture the variability in the analytical chain. PMID:27149415

  2. Actuation method and apparatus, micropump, and PCR enhancement method

    DOEpatents

    Ullakko, Kari; Mullner, Peter; Hampikian, Greg; Smith, Aaron

    2015-07-28

    An actuation apparatus includes at least one magnetic shape memory (MSM) element containing a material configured to expand and/or contract in response to exposure to a magnetic field. Among other things, the MSM element may be configured to pump fluid through a micropump by expanding and/or contracting in response to the magnetic field. The magnetic field may rotate about an axis of rotation and exhibit a distribution having a component substantially perpendicular to the axis of rotation. Further, the magnetic field distribution may include at least two components substantially orthogonal to one another lying in one or more planes perpendicular to the axis of rotation. The at least one MSM element may contain nickel, manganese, and gallium. A polymerase chain reaction (PCR) may be enhanced by contacting a PCR reagent and DNA material with the MSM element.

  3. Thermosiphon-based PCR reactor: experiment and modeling.

    PubMed

    Chen, Zongyuan; Qian, Shizhi; Abrams, William R; Malamud, Daniel; Bau, Haim H

    2004-07-01

    A self-actuated, flow-cycling polymerase chain reaction (PCR) reactor that takes advantage of buoyancy forces to continuously circulate reagents in a closed loop through various thermal zones has been constructed, tested, and modeled. The heating required for the PCR is advantageously used to induce fluid motion without the need for a pump. Flow velocities on the order of millimeters per second are readily attainable. In our preliminary prototype, we measured a cross-sectionally averaged velocity of 2.5 mm/s and a cycle time of 104 s. The flow velocity is nearly independent of the loop's length, making the device readily scalable. Successful amplifications of 700- and 305-bp fragments of Bacillus cereus genomic DNA have been demonstrated. Since the device does not require any moving parts, it is particularly suitable for miniature systems.

  4. Culture independent PCR: an alternative enzyme discovery strategy.

    PubMed

    Jacobsen, Jonas; Lydolph, Magnus; Lange, Lene

    2005-01-01

    Degenerate primers were designed for use in a culture-independent PCR screening of DNA from composite fungal communities, inhabiting residues of corn stovers and leaves. According to similarity searches and alignments amplified clone sequences affiliated with glycosyl hydrolase family 7 and glycosyl hydrolase family 45 though significant sequence divergence was observed. Glycosyl hydrolases from families 7 and 45 play a crucial role in biomass conversion to fuel ethanol. Research in this renewable energy source has two objectives: (i) To contribute to development of a renewable alternative to world's limited crude fossil oil reserves and (ii) to reduce air pollution. Amplification with 18S rDNA-specific primers revealed species within the ascomycetous orders Sordariales and Hypocreales as well as basidiomycetous order Agaricales to be present in these communities. Our study documents the value of culture-independent PCR in microbial diversity studies and could add to development of a new enzyme screening technology.

  5. Specific detection by PCR of Streptococcus agalactiae in milk.

    PubMed

    Martinez, G; Harel, J; Gottschalk, M

    2001-01-01

    The aim of this study was to develop a simple and specific method for direct detection of Streptococcus agalactiae from cow's milk. The method was based on polymerase chain reaction (PCR) using species-specific and universal primers derived from the 16S rRNA gene. The amplification product was verified by restriction endonuclease digest and sequencing. Specific identification was proven on a collection of 147 S. agalactiae isolates of bovine and human origin. In addition, 17 strains belonging to different bacterial species that potentially can be found in milk samples also tested negative. The PCR developed was used for direct detection of S. agalactiae in milk, using for the first time with gram-positive bacteria the nucleic acid-binding properties of diatomaceous earth. The test, which has high specificity, high sensitivity (100 cfu/mL), and can be carried out in less than 24 h, represents an innovative diagnostic tool for the detection of S. agalactiae in milk.

  6. Extraction of PCR-amplifiable genomic DNA from Bacillus anthracisspores

    SciTech Connect

    Torok, Tamas

    2003-05-19

    Bacterial endospore disruption and nucleic acid extractionresulting in DNA of PCR-amplifiable quality and quantity are not trivial.Responding to the needs of the Hazardous Materials Response Unit (HMRU),Laboratory Division, Federal Bureau of Investigation, protocols weredeveloped to close these gaps. Effectiveness and reproducibility of thetechniques were validated with laboratory grown pure spores of Bacillusanthracis and its close phylogenetic neighbors, and with spiked soils anddamaged samples.

  7. Evaluation of PCR Systems for Field Screening of Bacillus anthracis

    PubMed Central

    Ozanich, Richard M.; Colburn, Heather A.; Victry, Kristin D.; Bartholomew, Rachel A.; Arce, Jennifer S.; Heredia-Langner, Alejandro; Jarman, Kristin; Kreuzer, Helen W.

    2017-01-01

    There is little published data on the performance of hand-portable polymerase chain reaction (PCR) systems that can be used by first responders to determine if a suspicious powder contains a potential biothreat agent. We evaluated 5 commercially available hand-portable PCR instruments for detection of Bacillus anthracis. We used a cost-effective, statistically based test plan to evaluate systems at performance levels ranging from 0.85-0.95 lower confidence bound (LCB) of the probability of detection (POD) at confidence levels of 80% to 95%. We assessed specificity using purified genomic DNA from 13 B. anthracis strains and 18 Bacillus near neighbors, potential interference with 22 suspicious powders that are commonly encountered in the field by first responders during suspected biothreat incidents, and the potential for PCR inhibition when B. anthracis spores were spiked into these powders. Our results indicate that 3 of the 5 systems achieved 0.95 LCB of the probability of detection with 95% confidence levels at test concentrations of 2,000 genome equivalents/mL (GE/mL), which is comparable to 2,000 spores/mL. This is more than sufficient sensitivity for screening visible suspicious powders. These systems exhibited no false-positive results or PCR inhibition with common suspicious powders and reliably detected B. anthracis spores spiked into these powders, though some issues with assay controls were observed. Our testing approach enables efficient performance testing using a statistically rigorous and cost-effective test plan to generate performance data that allow users to make informed decisions regarding the purchase and use of field biodetection equipment. PMID:28192050

  8. Operational Evaluation of the Rapid Viability PCR Method for ...

    EPA Pesticide Factsheets

    Journal Article This research work has a significant impact on the use of the RV-PCR method to analyze post-decontamination environmental samples during an anthrax event. The method has shown 98% agreement with the traditional culture based method. With such a success, this method, upon validation, will significantly increase the laboratory throughput/capacity to analyze a large number of anthrax event samples in a relatively short time.

  9. Ultrasensitive Antibody Detection by Agglutination-PCR (ADAP)

    PubMed Central

    2016-01-01

    Antibodies are widely used biomarkers for the diagnosis of many diseases. Assays based on solid-phase immobilization of antigens comprise the majority of clinical platforms for antibody detection, but can be undermined by antigen denaturation and epitope masking. These technological hurdles are especially troublesome in detecting antibodies that bind nonlinear or conformational epitopes, such as anti-insulin antibodies in type 1 diabetes patients and anti-thyroglobulin antibodies associated with thyroid cancers. Radioimmunoassay remains the gold standard for these challenging antibody biomarkers, but the limited multiplexability and reliance on hazardous radioactive reagents have prevented their use outside specialized testing facilities. Here we present an ultrasensitive solution-phase method for detecting antibodies, termed antibody detection by agglutination-PCR (ADAP). Antibodies bind to and agglutinate synthetic antigen–DNA conjugates, enabling ligation of the DNA strands and subsequent quantification by qPCR. ADAP detects zepto- to attomoles of antibodies in 2 μL of sample with a dynamic range spanning 5–6 orders of magnitude. Using ADAP, we detected anti-thyroglobulin autoantibodies from human patient plasma with a 1000-fold increased sensitivity over an FDA-approved radioimmunoassay. Finally, we demonstrate the multiplexability of ADAP by simultaneously detecting multiple antibodies in one experiment. ADAP’s combination of simplicity, sensitivity, broad dynamic range, multiplexability, and use of standard PCR protocols creates new opportunities for the discovery and detection of antibody biomarkers. PMID:27064772

  10. PCR detection of aflatoxin producing fungi and its limitations.

    PubMed

    Levin, Robert E

    2012-05-01

    Unlike bacterial toxins that are primarily peptides and are therefore encoded by a single gene, fungal toxins such as the aflatoxins are multi-ring structures and therefore require a sequence of structural genes for their biological synthesis. There is therefore no specific PCR for any one of the four biologically produced aflatoxins. Unfortunately, the structural genes presently in use for PCR detection of aflatoxin producing fungi are also involved in the synthesis of other fungal toxins such as sterigmatocystin by Aspergillus versicolor and Aspergillus nidulans and therefore lack absolute specificity for aflatoxin producing fungi (Table 1). In addition, the genomic presence of several structural genes involved in aflatoxin biosynthesis does not guarantee the production of aflatoxin by all isolates of Aspergillus flavus and Aspergillus parasiticus. The most widely used DNA target regions for discriminating Aspergillus species are those of the rDNA complex, mainly the internal transcribed spacer regions 1 and 2 (ITS1 and ITS2) and the variable regions in the 5'-end of the 28S rRNA gene. Since these sequence regions are unrelated to the structural genes involved in aflatoxin biosynthesis there successful amplification can be used for species identification but do not confirm aflatoxin production. This review therefore presents the various approaches and limitations in the use of the PCR in attempting to detect aflatoxin producing fungi.

  11. STITCHER 2.0: primer design for overlapping PCR applications.

    PubMed

    O'Halloran, Damien M; Uriagereka-Herburger, Isabel; Bode, Katrin

    2017-03-30

    Overlapping polymerase chain reaction (PCR) is a common technique used by researchers in very diverse fields that enables the user to 'stitch' individual pieces of DNA together. Previously, we have reported a web based tool called STITCHER that provides a platform for researchers to automate the design of primers for overlapping PCR applications. Here we present STITCHER 2.0, which represents a substantial update to STITCHER. STITCHER 2.0 is a newly designed web tool that automates the design of primers for overlapping PCR. Unlike STITCHER, STITCHER 2.0 considers diverse algorithmic parameters, and returns multiple result files that include a facility for the user to draw their own primers as well as comprehensive visual guides to the user's input, output, and designed primers. These result files provide greater control and insight during experimental design and troubleshooting. STITCHER 2.0 is freely available to all users without signup or login requirements and can be accessed at the following webpage: www.ohalloranlab.net/STITCHER2.html.

  12. Electrochemiluminescence-PCR detection of genetically modified organisms

    NASA Astrophysics Data System (ADS)

    Liu, Jinfeng; Xing, Da; Shen, Xingyan; Zhu, Debin

    2005-01-01

    The detection methods for genetically modified (GM) components in foods have been developed recently. But many of them are complicated and time-consuming; some of them need to use the carcinogenic substance, and can"t avoid false-positive results. In this study, an electrochemiluminescence polymerase chain reaction (ECL-PCR) method for detection GM tobaccos is proposed. The Cauliflower mosaic virus 35S (CaMV35S) promoter was amplified by PCR, Then hybridized with a Ru(bpy)32+ (TBR)-labeled and a biotinylated probe. The hybridization products were captured onto streptavidin-coated paramagnetic beads, and detected by measuring the electrochemiluminescence (ECL) signal of the TBR label. Whether the tobaccos contain GM components was discriminated by detecting the ECL signal of CaMV35S promoter. The experiment results show that the detection limit for CaMV35S promoter is 100 fmol, and the GM components can be clearly identified in GM tobaccos. The ECL-PCR method provide a new means in GMOs detection due to its safety, simplicity and high efficiency.

  13. Nested PCR for detection of HSV-1 in oral mucosa

    PubMed Central

    Jalouli, Miranda-Masoumeh; Jalouli, Jamshid; Hasséus, Bengt; Öhman, Jenny; Hirsch, Jan-Michaél

    2015-01-01

    Background It has been estimated that 15%-20% of human tumours are driven by infection and inflammation, and viral infections play an important role in malignant transformation. The evidence that herpes simplex virus type 1 (HSV-1) could be involved in the aetiology of oral cancer varies from weak to persuasive. This study aimed to investigate by nested PCR (NPCR) the prevalence of HSV-1 in samples from normal oral mucosa, oral leukoplakia, and oral squamous cell carcinoma (OSCC). Material and Methods We investigated the prevalence of HSV-1 in biopsies obtained from 26 fresh, normal oral mucosa from healthy volunteers as well as 53 oral leukoplakia and 27 OSCC paraffin-embedded samples. DNA was extracted from the specimens and investigated for the presence of HSV-1 by nested polymerase chain reaction (NPCR) and DNA sequencing. Results HSV-1 was detected in 14 (54%) of the healthy samples, in 19 (36%) of the oral leukoplakia samples, and in 14 (52%) of the OSCC samples. The differences were not statistically significant. Conclusions We observed a high incidence of HSV-1 in healthy oral mucosa, oral leukoplakia, and OSCC tissues. Thus, no connection between OSCC development and presence of HSV-1 was detected. Key words:HSV-1, nested PCR, PCR. PMID:26449432

  14. Sex identification from exfoliated primary teeth--a PCR study.

    PubMed

    Kumar, Manju Gopa; Hegde, Amitha M

    2005-01-01

    Teeth endure postmortem degradation and extreme changes in ambient temperature and pressure better than most human tissues. In the present day scenario the growing number of crime against children in the form of battering, physical/sexual abuse, abduction and kidnapping, the use of exfoliated primary teeth, become many times the only evidence available at the crime scene. Despite exposure of the body to burial, mutilation, explosion or incineration, it is usually possible to extract DNA from pulp tissue of tooth with sufficient quality and quantity. Hence the present study was undertaken to find out the sex of a child from exfoliated/extracted deciduous teeth using a Polymerase Chain reaction (PCR) based analysis. Tooth samples were stored in room temperature after double coding for various periods. Dental pulp tissue was collected from each sample and DNA was isolated by proteinase-k digestion and phenol chloroform extraction methods. PCR amplification was done with two sets of oligonucleiotide primers. Amplification of X (131bp) and Y-specific sequences (172bp) in males and that of the X-specific sequence in females was observed and compared with the template DNA showing male and female controls. Determination of sexes of all freshly collected samples within 24 hours and after 1 month of extraction respectively gave 100% result. However, PCR was not found to be an effective method for sex determination after 6 months post extraction.

  15. Development of a multiplex PCR for identification of vineyard mealybugs.

    PubMed

    Daane, Kent M; Middleton, Mathew C; Sforza, René; Cooper, Monica L; Walton, Vaughn M; Walsh, Douglas B; Zaviezo, Tania; Almeida, Rodrigo P P

    2011-12-01

    A simple molecular tool was developed and tested to identify seven mealybug species found in North American vineyards: Pseudococcus maritimus Ehrhorn, Pseudococcus viburni (Signoret), Pseudococcus longispinus (Targioni-Tozzeti), Pseudococcus calceolariae (Maskell), Planococcus ficus (Signoret), Planococcus citri (Risso), and Ferrisia gilli Gullan. The developed multiplex PCR is based on the mitochondrial cytochrome c oxidase subunit one gene. In tests, this single-step multiplex PCR correctly identified 95 of 95 mealybug samples, representing all seven species and collected from diverse geographic regions. To test the sensitivity, single specimen samples with different Pl. ficus developmental stages (egg to adult female and adult male) were processed PCR and the resulting output provided consistent positive identification. To test the utility of this protocol for adult males caught in sex baited pheromone traps, Pl. ficus adult males were placed in pheromone traps, aged at a constant temperature of 26±2°C, and processed with the multiplex each day thereafter for 8 d. Results showed consistent positive identification for up to 6 d (range, 6-8 d). Results are discussed with respect to the usefulness of this molecular tool for the identification of mealybugs in pest management programs and biosecurity of invasive mealybugs.

  16. Identification of Candida spp. by phenotypic tests and PCR

    PubMed Central

    Marinho, Sandra Aparecida; Teixeira, Alice Becker; Santos, Otávio Silveira; Cazanova, Ricardo Flores; Ferreira, Carlos Alexandre Sanchez; Cherubini, Karen; de Oliveira, Sílvia Dias

    2010-01-01

    The correct identification of Candida species is of great importance, as it presents prognostic and therapeutical significance, allowing an early and appropriate antifungical therapy. The purpose of this study was to identify isolates of Candida spp. from oral mucosa of 38 patients with oral candidosis evaluated in 2004 by phenotypic methods and PCR, discriminating C. albicans from the other Candida species. The tests used for phenotypic analysis were germ-tube and chlamydoconidia production, culture in CHROMAgar™ Candida, carbohydrate assimilation test, growth at 45ºC and culture in Tween 80 agar. Genotypic confirmation was performed by PCR. Phenotypic tests showed that 63.2% strains formed germ-tubes, 73.7% produced chlamydoconidia, and 63.2% showed green colonies in chromogenic medium, presumptively indicating C. albicans or C. dubliniensis. The carbohydrate assimilation test confirmed these results. A total of 21% strains were identified as C. krusei and 13.2% were indicative of C. tropicalis. Of these later strains, three produced chlamydoconidia. The association of other phenotypic tests with culture in Tween 80 agar identified 95.8% of strains as C. albicans and 4.2% as C. dubliniensis. All 24 strains indicative of C. albicans and C. dubliniensis were confirmed by PCR as C. albicans. PMID:24031493

  17. [PCR-based diagnosis of mucormycosis in tissue samples].

    PubMed

    Bialek, R; Zelck, U E

    2013-11-01

    Mucormycosis is characterized by a rapid, often fatal progression. Early diagnosis of invasive mucormycosis is the key for timely therapeutic intervention and improved survival. Contrary to the more prevalent aspergillosis, effective antifungal therapy of mucormycosis is mainly limited to amphotericin B. Given the importance to guide the timely initiation of amphotericin B and possible surgical intervention, rapid and specific identification of fungal hyphae is essential. Conventional histopathology depends on abundance and morphology of the fungi as well as on the skills of the personnel, and usually shows an accuracy of 80 %. PCR assays targeting fungal ribosomal genes to identify Mucorales at least at genus level increase sensitivity, allow a rapid identification as well as detection of double mold infections. Thus, PCR assays are beneficial to complement existing approaches. They are recommended to rapidly specify tissue diagnosis and accurate identification of fungi. This will help to guide effective therapy and thereby, survival will increase. Retrospective analyses of mucormycosis by PCR help to evaluate therapeutic interventions and will optimize treatment options.

  18. Simultaneous multiplex PCR detection of seven cucurbit-infecting viruses.

    PubMed

    Kwon, Ji Yeon; Hong, Jin Sung; Kim, Min Jea; Choi, Sun Hee; Min, Byeong Eun; Song, Eun Gyeong; Kim, Hyun Hee; Ryu, Ki Hyun

    2014-09-01

    Two multiplex polymerase chain reaction (PCR) systems using dual priming oligonucleotide (DPO) primers were developed for the simultaneous detection of seven cucurbit-infecting viruses. One system allows for the detection of papaya ringspot virus, watermelon mosaic virus, and zucchini yellow mosaic virus, whereas the other permits the detection of cucumber green mottle mosaic virus, cucumber fruit mottle mosaic virus, kyuri green mottle mosaic virus, and zucchini green mottle mosaic virus. Viral species-specific DPO primers developed in this study detected as little as 10 fg/μl of viral RNA under monoplex conditions and 10 pg/μl of viral RNA under multiplex conditions. Multiplex PCR using the DPO primer sets was capable of amplifying viral genes at annealing temperatures ranging from 53 °C to 63 °C. Whereas the use of conventional primers gave rise to non-specific bands, the DPO primers detected target viral genes in the absence of non-specific amplification. When these DPO multiplex primer sets were applied to virus-infected cucurbit samples obtained in the field, multiple infection as well as single infection was accurately identified. This novel approach could also detect multiple viruses in infected seeds. The reliability of multiplex PCR systems using DPO primers for plant virus detection is discussed.

  19. Rapid diagnosis of Mycobacterium tuberculosis bacteremia by PCR.

    PubMed Central

    Folgueira, L; Delgado, R; Palenque, E; Aguado, J M; Noriega, A R

    1996-01-01

    A method based on DNA amplification and hybridization has been used for the rapid detection of Mycobacterium tuberculosis in blood samples from 38 hospitalized patients (15 human immunodeficiency virus [HIV] positive and 23 HIV negative) in whom localized or disseminated forms of tuberculosis were suspected. In 32 of these patients, the diagnosis of tuberculosis was eventually confirmed by conventional bacteriological or histological procedures. M. tuberculosis DNA was detected with the PCR technique in the peripheral blood mononuclear cells from 9 of 11 (82%) HIV-infected patients and in 7 of 21 (33%) HIV-negative patients (P < 0.01), while M. tuberculosis blood cultures were positive in 1 of 8 (12.5%) and 1 of 18 (5.5%) patients, respectively. PCR was positive in all cases with disseminated disease in both HIV-negative and HIV-positive patients and also in the HIV-positive patients with extrapulmonary tuberculosis. Seven samples from patients with documented illness other than tuberculosis and 12 specimens from healthy volunteers, including seven volunteers with a recent positive purified protein derivative test, were used as controls and had a negative PCR. These results suggest that detection of M. tuberculosis DNA in peripheral blood mononuclear cells may be a useful tool for rapid diagnosis of disseminated and extrapulmonary forms of tuberculosis, especially in an HIV-positive population. PMID:8904404

  20. STITCHER 2.0: primer design for overlapping PCR applications

    PubMed Central

    O’Halloran, Damien M.; Uriagereka-Herburger, Isabel; Bode, Katrin

    2017-01-01

    Overlapping polymerase chain reaction (PCR) is a common technique used by researchers in very diverse fields that enables the user to ‘stitch’ individual pieces of DNA together. Previously, we have reported a web based tool called STITCHER that provides a platform for researchers to automate the design of primers for overlapping PCR applications. Here we present STITCHER 2.0, which represents a substantial update to STITCHER. STITCHER 2.0 is a newly designed web tool that automates the design of primers for overlapping PCR. Unlike STITCHER, STITCHER 2.0 considers diverse algorithmic parameters, and returns multiple result files that include a facility for the user to draw their own primers as well as comprehensive visual guides to the user’s input, output, and designed primers. These result files provide greater control and insight during experimental design and troubleshooting. STITCHER 2.0 is freely available to all users without signup or login requirements and can be accessed at the following webpage: www.ohalloranlab.net/STITCHER2.html. PMID:28358011

  1. A PCR-based linkage map of human chromosome 1

    SciTech Connect

    Engelstein, M.; Hudson, T.J.; Lane, J.M.; Lee, M.K.; Dracopoli, C. ); Leverone, B.; Landes, G.M. ); Peltonen, L. ); Weber, J.L. )

    1993-02-01

    A genetic linkage map of human chromosome 1 based entirely on PCR-typable markers has been developed using 38 simple sequence repeat (SSR) polymorphisms. These SSRs include 36 dinucleotide repeats and 2 tetranucleotide repeats. The average heterozygosity at these markers was 0.73 and ranged form 0.52 to 0.95. Multipoint linkage analysis was used to develop a map of these 38 markers in which the relative placement of each locus is supported by likelihood odds > 1000:1. This PCR-based map was anchored at the centromere by the D1Z5 [alpha]-satellite polymorphism, and the ends of the map were defined by D1Z2 and D1S68, which are the most distal loci in the CEPH consortium map of chromosome 1. The sex-averaged, male, and female maps extend for 328, 273, and 409 cM, respectively. The average distance between markers on the sex-averaged map is 8 cM, and the largest interval is 32 cM. This map of highly informative PCR-based markers will provide a rapid means of screening human chromosome 1 for the presence of disease genes. 36 refs., 4 figs., 4 tabs.

  2. Improved purification and PCR amplification of DNA from environmental samples.

    PubMed

    Arbeli, Ziv; Fuentes, Cilia L

    2007-07-01

    Purification and PCR amplification procedures for DNA extracted from environmental samples (soil, compost, and river sediment) were improved by introducing three modifications: precipitation of DNA with 5% polyethylene glycol 8000 (PEG) and 0.6 M NaCl; filtration with a Sepharose 4B-polyvinylpolypyrrolidone (PVPP) spin column; and addition of skim milk (0.3% w/v) to the PCR reaction solution. Humic substances' concentration after precipitation with 5% PEG was 2.57-, 5.3-, and 78.9-fold lower than precipitation with 7.5% PEG, 10% PEG, and isopropanol, respectively. After PEG precipitation, Sepharose, PVPP and the combined (Sepharose-PVPP) column removed 92.3%, 89.5%, and 98%, respectively, of the remaining humic materials. Each of the above-mentioned modifications improved PCR amplification of the 16S rRNA gene. DNA extracted by the proposed protocol is cleaner than DNA extracted by a commercial kit. Nevertheless, the improvement of DNA purification did not improve the detection limit of atrazine degradation gene atzA.

  3. Colorimetric Integrated PCR Protocol for Rapid Detection of Vibrio parahaemolyticus

    PubMed Central

    Cheng, Kewen; Pan, Daodong; Teng, Jun; Yao, Li; Ye, Yingwang; Xue, Feng; Xia, Fan; Chen, Wei

    2016-01-01

    Rapid detection of pathogens is of great significance for food safety and disease diagnosis. A new colorimetric method for rapid and easy detection of Vibrio parahaemolyticus (V. parahaemolyticus or Vp) has been developed in this research. A specific sequence was designed and integrated with the forward primer for molecular detection of Vp. This specific sequence was tested and treated as the horseradish peroxidase (HRP)-mimicking DNAzyme and could be amplified during the polymerase chain reaction (PCR) process. The products of PCR including the sequence of HRP-mimicking DNAzyme could produce the distinguished color in the presence of catalysis substrates. The optical signal of the catalysis reaction, which is in a linear relationship with the initial template of Vp, could be determined with the naked eye or measured with Ultraviolet-visible (UV-vis) for qualitative and quantitative detections, respectively. Based on the optical signal intensity, rapid and easy detection of Vp was successfully achieved with satisfied sensitivity and specificity. Furthermore, the detection of tdh, trh, tlh and toxR virulence genes of two Vp species (Vp 33847 and Vp 17802) were all performed successfully with this developed colorimetric integrated PCR protocol, which demonstrated potential applicability for the rapid detection of other bacteria. PMID:27690041

  4. Monitoring infection: from blood culture to polymerase chain reaction (PCR).

    PubMed

    Book, Malte; Lehmann, Lutz Eric; Zhang, XiangHong; Stüber, Frank

    2013-06-01

    In patients with sepsis, diagnosis of blood stream infection (BSI) is a key concern to the therapist. Direct verification of pathogens in the blood stream executed by blood cultures (BC) still is regarded as the gold standard up to date. The quickest possible initiation of an appropriate antimicrobial therapy is a cornerstone of an effective therapy. Moreover, in this view BC can also serve to identify antimicrobial agents to target the pathogen. However, when employing BC the time needed until microbiological results are available ranges from 24 up to 72 h. Moreover, infections caused by multiple pathogens often remain undetected and concurrent antibiotic therapy may lower the overall sensitivity. Alternative pathogen characterization can be performed by polymerase chain reaction (PCR) based amplification methods. Results using PCR can be obtained within 6-8 h. Therefore, the time delay until an appropriate therapy can be reduced enormously. Moreover, these methods have the potential to enhance the sensitivity in the diagnosis of blood stream infections. Therefore, PCR based methods might be a valuable adjunct to present procedures of diagnosing bacteraemia.

  5. Detection and counting of Nitrobacter populations in soil by PCR.

    PubMed Central

    Degrange, V; Bardin, R

    1995-01-01

    Although the biological conversion of nitrite to nitrate is a well-known process, studies of Nitrobacter populations are hindered by their physiological characteristics. This report describes a new method for detecting and counting Nitrobacter populations in situ with the PCR. Two primers from the 16S rRNA gene were used to generate a 397-bp fragment by amplification of Nitrobacter species DNA. No signal was detected from their phylogenetic neighbors or the common soil bacteria tested. Extraction and purification steps were optimized for minimal loss and maximal purity of soil DNA. The detection threshold and accuracy of the molecular method were determined from soil inoculated with 10, 10(2), or 10(3) Nitrobacter hamburgensis cells per g of soil. Counts were also done by the most-probable-number (MPN)-Griess and fluorescent antibody methods. PCR had a lower detection threshold (10(2) Nitrobacter cells per g of soil) than did the MPN-Griess or fluorescent antibody method. When PCR amplification was coupled with the MPN method, the counting rate reached 65 to 72% of inoculated Nitrobacter cells. Tested on nonsterile soil, this rapid procedure was proved efficient. PMID:7793930

  6. Species identification in meat products using real-time PCR.

    PubMed

    Jonker, K M; Tilburg, J J H C; Hagele, G H; de Boer, E

    2008-05-01

    One of the most convenient methods for the identification of animal species in processed meat products is the examination of DNA sequences. Real-time polymerase chain reaction (qPCR) techniques are particularly suitable because even small fragments of DNA formed during heat processing of the meat can be amplified and identified. A real-time PCR method has been developed and evaluated for the identification of processed meat products. In test mixtures containing beef, pork, horse, mutton, chicken and turkey, it was possible to identify these species down to a level of 0.05%. By adjusting the number of cycles, it was possible to detect levels as low as 0.01% of these species. Cross-reactivity between these species was not found, except for pure horsemeat (250 ng DNA) in the assay for turkey meat. Cross-reactivity of deer, roe, ostrich, kangaroo, goat, domestic duck, mallard, goose, pigeon, guinea fowl, quail and pheasant was also investigated and it was found that amounts as high as 250 ng DNA of these species in the reaction vial did not result in (false) positive signals except for amounts higher than 125 ng deer DNA and higher than 50 ng pigeon DNA in the determination of chicken and beef, respectively. More than 150 meat samples were examined using DNA hybridization and real-time PCR. A comparison of the results showed a better performance of the real-time procedure compared to DNA hybridization.

  7. Direct-to-PCR tissue preservation for DNA profiling.

    PubMed

    Sorensen, Amy; Berry, Clare; Bruce, David; Gahan, Michelle Elizabeth; Hughes-Stamm, Sheree; McNevin, Dennis

    2016-05-01

    Disaster victim identification (DVI) often occurs in remote locations with extremes of temperatures and humidities. Access to mortuary facilities and refrigeration are not always available. An effective and robust DNA sampling and preservation procedure would increase the probability of successful DNA profiling and allow faster repatriation of bodies and body parts. If the act of tissue preservation also released DNA into solution, ready for polymerase chain reaction (PCR), the DVI process could be further streamlined. In this study, we explored the possibility of obtaining DNA profiles without DNA extraction, by adding aliquots of preservative solutions surrounding fresh human muscle and decomposing human muscle and skin tissue samples directly to PCR. The preservatives consisted of two custom preparations and two proprietary solutions. The custom preparations were a salt-saturated solution of dimethyl sulfoxide (DMSO) with ethylenediaminetetraacetic (EDTA) and TENT buffer (Tris, EDTA, NaCl, Tween 20). The proprietary preservatives were DNAgard (Biomatrica(®)) and Tissue Stabilising Kit (DNA Genotek). We obtained full PowerPlex(®) 21 (Promega) and GlobalFiler(®) (Life Technologies) DNA profiles from fresh and decomposed tissue preserved at 35 °C for up to 28 days for all four preservatives. The preservative aliquots removed from the fresh muscle tissue samples had been stored at -80 °C for 4 years, indicating that long-term archival does not diminish the probability of successful DNA typing. Rather, storage at -80 °C seems to reduce PCR inhibition.

  8. Enzymological considerations for a theoretical description of the quantitative competitive polymerase chain reaction (QC-PCR).

    PubMed

    Schnell, S; Mendoza, C

    1997-02-21

    The enzymological principles of the polymerase chain reaction (PCR) and of the quantitative competitive PCR (QC-PCR) are developed, proposing a theoretical framework that will facilitate quantification in experimental methodologies. It is demonstrated that the specificity of the QC-PCR, i.e. the ratio of the target initial velocity to that of the competitor template, remains constant not only during a particular amplification but also for increasing initial competitor concentrations. Linear fitting procedures are thus recommended that will enable a quantitative estimate of the initial target concentration. Finally, expressions for the efficiency of the PCR and QC-PCR are derived that are in agreement with previous experimental inferences.

  9. Nuclear DNA PCR-RFLPs that distinguish African and European honey bee groups of subspecies. II: Conversion of long PCR markers to standard PCR.

    PubMed

    Suazo, Alonso; Hall, H Glenn

    2002-08-01

    Nuclear DNA PCR-RFLPs previously found in amplifications of three long (> 5 kbp) anonymous regions of DNA were made analyzable using standard PCR procedures. RFLP analyses were simplified by restricting the amplifications to sections, within each locus, that contained most of the informative polymorphic sites. AluI digests of locus L-1 section 2 (L-1S2) revealed three suballeles of which one was African-specific (Apis mellifera scutellata Lepeletier) and one was east European-predominant (A. m. ligustica Spinola, A. m. carnica Pollman, and A. m. caucasica Gorbachev). Alleles found originally at locus L-2 with AvaI were determined in RFLP analysis of two sections, L-2S1int and L-2S2, resulting in two African-specific and two east European-predominant suballeles. Suballele identity was determined by the combination of banding patterns from both fragments. Polymorphisms revealed by HaeIII in locus L-2 were analyzed in amplifications and digests of L-2SM1int. an 830 bpfragment within L-2S1. Seven suballeles were found of which two were African-specific and three were east European-specific or predominant, including one suballele specific to the east European subspecies A. m. caucasica. In locus L-5, RFLPs were detected with HaeIII, DdeI, and SpeI. HaeIII polymorphisms were analyzed by amplification and digestion offragments L-5S1xt and L-5S1ter: Five suballeles were found of which three were African-specific and one east European-predominant. For DdeI, all five alleles originally found with long PCR could be identified in RFLP analyses of three sections. Two African-specific, one east European-specific, and one west European-predominant (A. m. mellifera L. and A. m. iberica Goetze) suballeles were found. A west European-predominant suballele was also found in RFLP analysis of L-5S3 with SpeI. Allele frequency data from Old World and US. populations are presented.

  10. Development of Nested PCR, Multiplex PCR, and Loop-Mediated Isothermal Amplification Assays for Rapid Detection of Cylindrocladium scoparium on Eucalyptus

    PubMed Central

    Qiao, Tian-Min; Zhang, Jing; Li, Shu-Jiang; Han, Shan; Zhu, Tian-Hui

    2016-01-01

    Eucalyptus dieback disease, caused by Cylindrocladium scoparium, has occurred in last few years in large Eucalyptus planting areas in China and other countries. Rapid, simple, and reliable diagnostic techniques are desired for the early detection of Eucalyptus dieback of C. scoparium prior to formulation of efficient control plan. For this purpose, three PCR-based methods of nested PCR, multiplex PCR, loop-mediated isothermal amplification (LAMP) were developed for detection of C. scoparium based on factor 1-alpha (tef1) and beta-tubulin gene in this study. All of the three methods showed highly specific to C. scoparium. The sensitivities of the nested PCR and LAMP were much higher than the multiplex PCR. The sensitivity of multiplex PCR was also higher than regular PCR. C. scoparium could be detected within 60 min from infected Eucalyptus plants by LAMP, while at least 2 h was needed by the rest two methods. Using different Eucalyptus tissues as samples for C. scoparium detection, all of the three PCR-based methods showed much better detection results than regular PCR. Base on the results from this study, we concluded that any of the three PCR-based methods could be used as diagnostic technology for the development of efficient strategies of Eucalyptus dieback disease control. Particularly, LAMP was the most practical method in field application because of its one-step and rapid reaction, simple operation, single-tube utilization, and simple visualization of amplification products. PMID:27721691

  11. Development of Nested PCR, Multiplex PCR, and Loop-Mediated Isothermal Amplification Assays for Rapid Detection of Cylindrocladium scoparium on Eucalyptus.

    PubMed

    Qiao, Tian-Min; Zhang, Jing; Li, Shu-Jiang; Han, Shan; Zhu, Tian-Hui

    2016-10-01

    Eucalyptus dieback disease, caused by Cylindrocladium scoparium, has occurred in last few years in large Eucalyptus planting areas in China and other countries. Rapid, simple, and reliable diagnostic techniques are desired for the early detection of Eucalyptus dieback of C. scoparium prior to formulation of efficient control plan. For this purpose, three PCR-based methods of nested PCR, multiplex PCR, loop-mediated isothermal amplification (LAMP) were developed for detection of C. scoparium based on factor 1-alpha (tef1) and beta-tubulin gene in this study. All of the three methods showed highly specific to C. scoparium. The sensitivities of the nested PCR and LAMP were much higher than the multiplex PCR. The sensitivity of multiplex PCR was also higher than regular PCR. C. scoparium could be detected within 60 min from infected Eucalyptus plants by LAMP, while at least 2 h was needed by the rest two methods. Using different Eucalyptus tissues as samples for C. scoparium detection, all of the three PCR-based methods showed much better detection results than regular PCR. Base on the results from this study, we concluded that any of the three PCR-based methods could be used as diagnostic technology for the development of efficient strategies of Eucalyptus dieback disease control. Particularly, LAMP was the most practical method in field application because of its one-step and rapid reaction, simple operation, single-tube utilization, and simple visualization of amplification products.

  12. Nucleic acid extraction from polluted estuarine water for detection of viruses and bacteria by PCR and RT-PCR analysis.

    PubMed

    Petit, F; Craquelin, S; Guespin-Michel, J; Buffet-Janvresse, C

    1999-03-01

    We describe an extraction protocol for genomic DNA and RNA of both viruses and bacteria from polluted estuary water. This procedure was adapted to the molecular study of microflora of estuarine water where bacteria and viruses are found free, forming low-density biofilms, or intimately associated with organo-mineral particles. The sensitivity of the method was determined with seeded samples for RT-PCR and PCR analysis of viruses (10 virions/mL), and bacteria (1 colony-forming unit mL). We report an example of molecular detection of both poliovirus and Salmonella in the Seine estuary (France) and an approach to studying their association with organo-mineral particles.

  13. Quantitative real-time PCR (qPCR) for the detection and quantification of dactylogyrid parasites infecting Lutjanus guttatus.

    PubMed

    Soler-Jiménez, L C; García-Gasca, A; Fajer-Ávila, E J

    2017-03-07

    Severe infections of the spotted rose snapper Lutjanus guttatus resulting from dactylogyrid monogeneans present a risk to aquaculture. Currently, the diagnosis of this infection requires the morphological identification and manual quantification of parasites. Based on the characterization of the 28S rRNA gene of dactylogyrid species present in L. guttatus, specific primers were designed for real-time polymerase chain reaction (qPCR) using EvaGreen® chemistry. The standard curve method estimated the number of dactylogyrids accurately. A total of 85 gill samples from cage-cultured fish infected with dactylogyrids were analysed. The estimated number of dactylogyrids using this molecular method was very similar to the manual count that was performed initially. The standardized qPCR approach will be helpful as a complementary method for the early routine monitoring of dactylogyrid infections and for epidemiological studies in which a high number of fish must be studied.

  14. Biofunctionalization of Polyoxometalates with DNA Primers, Their Use in the Polymerase Chain Reaction (PCR) and Electrochemical Detection of PCR Products.

    PubMed

    Debela, Ahmed M; Ortiz, Mayreli; Beni, Valerio; Thorimbert, Serge; Lesage, Denis; Cole, Richard B; O'Sullivan, Ciara K; Hasenknopf, Bernold

    2015-12-01

    The bioconjugation of polyoxometalates (POMs), which are inorganic metal oxido clusters, to DNA strands to obtain functional labeled DNA primers and their potential use in electrochemical detection have been investigated. Activated monooxoacylated polyoxotungstates [SiW11 O39 {Sn(CH2 )2 CO}](8-) and [P2 W17 O61 {Sn(CH2 )2 CO}](6-) have been used to link to a 5'-NH2 terminated 21-mer DNA forward primer through amide coupling. The functionalized primer was characterized by using a battery of techniques, including electrophoresis, mass spectrometry, as well as IR and Raman spectroscopy. The functionality of the POM-labeled primers was demonstrated through hybridization with a surface-immobilized probe. Finally, the labeled primers were successfully used in the polymerase chain reaction (PCR) and the PCR products were characterized by using electrophoresis.

  15. Simplified diagnosis of malaria infection: GFM/PCR/ELISA a simplified nucleic acid amplification technique by PCR/ELISA.

    PubMed

    Machado, R L; Garret, D O; Adagu, I S; Warhurst, D C; Póvoa, M M

    1998-01-01

    We report an adaptation of a technique for the blood sample collection (GFM) as well as for the extraction and amplification of Plasmodium DNA for the diagnosis of malaria infection by the PCR/ELISA. The method of blood sample collection requires less expertise and saves both time and money, thus reducing the cost by more than half. The material is also suitable for genetic analysis in either fresh or stored specimens prepared by this method.

  16. Detection of feline calicivirus, feline herpesvirus 1 and Chlamydia psittaci mucosal swabs by multiplex RT-PCR/PCR.

    PubMed

    Sykes, J E; Allen, J L; Studdert, V P; Browning, G F

    2001-07-26

    A single tube, multiplex reverse transcription (RT)-polymerase chain reaction (PCR)/PCR assay was developed for detection of feline herpesvirus 1 (FHV1), Chlamydia psittaci and feline calicivirus (FCV) in cats with upper respiratory tract disease (URTD), incorporating a simple, rapid extraction procedure capable of extracting both DNA and RNA. The assay was found to be as sensitive in vitro as simplex assays that have previously been shown to be as sensitive as, or more sensitive than, culture for each pathogen in experimentally infected cats. Conjunctival alone or both conjunctival and oropharyngeal swabs were collected from cats in 104 households with URTD. FHV1 was detected in 18 (17.3%) and C. psittaci was detected in 12 (11.5%) households. The prevalence of C. psittaci was not significantly different to that determined using a duplex PCR assay for C. psittaci and FHV1. The prevalence of FCV was affected by sample storage temperature. Of samples stored at -70 degrees C, 0/31 were positive for FCV but FCV was detected in 10/73 (13.7%) samples stored at 4 degrees C (P=0.006). Of the samples stored at 4 degrees C, 3/19 (15.8%) conjunctival swabs were positive for FCV and 6/32 (18.8%) oropharyngeal/conjunctival swabs were positive for FCV (P=0.79). The potential utility of restriction endonuclease analysis of RT-PCR products resulting from amplification of the hypervariable region of the capsid protein gene of FCV in field samples, without prior cultivation, was also examined. The assay may have considerable importance for diagnosis and epidemiological surveys of feline upper respiratory tract pathogens.

  17. Efficacy comparison of adjuvants in PcrV vaccine against Pseudomonas aeruginosa pneumonia.

    PubMed

    Hamaoka, Saeko; Naito, Yoshifumi; Katoh, Hideya; Shimizu, Masaru; Kinoshita, Mao; Akiyama, Koichi; Kainuma, Atsushi; Moriyama, Kiyoshi; Ishii, Ken J; Sawa, Teiji

    2017-02-01

    Vaccination against the type III secretion system of P. aeruginosa is a potential prophylactic strategy for reducing the incidence and improving the poor prognosis of P. aeruginosa pneumonia. In this study, the efficacies of three different adjuvants, Freund's adjuvant (FA), aluminum hydroxide (alum) and CpG oligodeoxynucleotide (ODN), were examined from the viewpoint of inducing PcrV-specific immunity against virulent P. aeruginosa. Mice that had been immunized intraperitoneally with recombinant PcrV formulated with one of the above adjuvants were challenged intratracheally with a lethal dose of P. aeruginosa. The PcrV-FA immunized group attained a survival rate of 91%, whereas the survival rates of the PcrV-alum and PcrV-CpG groups were 73% and 64%, respectively. In terms of hypothermia recovery after bacterial instillation, PcrV-alum was the most protective, followed by PcrV-FA and PcrV-CpG. The lung edema index was lower in the PcrV-CpG vaccination group than in the other groups. PcrV-alum immunization was associated with the greatest decrease in myeloperoxidase in infected lungs, and also decreased the number of lung bacteria to a similar number as in the PcrV-FA group. There was less neutrophil recruitment in the lungs of mice vaccinated with PcrV-alum or PcrV-CpG than in those of mice vaccinated with PcrV-FA or PcrV alone. Overall, in terms of mouse survival the PcrV-CpG vaccine, which could be a relatively safe next-generation vaccine, showed a comparable effect to the PcrV-alum vaccine.

  18. Single-step PCR in molecular diagnosis of hepatitis C virus infection.

    PubMed Central

    Farma, E; Boeri, E; Bettini, P; Repetto, C M; McDermott, J; Lillo, F B; Varnier, O E

    1996-01-01

    The diagnostic utility of two PCR systems and three PCR detection methods for hepatitis C virus (HCV) RNA was evaluated in serum samples. A nested PCR was considered the reference assay and was compared with two single-step PCR methods: the first is based on the detection of PCR products by liquid hybridization with a 32P-end-labeled probe, and the second is the Roche Amplicor colorimetric assay using microwell plate hybridization with a specific nucleic acid probe. Using the Pelicheck HCV RNA Eurohep genotype 1 proficiency panel, our laboratory achieved medium-high levels of performance with all three methods. The highest sensitivity was, however, observed with the isotopic single-step PCR (ss-PCR) method. The analytical sensitivity of ss-PCR with isotopic detection and ss-PCR with colorimetric detection was identical to that of nested PCR, with a 100% result concordance. Comparison of ss-PCR with enzyme-linked immunosorbent and RIBA assays in the analysis of clinical samples showed a high concordance. ss-PCR methods appear more suitable for diagnostic application. Nevertheless, HCV RNA PCR cannot be considered a screening assay; it should be requested in the presence of reactive serology or specific clinical symptomatology with altered liver parameters, and it is a potential tool for the follow-up of patients with HCV infection. PMID:8940466

  19. Broad-range real time PCR and DNA sequencing for the diagnosis of bacterial meningitis.

    PubMed

    Deutch, Susanna; Pedersen, Lisbeth N; Pødenphant, Lone; Olesen, Rikke; Schmidt, Michael B; Møller, Jens K; Ostergaard, Lars

    2006-01-01

    Rapid aetiological diagnosis of bacterial meningitis is crucial for the early targeting of antimicrobial and adjuvant therapy. Broad-range polymerase chain reaction (PCR) targeting the 16S rRNA gene allows aetiological diagnosis of bacterial meningitis when applied to cerebrospinal fluid (CSF). We assessed the additional diagnostic effect of applying a novel broad-range real time PCR and subsequent DNA sequencing to culture, microscopy, and broad-range conventional PCR on CSF in patients with suspected bacterial meningitis. Broad-range conventional PCR and broad-range real time PCR with subsequent DNA sequencing were applied to 206 CSF specimens collected consecutively from 203 patients aged 6 d to 86 y. Patients' charts were reviewed for clinical information. 17 pathogens were identified by PCR and DNA sequencing or culture. Three specimens were negative by culture but positive by broad-range real time PCR. Three specimens were positive by culture but negative by broad-range real time PCR. Compared with culture, the sensitivity of broad-range real time PCR was 86%, and the specificity 98%. Conventional PCR resulted in a sensitivity of 64% and specificity of 98%. Broad-range real time PCR was generally comparable to culture of CSF and may be a useful supplement, particularly when antimicrobial therapy has been administered. Broad-range real time PCR was more sensitive than broad-range conventional PCR and microscopy.

  20. Comparison of standard, quantitative and digital PCR in the detection of enterotoxigenic Bacteroides fragilis.

    PubMed

    Purcell, Rachel V; Pearson, John; Frizelle, Frank A; Keenan, Jacqueline I

    2016-09-30

    Gut colonization with enterotoxigenic Bacteroides fragilis (ETBF) appears to be associated with the development of colorectal cancer. However, differences in carriage rates are seen with various testing methods and sampling sites. We compared standard PCR, SYBR green and TaqMan quantitative PCR (qPCR) and digital PCR (dPCR) in detecting the B. fragilis toxin (bft) gene from cultured ETBF, and from matched luminal and faecal stool samples from 19 colorectal cancer patients. Bland-Altman analysis found that all three quantitative methods performed comparably in detecting bft from purified bacterial DNA, with the same limits of detection (<1 copy/μl). However, SYBR qPCR under-performed compared to TaqMan qPCR and dPCR in detecting bft in clinical stool samples; 13/38 samples were reported positive by SYBR, compared to 35 and 36 samples by TaqMan and dPCR, respectively. TaqMan qPCR and dPCR gave bft copy numbers that were 48-fold and 75-fold higher for the same samples than SYBR qPCR, respectively (p < 0.001). For samples that were bft-positive in both fecal and luminal stools, there was no difference in relative abundance between the sites, by any method tested. From our findings, we recommend the use of TaqMan qPCR as the preferred method to detect ETBF from clinical stool samples.

  1. Development of an RNA extraction protocol for detection of waterborne viruses by reverse transcriptase quantitative PCR (RT-qPCR).

    PubMed

    Jothikumar, N; Sobsey, M D; Cromeans, T L

    2010-10-01

    RNA extraction from environmental samples yields frequently an RNA preparation containing inhibitors of molecular reactions. Commercial RNA extraction kits commonly permit extraction of only 0.1-0.2 ml sample volume. An RNA extraction buffer (RNAX buffer) was formulated for the extraction of viral RNA from 4.0 ml using a silica column based protocol. To evaluate the RNAX buffer based protocol, we used hepatitis A virus (HAV) and coxsackievirus B3 (CVB3) to monitor the RNA extraction efficiency from environmental samples. For evaluation of viral RNA recovery from water concentrates which were prepared from river and pond water by PEG concentration, serial ten fold dilutions of two waterborne viruses were added to the water concentrates for evaluation by quantitative detection. Quantitative recovery of HAV and CVB3 was determined by reverse transcriptase quantitative real-time PCR (RT-qPCR). The extracted RNA was compatible with RT-qPCR and sensitivity of detection of 0.8PFU per reaction was found with RNAX buffer and the developed protocol. This level of sensitivity was obtained using viral RNA extracted from 4.0 ml of an inoculated water sample concentrate. The RNAX buffer developed in this study could be applicable to the detection of other pathogens in water and food.

  2. Senior Thai fecal microbiota comparison between vegetarians and non-vegetarians using PCR-DGGE and real-time PCR.

    PubMed

    Ruengsomwong, Supatjaree; Korenori, Yuki; Sakamoto, Naoshige; Wannissorn, Bhusita; Nakayama, Jiro; Nitisinprasert, Sunee

    2014-08-01

    The fecal microbiotas were investigated in 13 healthy Thai subjects using polymerase chain reaction denaturing gradient gel electrophoresis (PCR-DGGE). Among the 186 DNA bands detected on the polyacrylamide gel, 37 bands were identified as representing 11 species: Bacteroides thetaiotaomicron, Bacteroides ovatus, Bacteroides uniformis, Bacteroides vulgatus, Clostridium colicanis, Eubacterium eligenes, E. rectale, Faecalibacterium prausnitzii, Megamonas funiformis, Prevotella copri, and Roseburia intestinalis, belonging mainly to the groups of Bacteroides, Prevotella, Clostridium, and F. prausnitzii. A dendrogram of the PCR-DGGE divided the subjects; vegetarians and non-vegetarians. The fecal microbiotas were also analyzed using a quantitative real-time PCR focused on Bacteroides, Bifidobacterium, Enterobacteriaceae, Clostrium coccoides-Eubacterium rectale, C. leptum, Lactobacillus, and Prevotella. The nonvegetarian and vegetarian subjects were found to have significant differences in the high abundance of the Bacteroides and Prevotella genera, respectively. No significant differences were found in the counts of Bifidabacterium, Enterobacteriaceae, C. coccoides-E. rectale group, C. leptum group, and Lactobacillus. Therefore, these findings on the microbiota of healthy Thais consuming different diets could provide helpful data for predicting the health of South East Asians with similar diets.

  3. Validation of Reference Genes for Real-Time Quantitative PCR (qPCR) Analysis of Avibacterium paragallinarum

    PubMed Central

    Wen, Shuxiang; Chen, Xiaoling; Xu, Fuzhou; Sun, Huiling

    2016-01-01

    Real-time quantitative reverse transcription PCR (qRT-PCR) offers a robust method for measurement of gene expression levels. Selection of reliable reference gene(s) for gene expression study is conducive to reduce variations derived from different amounts of RNA and cDNA, the efficiency of the reverse transcriptase or polymerase enzymes. Until now reference genes identified for other members of the family Pasteurellaceae have not been validated for Avibacterium paragallinarum. The aim of this study was to validate nine reference genes of serovars A, B, and C strains of A. paragallinarum in different growth phase by qRT-PCR. Three of the most widely used statistical algorithms, geNorm, NormFinder and ΔCT method were used to evaluate the expression stability of reference genes. Data analyzed by overall rankings showed that in exponential and stationary phase of serovar A, the most stable reference genes were gyrA and atpD respectively; in exponential and stationary phase of serovar B, the most stable reference genes were atpD and recN respectively; in exponential and stationary phase of serovar C, the most stable reference genes were rpoB and recN respectively. This study provides recommendations for stable endogenous control genes for use in further studies involving measurement of gene expression levels. PMID:27942007

  4. Type A influenza virus detection from horses by real-time RT-PCR and insulated isothermal RT-PCR.

    PubMed

    Balasuriya, Udeni B R

    2014-01-01

    Equine influenza (EI) is a highly contagious disease of horses caused by the equine influenza virus (EIV) H3N8 subtype. EI is the most important respiratory virus infection of horses and can disrupt major equestrian events and cause significant economic losses to the equine industry worldwide. Influenza H3N8 virus spreads rapidly in susceptible horses and can result in very high morbidity within 24-48 h after exposure to the virus. Therefore, rapid and accurate diagnosis of EI is critical for implementation of prevention and control measures to avoid the spread of EIV and to reduce the economic impact of the disease. The probe-based real-time reverse transcriptase polymerase chain reaction (rRT-PCR) assays targeting various EIV genes are reported to be highly sensitive and specific compared to the Directigen Flu A(®) test and virus isolation in embryonated hens' eggs. Recently, a TaqMan(®) probe-based insulated isothermal RT-PCR (iiRT-PCR) assay for the detection of EIV H3N8 subtype has been described. These molecular based diagnostic assays provide a fast and reliable means of EIV detection and disease surveillance.

  5. Determinación de la orientación global SAO-Hipparcos mediante una expansión en armónicos vectoriales

    NASA Astrophysics Data System (ADS)

    Cionco, R. G.; Vucetich, H.; Orellana, R.; Arias, E. F.

    En base a las diferencias de posición y movimientos propios de 101352 estrellas con posición SAO observadas por HIPPARCOS y utilizando la naturaleza vectorial de esas diferencias, determinamos 6 parámetros de orientación global (3 de rotación y 3 de desplazamiento axial), para los sistemas de referencia asociados a los marcos mencionados, mediante una descomposición en serie de armónicos vectoriales ortogonales.

  6. Data Analysis of Transcriptomic Sequences and qPCR Validations for Microbial Communities during Algal Blooms

    EPA Pesticide Factsheets

    A training opportunity is open to a highly microbial-research-motivated student to conduct sequence analysis, explore novel genes and metabolic pathways, validate resultant findings using qPCR/RT-qPCR and summarize the findings

  7. Rapid detection and biovar differentiation of Ureaplasma urealyticum in clinical specimens by PCR.

    PubMed

    Teng, L J; Ho, S W; Ho, H N; Liaw, S J; Lai, H C; Luh, K T

    1995-07-01

    On the basis of the nucleotide sequence of the multiple-banded (MB) antigen genes of Ureaplasma urealyticum, a polymerase chain reaction (PCR) technique was developed for rapid detection and biovar differentiation of U. urealyticum in a total of 100 urogenital specimens from 50 female patients. Positive PCR UM-1 amplification was found in 28 cervical swabs and 31 urine samples. Overall agreement between PCR and culture was 95%. Members of the two biovars of U. urealyticum could be distinguished by the size of the PCR UM-1 amplification products. Biovar differentiation was also demonstrated by two additional sets of PCRs: PCR UM-2 and UM-3. The PCR UM-2 was used to amplify biovar 1, while PCR UM-3 amplified biovar 2 specifically. The results indicated that use of the MB antigen gene as a target for PCR amplification could provide rapid and specific detection and biotyping of ureaplasma DNA in urogenital samples.

  8. Comprehensive GMO detection using real-time PCR array: single-laboratory validation.

    PubMed

    Mano, Junichi; Harada, Mioko; Takabatake, Reona; Furui, Satoshi; Kitta, Kazumi; Nakamura, Kosuke; Akiyama, Hiroshi; Teshima, Reiko; Noritake, Hiromichi; Hatano, Shuko; Futo, Satoshi; Minegishi, Yasutaka; Iizuka, Tayoshi

    2012-01-01

    We have developed a real-time PCR array method to comprehensively detect genetically modified (GM) organisms. In the method, genomic DNA extracted from an agricultural product is analyzed using various qualitative real-time PCR assays on a 96-well PCR plate, targeting for individual GM events, recombinant DNA (r-DNA) segments, taxon-specific DNAs, and donor organisms of the respective r-DNAs. In this article, we report the single-laboratory validation of both DNA extraction methods and component PCR assays constituting the real-time PCR array. We selected some DNA extraction methods for specified plant matrixes, i.e., maize flour, soybean flour, and ground canola seeds, then evaluated the DNA quantity, DNA fragmentation, and PCR inhibition of the resultant DNA extracts. For the component PCR assays, we evaluated the specificity and LOD. All DNA extraction methods and component PCR assays satisfied the criteria set on the basis of previous reports.

  9. Comparison of a PCR assay in whole blood and serum specimens for canine brucellosis diagnosis.

    PubMed

    Keid, L B; Soares, R M; Vasconcellos, S A; Salgado, V R; Megid, J; Richtzenhain, L J

    2010-07-17

    The performance of a serum PCR assay was compared with that of a blood PCR assay for the diagnosis of canine brucellosis caused by Brucella canis in 72 dogs. The dogs were classified into three groups (infected, non-infected and suspected brucellosis) according to the results of blood culture and serological tests. The sensitivities of blood PCR and serum PCR were, respectively, 97.14 per cent and 25.71 per cent. The specificities of both were 100 per cent. In the group of dogs with suspected brucellosis, three were positive by blood PCR and none was positive by serum PCR. Serum PCR showed little value for the direct diagnosis of canine brucellosis as the assay had low diagnostic sensitivity and fewer positive dogs were detected by this test than by blood culture, blood PCR, rapid slide agglutination test (RSAT) and RSAT with 2-mercaptoethanol.

  10. Real-Time PCR in Clinical Microbiology: Applications for Routine Laboratory Testing

    PubMed Central

    Espy, M. J.; Uhl, J. R.; Sloan, L. M.; Buckwalter, S. P.; Jones, M. F.; Vetter, E. A.; Yao, J. D. C.; Wengenack, N. L.; Rosenblatt, J. E.; Cockerill, F. R.; Smith, T. F.

    2006-01-01

    Real-time PCR has revolutionized the way clinical microbiology laboratories diagnose many human microbial infections. This testing method combines PCR chemistry with fluorescent probe detection of amplified product in the same reaction vessel. In general, both PCR and amplified product detection are completed in an hour or less, which is considerably faster than conventional PCR detection methods. Real-time PCR assays provide sensitivity and specificity equivalent to that of conventional PCR combined with Southern blot analysis, and since amplification and detection steps are performed in the same closed vessel, the risk of releasing amplified nucleic acids into the environment is negligible. The combination of excellent sensitivity and specificity, low contamination risk, and speed has made real-time PCR technology an appealing alternative to culture- or immunoassay-based testing methods for diagnosing many infectious diseases. This review focuses on the application of real-time PCR in the clinical microbiology laboratory. PMID:16418529

  11. 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...

  12. Development and validation of two SYBR green PCR assays and a multiplex real-time PCR for the detection of Shiga toxin-producing Escherichia coli in meat.

    PubMed

    Brusa, Victoria; Galli, Lucía; Linares, Luciano H; Ortega, Emanuel E; Lirón, Juan P; Leotta, Gerardo A

    2015-12-01

    Shiga toxin-producing Escherichia coli (STEC) are recognized as food-borne pathogens. We developed and validated two SYBR green PCR (SYBR-PCR) and a real-time multiplex PCR (RT-PCR) to detect stx1 and stx2 genes in meat samples, and compared these techniques in ground beef samples from retail stores. One set of primers and one hydrolysis probe were designed for each stx gene. For RT-PCR, an internal amplification control (IAC) was used. All PCR intra-laboratory validations were performed using pure strains and artificially contaminated ground beef samples. A total of 50 STEC and 30 non-STEC strains were used. Naturally contaminated ground beef samples (n=103) were obtained from retail stores and screened with SYBR-PCR and RT-PCR, and stx-positive samples were processed for STEC isolation. In the intra-laboratory validation, each PCR obtained a 1×10(2) CFU mL(-1) limit of detection and 100% inclusivity and exclusivity. The same results were obtained when different laboratory analysts in alternate days performed the assay. The level of agreement obtained with SYBR-PCR and RT-PCR was kappa=0.758 and 0.801 (P<0.001) for stx1 and stx2 gene detection, respectively. Two PCR strategies were developed and validated, and excellent performance with artificially contaminated ground beef samples was obtained. However, the efforts made to isolate STEC from retail store samples were not enough. Only 11 STEC strains were isolated from 35 stx-positive ground beef samples identically detected by all PCRs. The combination of molecular approaches based on the identification of a virulence genotypic profile of STEC must be considered to improve isolation.

  13. Development of TaqMan probe-based insulated isothermal PCR (iiPCR) for sensitive and specific on-site pathogen detection.

    PubMed

    Tsai, Yun-Long; Wang, Hwa-Tang Thomas; Chang, Hsiao-Fen Grace; Tsai, Chuan-Fu; Lin, Ching-Ko; Teng, Ping-Hua; Su, Chen; Jeng, Chien-Chung; Lee, Pei-Yu

    2012-01-01

    Insulated isothermal PCR (iiPCR), established on the basis of Ralyeigh-Bénard convection, is a rapid and low-cost platform for nucleic acid amplification. However, the method used for signal detection, namely gel electrophoresis, has limited the application of iiPCR. In this study, TaqMan probe-based iiPCR system was developed to obviate the need of post-amplification processing. This system includes an optical detection module, which was designed and integrated into the iiPCR device to detect fluorescent signals generated by the probe. TaqMan probe-iiPCR assays targeting white spot syndrome virus (WSSV) and infectious myonecrosis virus were developed for preliminary evaluation of this system. Significant elevation of fluorescent signals was detected consistently among positive iiPCR reactions in both assays, correlating with amplicon detection by gel electrophoresis analysis. After condition optimization, a threshold value of S/N (fluorescent intensity(after)/fluorescent intensity(before)) for positive reactions was defined for WSSV TaqMan probe-iiPCR on the basis of 20 blank reactions. WSSV TaqMan probe-iiPCR generated positive S/Ns from as low as 10(1) copies of standard DNA and lightly infected Litopenaeus vannamei. Compared with an OIE-certified nested PCR, WSSV TaqMan probe-iiPCR showed a sensitivity of 100% and a specificity of 96.67% in 120 WSSV-free or lightly infected shrimp samples. Generating positive signals specifically and sensitively, TaqMan probe-iiPCR system has a potential as a low-cost and rapid on-site diagnostics method.

  14. The PCR-GLOBWB global hydrological reanalysis product

    NASA Astrophysics Data System (ADS)

    Bierkens, M. F.; Wanders, N.; Sutanudjaja, E.; Van Beek, L. P.

    2013-12-01

    Accurate and long time series of hydrological data are important for understanding land surface water and energy budgets in many parts of the world, as well as for improving real-time hydrological monitoring and climate change anticipation. The ultimate goal of the present work is to produce a multi-decadal land surface hydrological reanalysis with retrospective and updated hydrological states and fluxes that are constrained to available in-situ river discharge measurements. Here we used PCR-GLOBWB (van Beek et al., 2011), which is a large-scale hydrological model intended for global to regional studies. PCR-GLOBWB provides a grid-based representation of terrestrial hydrology with a typical spatial resolution of approximately 50×50 km (currently 0.5° globally) on a daily basis. For each grid cell, PCR-GLOBWB is basically a leaky bucket type of water balance model with a process-based simulation of moisture storage in two vertically stacked soil layers as well as the water exchange between the soil and the atmosphere and the underlying groundwater reservoir. Exchange to the atmosphere comprises precipitation, evaporation and transpiration, as well as snow accumulation and melt, which are all simulated by considering vegetation phenology and sub-grid distributions of elevation, land cover and soil saturation distribution. The model thus includes detailed schemes for runoff-infiltration partitioning, interflow, groundwater recharge and baseflow, as well as river routing of discharge. . By embedding the PCR-GLOBWB model in an Ensemble Kalman Filter framework, we calibrated the model parameters based on the discharge observations from the Global Runoff Data Centre. The parameters calibrated are related to snow module, runoff-infiltration partitioning, groundwater recharge, channel discharge and baseflow processes, as well as pre-factors to correct forcing precipitation fields due to local topographic and orographic effects. Results show that the model parameters can

  15. The PCR-GLOBWB global hydrological reanalysis product

    NASA Astrophysics Data System (ADS)

    Wanders, Niko; Bierkens, Marc; Sutanudjaja, Edwin; van Beek, Rens

    2014-05-01

    Accurate and long time series of hydrological data are important for understanding land surface water and energy budgets in many parts of the world, as well as for improving real-time hydrological monitoring and climate change anticipation. The ultimate goal of the present work is to produce a multi-decadal "land surface hydrological reanalysis" dataset with retrospective and updated hydrological states and fluxes that are constrained to available in-situ river discharge measurements. Here we use PCR-GLOBWB (van Beek et al., 2011), which is a large-scale hydrological model intended for global to regional studies. PCR-GLOBWB provides a grid-based representation of terrestrial hydrology with a typical spatial resolution of approximately 50×50 km (currently 0.5° globally) on a daily basis. For each grid cell, PCR-GLOBWB simulates moisture storage in two vertically stacked soil layers as well as the water exchange between the soil and the atmosphere and the underlying groundwater reservoir. Exchange to the atmosphere comprises precipitation, evaporation and transpiration, as well as snow accumulation and melt, which are all simulated by considering vegetation phenology and sub-grid variations of elevation, land cover and soil saturation distribution. The model includes improved schemes for runoff-infiltration partitioning, interflow, groundwater recharge and baseflow, as well as river routing of discharge. It also dynamically simulates water storage in reservoirs, water demand and the withdrawal, allocation and consumptive use of surface water and groundwater resources. By embedding the PCR-GLOBWB model in an Ensemble Kalman Filter framework, we calibrate the model parameters based on the discharge observations from the Global Runoff Data Centre. The parameters calibrated are related to snow accumulation and melt, runoff-infiltration partitioning, groundwater recharge, channel discharge and baseflow processes, as well as pre-factors to correct forcing precipitation

  16. Comparative PCR-based fingerprinting of Vibrio cholerae isolated in Malaysia.

    PubMed

    Shuan Ju Teh, Cindy; Thong, Kwai Lin; Osawa, Ro; Heng Chua, Kek

    2011-01-01

    Vibrio cholerae, the causative agent of cholera, is endemic in many parts of the world, especially in countries poor in resources. Molecular subtyping of V. cholerae is useful to trace the regional spread of a clone or multidrug-resistant strains during outbreaks of cholera. Current available PCR-based fingerprinting methods such as Random Amplified Polymorphic DNA (RAPD)-PCR, Enterobacterial Repetitive Intergenic Consensus Sequence (ERIC)-PCR, and Repetitive Extragenic Palindromic (REP)-PCR were used to subtype V. cholerae. However, there are problems for inter-laboratory comparison as these PCR methods have their own limitations especially when different PCR methods have been used for molecular typing. In this study, a Vibrio cholerae Repeats-PCR (VCR-PCR) approach which targets the genetic polymorphism of the integron island of Vibrios was used and compared with other PCR-based fingerprinting methods in subtyping. Forty-three V. cholerae of different serogroups from various sources were tested. The PCR-fingerprinting approaches were evaluated on typeability, reproducibility, stability and discriminatory power. Overall, Malaysian non-O1/non-O139 V. cholerae were more diverse than O1 strains. Four non-O1/non-O139 strains were closely related with O1 strains. The O139 strain in this study shared similarity with strains of both O1 and non-O1/non-O139 serogroups. ERIC-PCR was the most discriminative approach (D value = 0.996). VCR-PCR was useful in discriminating non-O1/non-O139 strains. RAPD-PCR and REP-PCR were less suitable for efficient subtyping purposes as they were not reproducible and lacked stability. The combination of the ERIC-PCR and VCR-PCR may overcome the inadequacy of any one approach and hence provide more informative data.

  17. A specific real-time PCR assay for the detection of Bordetella pertussis.

    PubMed

    Vincart, Benoit; De Mendonça, Ricardo; Rottiers, Sylvianne; Vermeulen, Françoise; Struelens, Marc J; Denis, Olivier

    2007-07-01

    A novel real-time PCR (RT-PCR) assay was developed for detection of Bordetella pertussis in respiratory specimens by targeting the pertactin gene. In vitro evaluation with reference strains and quality control samples showed analytical sensitivity equivalent to and specificity superior to those of PCR assays which target the IS481 element. The pertactin-based RT-PCR assay offers better discrimination between B. pertussis and other Bordetella species than previously described assays.

  18. Detection and strain identification of Actinobacillus actinomycetemcomitans by nested PCR.

    PubMed Central

    Leys, E J; Griffen, A L; Strong, S J; Fuerst, P A

    1994-01-01

    By using PCR, Actinobacillus actinomycetemcomitans strains were identified directly from plaque samples without the need to isolate or culture bacteria. DNA fragments were generated by a nested, two-step PCR amplification of the ribosomal spacer region between the 16S and 23S rRNA genes. For the first amplification, primers homologous to sequences common to all bacterial species were used. This was followed by a second amplification with primers specific to A. actinomycetemcomitans. The ribosomal DNA spacer region was amplified from as few as 10 bacterial cells within a total population of 10(8) cells (0.00001%), and cross-reactivity between species was not observed. DNA fragments specific for Porphyromonas gingivalis were generated from the same samples by using a P. gingivalis-specific primer, and equivalent sensitivity and specificity were observed. A. actinomycetemcomitans was detected in 60% and P. gingivalis was detected in 79% of 52 subjects tested. Sequence analysis of the spacer region DNA fragment for A. actinomycetemcomitans gave precise strain identification, producing unique sequences for seven reference strains and identification of nine plaque-derived isolates. A phylogenetic tree based on quantitative sequence relationships was constructed. Two-step PCR amplification directly from plaque samples combined with sequence analysis of the ribosomal DNA spacer region provides a sensitive assay for detection and strain identification of multiple species directly from a single plaque sample. This simplified approach provides a practical method for large-scale studies on the transmission and pathogenicity of periodontitis-associated bacteria. Images PMID:8051258

  19. Multiplex PCR testing for nine different sexually transmitted infections.

    PubMed

    Kriesel, John D; Bhatia, Amiteshwar S; Barrus, Cammie; Vaughn, Mike; Gardner, Jordan; Crisp, Robert J

    2016-12-01

    Current sexually transmitted infection (STI) testing is not optimal due to delays in reporting or missed diagnoses due to a lack of comprehensive testing. The FilmArray® (BioFire Diagnostics, LLC, Salt Lake City, Utah) is a user-friendly, fully automated, multiplex PCR system that is being developed for rapid point-of-care use. A research-use-only STI panel including multiple PCR primer sets for each organism was designed to detect Chlamydia trachomatis, Neisseria gonorrhoeae, Treponema pallidum, Trichomonas vaginalis, Mycoplasma genitalium, Ureaplasma urealyticum, Haemophilus ducreyi, and herpes simplex virus (HSV) types 1 and 2. Standard clinical testing included Gram stain, nucleic acid amplification, wet mount examination, herpes simplex virus culture, and syphilis IgG. Standard clinical tests were not available for all the organisms tested by the FilmArray STI panel. Two hundred and ninety-five clinical specimens from 190 subjects were directly compared to standard testing. Urine (n = 146), urethral/cervical swabs (31), oral swabs (60), rectal swabs (43), and ulcer swabs (15) were tested. Among the tested samples, FilmArray detected C. trachomatis in 39 (13%), N. gonorrhoeae in 20 (7%), T. vaginalis in nine (3%), HSV 1 in five (2%), HSV 2 in five (2%), U. urealyticum in 36 (12%), M. genitalium in eight (3%), and T. pallidum in 11 (4%). Concordance between the FilmArray STI panel and standard nucleic acid amplification testing for C. trachomatis was 98% and for N. gonorrhoeae was 97%. Multiplex PCR STI testing has the potential to improve public health by providing rapid, sensitive, and reliable results within the clinic or nearby laboratory.

  20. PCR and Genotyping for HPV in Cervical Cancer Patients

    PubMed Central

    Prakash, Pradyot; Patne, Shashikant C U; Singh, Ashish Kumar; Kumar, Mohan; Mishra, Mukti Nath; Gulati, Anil Kumar

    2016-01-01

    Aims: To devise nested multiplex polymerase chain reaction (NMPCR) protocol for detection of mucosal human papilloma viruses (HPVs) and typing of HPV-16 and -18 in formalin-fixed, paraffin-embedded (FFPE) tissues of carcinoma cervix (CaCx). Settings and Design: Cross-sectional observational study. Materials and Methods: NMPCR was done for simultaneous detection of HPV, targeting 134 bp L1 capsid gene employing GP+/mGP+ primers and typing of genotypes-16 and -18, targeting E6/E7 gene from 34 FFPE tissue blocks of CaCx and cervical intraepithelial neoplasia (CIN). Detection of 142 bp consensus sequence of L1 capsid gene was performed by nested PCR employing MY/GP+ primers. Sequencing of selected PCR amplicons of the later protocol obtained from control cell line DNA and 5 select samples were done for validation of the NMPCR protocol. Statistical Analysis Used: Calculation of percentage from the Microsoft Excel Software. Results: Of 26 FFPE samples of CaCx, 17 (65.3%) samples were found positive for HPV by NMPCR. Amplicons of 142 bp L1 capsid gene employing MY/GP+ primers were observed in 11 (42.3%) samples of CaCx. Nearly 25% samples of CIN were positive for HPV. On sequence analysis, it was observed that the sample typed as HPV-16 by NMPCR was found to be the same on sequencing of amplicons obtained after MY/GP+ nested PCR. Conclusions: This study indicates the usefulness of our NMPCR protocol for detection of mucosal HPVs and typing of HPV-16 and -18 from FFPE tissue samples of CaCx. The NMPCR protocol may be used to detect HPV and type common genotypes-16 and -18 in fresh tissue of cervical biopsy or scrape samples for screening of CaCx. PMID:27621560

  1. PCR-based polymorphisms in neurofibromatosis type 1 (NFI)

    SciTech Connect

    Lai, P.S.; Chee, S.; Low, P.S.

    1994-09-01

    Neurofibromatosis type 1 (NF1) is one of the most common genetic disorders in humans with an incidence of 1 in 3,000. The NF1 gene is located on chromosome 17q 11.2 and encodes an ubiquitously expressed transcript of about 13kb. Direct mutation detection is difficult in this disorder due to the large gene size, high mutation rate and variety of mutations. We have studied the allele frequencies of seven PCR-based polymorphisms. Six of the probes used flank the NF1 gene, namely p11.3C4.2/Msp I (proximal), pEW206/Msp I (distal), p2.f9.8/Rsa I (distal), pEW207/Bgl II (distal), pEW207/Hind III (distal) and pHHH202/Rsa I (proximal). An intragenic RFLP, pEvi 2B-B/Eco R1 polymorphism in intron 27, was also analyzed by PCR. Allele frequencies for 48 normal unrelated individuals were obtained as follows: A1 = 0.40, A2 = 0.6 (p11.3C4.2/Msp I), A1 = 0.44, A2 = 0.56 (pEW206/Msp I), A1 = 0.17, A2 = 0.83 (p2.F9.8/Rsa I), A1 = 0.64, A2 = 0.36 (pEW207/Bgl I), A1 = 0.45, A2 = 0.55 (pEvi 2B-B/Eco RI). Heterozygosity rates of the alleles ranged from 20.8% to 51.7%. Using a combination of these markers, seven local families with NF1 were studied. Normal Mendelian segregation of alleles was observed in these families and no recombination was detected so far. These PCR-based markers were found to be useful for linkage analysis in our families.

  2. Comparison of multilocus sequence typing (MLST) and repetitive sequence-based PCR (rep-PCR) fingerprinting for differentiation of Campylobacter jejuni isolated from broiler in Chiang Mai, Thailand.

    PubMed

    Patchanee, Prapas; Chokboonmongkol, Chomporn; Zessin, Karl-Hans; Alter, Thomas; Pornaem, Sarinya; Chokesajjawatee, Nipa

    2012-11-01

    We compared rapid fingerprinting using repetitive sequence-based PCR (rep-PCR) for subtyping Campylobacter jejuni isolates to the widely used multilocus sequence typing (MLST). Representative C. jejuni isolates (n = 16) from broilers were analyzed using MLST and rep-PCR. Both techniques demonstrated an equal discriminatory power of 0.8917, and 9 subgroups were identified. Clonal identification of all 16 isolates was identical for both techniques. The rep-PCR as described in this study may be used as a rapid and cost-effective alternative for subtyping of C. jejuni isolates, or as an effective screening tool in large epidemiological studies.

  3. Linear-After-The-Exponential (LATE)–PCR: An advanced method of asymmetric PCR and its uses in quantitative real-time analysis

    PubMed Central

    Sanchez, J. Aquiles; Pierce, Kenneth E.; Rice, John E.; Wangh, Lawrence J.

    2004-01-01

    Conventional asymmetric PCR is inefficient and difficult to optimize because limiting the concentration of one primer lowers its melting temperature below the reaction annealing temperature. Linear-After-The-Exponential (LATE)–PCR describes a new paradigm for primer design that renders assays as efficient as symmetric PCR assays, regardless of primer ratio. LATE-PCR generates single-stranded products with predictable kinetics for many cycles beyond the exponential phase. LATE-PCR also introduces new probe design criteria that uncouple hybridization probe detection from primer annealing and extension, increase probe reliability, improve allele discrimination, and increase signal strength by 80–250% relative to symmetric PCR. These improvements in PCR are particularly useful for real-time quantitative analysis of target numbers in small samples. LATE-PCR is adaptable to high throughput applications in fields such as clinical diagnostics, biodefense, forensics, and DNA sequencing. We showcase LATE-PCR via amplification of the cystic fibrosis CFΔ508 allele and the Tay-Sachs disease TSD 1278 allele from single heterozygous cells. PMID:14769930

  4. Comparative evaluation of the nested ITS PCR against the 18S PCR-RFLP in a survey of bovine trypanosomiasis in Kwale County, Kenya.

    PubMed

    Odongo, Steven; Delespaux, Vincent; Ngotho, Maina; Bekkele, Serkalem Mindaye; Magez, Stefan

    2016-09-01

    We compared the nested internal transcribed spacer (ITS) PCR and the 18S PCR-RFLP (restriction-fragment length polymorphism) pan-trypanosome assays in a cross-sectional survey of bovine trypanosomiasis in 358 cattle in Kwale County, Kenya. The prevalence of trypanosomiasis as determined by the nested ITS PCR was 19.6% (70/358) and by 18S PCR-RFLP was 16.8% (60/358). Of the pathogenic trypanosomes detected, the prevalence of Trypanosoma congolense and Trypanosoma vivax was greater than that of Trypanosoma simiae The nested ITS PCR detected 83 parasite events, whereas the 18S PCR-RFLP detected 64; however, overall frequencies of infections and the parasite events detected did not differ between the assays (χ(2) = 0.8, df = 1, p > 0.05 and χ(2) = 2.5, df = 1, p > 0.05, respectively). The kappa statistic (0.8) showed good agreement between the tests. The nested ITS PCR and the 18S PCR-RFLP had comparable sensitivity, although the nested ITS PCR was better at detecting mixed infections (χ(2) = 5.4, df = 1, p < 0.05).

  5. [Differentiation of geographic biovariants of smallpox virus by PCR].

    PubMed

    Babkin, I V; Babkina, I N

    2010-01-01

    Comparative analysis of amino acid and nucleotides sequences of ORFs located in extended segments of the terminal variable regions in variola virus genome detected a promising locus for viral genotyping according to the geographic origin. This is ORF O1L of VARV. The primers were calculated for synthesis of this ORF fragment by PCR, which makes it possible to distinguish South America-Western Africa genotype from other VARV strains. Subsequent RFLP analysis reliably differentiated Asian strains from African strains (except Western Africa isolates). This method has been tested using 16 VARV strains from various geographic regions. The developed approach is simple, fast and reliable.

  6. PCR-based typing of IncC plasmids.

    PubMed

    Harmer, Christopher J; Hall, Ruth M

    IncC (A/C2) plasmids are known to play an important role in the spread of multiple antibiotic resistance determinants, including extended-spectrum β-lactamases and carbapenamases, amongst Gram negative bacterial populations. The ability to identify and track these plasmids is valuable in epidemiological and clinical studies. A recent comparative analysis of the backbones of sequenced IncC plasmids identified two distinct lineages, type 1 and type 2, with different evolutionary histories. Here, a simple PCR method to rapidly assign plasmids to one of these lineages by detecting variable regions in the backbone was developed. This PCR scheme uses two primer pairs to assign the plasmid to a lineage, and an additional two PCRs can be used to detect the i1 and i2 insertions, which are only found in type 2. PCRs were also developed to detect the presence or absence of the sul2-containing ARI-B island, which is found in some plasmids belonging to both type 1 and type 2, and the ARI-A island found in most type 1 plasmids. The PCR strategy was validated using sequenced type 1 plasmids pRMH760 and pDGO100, and the type 2 plasmid pSRC119-A/C, and a collection of non-IncC plasmids in Escherichia coli, Salmonella enterica, and Klebsiella pneumoniae backgrounds. An IncC plasmid detected in an antibiotic susceptible commensal E. coli isolate was examined and found to be a type 1, lacking any antibiotic resistance islands and missing a large backbone segment. Examination of pIP40a, an IncC plasmid isolated in Paris in 1969, by PCR revealed that it belongs to type 1 but lacks ARI-A. However, it includes both ends of the integrative element GIsul2, whereas only remnants of one end of this element are found in more recently isolated IncC plasmids. The sequence of pIP40a was determined and confirmed the assignment to type 1 and revealed the presence of a complete copy of GIsul2.

  7. Amelogenin sex determination by pyrosequencing of short PCR products.

    PubMed

    Tschentscher, Frank; Frey, Ulrich H; Bajanowski, Thomas

    2008-07-01

    We developed an assay, which allows the sex determination of human DNA samples by pyrosequencing of short PCR products. A 48/45-bp stretch including primers of the amelogenin gene with a 3-bp insertion on the Y chromosome was chosen for analysis. In an initial study, we correctly typed 50 male and 50 female DNA samples from unrelated donors. First experiments with forensic samples, which failed in conventional analyses, indicate that this approach might be an advantage when dealing with degraded DNA.

  8. Novel primers and PCR protocols for the specific detection and quantification of Sphingobium suberifaciens in situ

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The pathogen causing corky root on lettuce, Sphingobium suberifaciens, is recalcitrant to standard epidemiological methods. Primers were selected from 16S rDNA sequences useful for the specific detection and quantification of S. suberifaciens. Conventional (PCR) and quantitative (qPCR) PCR protocols...