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Sample records for lato mediante pcr

  1. 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.

  2. A novel PCR-RFLP assay for molecular characterization of Echinococcus granulosus sensu lato and closely related species in developing countries.

    PubMed

    Chaâbane-Banaoues, Raja; Oudni-M'rad, Myriam; M'rad, Selim; Amani, Hizem; Mezhoud, Habib; Babba, Hamouda

    2016-10-01

    Cystic echinococcosis, due to Echinococcus granulosus sensu lato (s. l.), currently affects three million people, especially in low-income countries and results in high livestock production loss. DNA-based methods demonstrated genetic variability of E. granulosus s. l., and five species were recognized to belong to the complex, including E. granulosus sensu stricto (s.s) (genotypes G1-G3), Echinococcus equinus (genotype G4), Echinococcus ortleppi (genotype G5), Echinococcus canadensis (genotypes G6-G10), and the lion strain Echinococcus felidis. The characterization of Echinococcus species responsible for human and animal echinococcosis is crucial to adapt the preventive measures against this parasitic disease. The sequencing approach is the gold standard for genotyping assays. Unfortunately, developing countries do not often have access to these techniques. Based on in silico RFLP tools, we described an accurate PCR-RFLP method for Echinococcus spp. characterization. The double digestion with the HaeIII and HinfI restriction enzymes of the PCR product from nad1 gene (1071 bp) led to a clear discrimination between E. granulosus s. l. and most closely related species (Echinococcus shiquicus and Echinococcus multilocularis).Molecular procedures and phylogenetic analysis confirmed the efficiency and the reproducibility of this simple and fast PCR-RFLP method. This technique is proved useful for fresh/unfixed and FF-PET tissues and enables large-scale molecular epidemiological screening in developing countries.

  3. Accurate identification of Candida parapsilosis (sensu lato) by use of mitochondrial DNA and real-time PCR.

    PubMed

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

    2012-07-01

    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.

  4. Three species of Borrelia burgdorferi sensu lato (B. burgdorferi sensu stricto, B afzelii, and B. garinii) identified from cerebrospinal fluid isolates by pulsed-field gel electrophoresis and PCR.

    PubMed Central

    Busch, U; Hizo-Teufel, C; Boehmer, R; Fingerle, V; Nitschko, H; Wilske, B; Preac-Mursic, V

    1996-01-01

    A total of 36 European Borrelia burgdorferi sensu lato cerebrospinal fluid isolates (mainly from southern Germany) were analyzed by pulsed-field gel electrophoresis (PFGE) for large restriction fragment pattern (LRFP) and linear plasmid profiles. Analyzing this large panel of isolates, we detected all three species of B. burgdorferi sensu lato pathogenic for humans in cerebrospinal fluid from patients with Lyme neuroborreliosis by PFGE typing after MluI digestion: 21 B. garinii (58%), 10 B. afzelii (28%), and 4 B. burgdorferi sensu stricto (11%) strains as well as 1 isolate with bands characteristic of both B. afzelii and B. garinii. Species classification by PFGE typing was confirmed by 16S rRNA-specific PCR. Eighteen isolates (11 B. garinii, 6 B. afzelii, and 1 B. burgdorferi sensu stricto isolate) were further characterized by LRFP with four different restriction enzymes (ApaI, KspI, SmaI, and XhoI). All B. afzelii isolates showed identical patterns for each restriction enzyme group. Considerable heterogeneity was demonstrated within the B. garinii group. Subsequent analysis of plasmid profiles revealed only marginal differences for B. afzelii strains but different patterns for B. garinii isolates. In one B. afzelii strain we found a linear plasmid of about 110 kbp not described before. LRFP analysis by PFGE is a suitable tool for the molecular characterization of B. burgdorferi sensu lato strains and allows determination not only of the species but also of the subtypes within B. garinii. PMID:8727878

  5. 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.

  6. 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. PMID:26999390

  7. 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

  8. 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. PMID:27061834

  9. Detection of Borrelia burgdorferi sensu lato DNA in the blood of wild bison from Białowieza primeval forest in eastern Poland.

    PubMed

    Adaszek, L; Dzięgiel, B; Krzysiak, M; Skrzypczak, M; Adaszek, M; Staniec, M; Wyłupek, D; Winiarczyk, S

    2014-01-01

    The aim of the present study was to investigate the occurrence of Borrelia burgdorferi sensu lato DNA in a group of 120 wild bison (Bison bonasus) from the Bialowieza Primeval Forest in eastern Poland. The PCR technique revealed the presence of 16S RNA of Borrelia burgdorferi sensu lato in the blood of 16 out of 120 examined animals. DNA amplification by means of primers SC1 and SC2 gave a product with a size of 300-bp. The sequences of the PCR products obtained showed 100% homology with each other and 100% homology with B. burgdorferi s.1. 16S RNA gene DQ111061. Results of this study suggest that wild bison are important in maintaining agents of Lyme borreliosis, and that studies of reservoir competence of this species are indicated.

  10. Re-identification of Aspergillus fumigatus sensu lato based on a new concept of species delimitation.

    PubMed

    Hong, Seung-Beom; Kim, Dae-Ho; Park, In-Cheol; Choi, Young-Joon; Shin, Hyeon-Dong; Samson, Robert

    2010-10-01

    The species concept of Aspergillus fumigatus sensu stricto has recently been defined by polyphasic taxonomy. Based on the new concept of species delimitations, 146 worldwide strains of Aspergillus fumigatus sensu lato were re-identified. Of those 146 strains, 140 (95.8%) could be identified as A. fumigatus sensu stricto, 3 (2.1%) as A. lentulus, and the remaining 3 strains as A. viridinutans complex, Neosartorya udagawae, and N. cf. nishimurae. Of 98 clinical strains, only 1 from dolphin nostril was identified as A. lentulus and not A. fumigatus sensu stricto. Random amplification of polymorphic DNA-polymerase chain reaction (RAPD-PCR) with primers PELF and URP1F produced nearly the same band patterns among 136 strains of A. fumigatus sensu stricto while discriminated the species from its related species. We also discussed about identification of several atypical A. fumigatus strains from clinical environments.

  11. Complete Genome Sequence of Bacillus cereus Sensu Lato Bacteriophage Bcp1.

    PubMed

    Schuch, Raymond; Pelzek, Adam J; Fazzini, Monica M; Nelson, Daniel C; Fischetti, Vincent A

    2014-01-01

    Bacillus cereus sensu lato organisms are an ecologically diverse group that includes etiologic agents of food poisoning, periodontal disease, and anthrax. The recently identified Bcp1 bacteriophage infects B. cereus sensu lato and is being developed as a therapeutic decontamination agent and diagnostic countermeasure. We announce the complete genome sequence of Bcp1.

  12. Molecular and microscopical evidence of Ehrlichia spp. and Borrelia burgdorferi sensu lato in patients, animals and ticks in the Czech Republic.

    PubMed

    Hulinska, D; Votypka, J; Plch, J; Vlcek, E; Valesová, M; Bojar, M; Hulinsky, V; Smetana, K

    2002-10-01

    We report moderately severe cases of human ehrlichiosis and a lethal one caused by human granulocytic Ehrlichia, the HGE agent, closely related to Ehrlichia phagocytophila and Ehrlichia equi. Their vector is the Ixodes ricinus tick, which also transmits Borrelia burgorferi sensu lato in central, west and east regions of the Czech Republic. The diagnosis was established by PCR with sequence analysis of the genes encoding 16S rRNA of Ehrlichia and with reverse hybridization by using enzyme linked immunosorbent assay with different covalently coupled probes to the activated plate. Ten out of 47 patients and 10 huntsmen were PCR positive and 7 of them seroconverted to the HGE. Coinfection of Ehrlichia phagocytophila with Borrelia burgdorferi sensu lato was detected in 3 patients. Ehrlichia spp., the HGE agent, was isolated and propagated only from one patient in the HL-60 promyelocytic cell line. The maintenance of Ehrlichia in culture and in patients was assayed also by immunocytological staining and electron microscopy. Sequence or hybridization analysis of PCR results in different wild mammals and birds showed significant sources of Ehrlichia fagocytophila in nature. Three variants of E. phagocytophila in wild roe deer and boars, as well as for the first time in birds, have been described. Cultures from the blood of horses, and from the spleen and kidney specimens of roes and boars, PCR positive for Ehrlichia spp., displayed a disappearing level of the pathogen or contamination with other bacteria. PMID:12437223

  13. Vector Capability of Xiphinema americanum sensu lato in California.

    PubMed

    Griesbach, J A; Maggenti, A R

    1989-10-01

    Seven field populations of Xiphinema americanum sensu lato from California's major agronomic areas were tested for their ability to transmit two nepoviruses, including the prune brownline, peach yellow bud, and grapevine yellow vein strains of tomato ringspot virus and the bud blight strain of tobacco ringspot virus. Two field populations transmitted all isolates, one population transmitted all tomato ringspot virus isolates but failed to transmit bud blight strain of tobacco ringspot virus, and the remaining four populations failed to transmit any virus. Only one population, which transmitted all isolates, bad been associated with field spread of a nepovirus. As two California populations of Xiphinema americanum sensu lato were shown to have the ability to vector two different nepoviruses, a nematode taxonomy based on a parsimony of virus-vector relationship is not practical for these populations. Because two California populations of X. americanum were able to vector tobacco ringspot virus, commonly vectored by X. americanum in the eastern United States, these western populations cannot be differentiated from eastern populations by vector capability tests using tobacco ringspot virus.

  14. Diversity of commensal Bacillus cereus sensu lato isolated from the common sow bug (Porcellio scaber, Isopoda).

    PubMed

    Swiecicka, Izabela; Mahillon, Jacques

    2006-04-01

    Although Bacillus cereus sensu lato are important both from an ecological and an economical point of view, little is known about their population structure, ecology, and relationships with other organisms. In the present work, the genotypic similarity of arthropod-borne B. cereus s.l. isolates, and their symbiotic relationship with the host are assessed. Bacilli of this group were recovered from the digestive tracts of sow bugs (Porcellio scaber) collected in three closely located sites. Their genotypic diversity was investigated using pulse-field gel electrophoresis (PFGE) following the whole-genome DNA digestions with NotI and AscI, and PCR amplification of virulence genes. The majority of the sow-bug Bacillus cereus sensu stricto isolates originating from the same but also from different sites displayed identical PFGE patterns, virulence gene content and enterotoxicity, indicating strong genetic and genomic relationships. The sow-bug Bacillus mycoides/Bacillus pseudomycoides strains displayed a higher diversity. The isopod-B. cereus s.l. relationship was also evaluated using antibiotic-resistant derivatives of B. cereus s.s., B. mycoides/B. pseudomycoides and Bacillus thuringiensis reintroduced into sow bugs. Both spores and vegetative cells of B. cereus s.l. were recovered from sow bugs over a 30-day period, strongly suggesting that these bacteria are natural residents of terrestrial isopods.

  15. Diversity of commensal Bacillus cereus sensu lato isolated from the common sow bug (Porcellio scaber, Isopoda).

    PubMed

    Swiecicka, Izabela; Mahillon, Jacques

    2006-04-01

    Although Bacillus cereus sensu lato are important both from an ecological and an economical point of view, little is known about their population structure, ecology, and relationships with other organisms. In the present work, the genotypic similarity of arthropod-borne B. cereus s.l. isolates, and their symbiotic relationship with the host are assessed. Bacilli of this group were recovered from the digestive tracts of sow bugs (Porcellio scaber) collected in three closely located sites. Their genotypic diversity was investigated using pulse-field gel electrophoresis (PFGE) following the whole-genome DNA digestions with NotI and AscI, and PCR amplification of virulence genes. The majority of the sow-bug Bacillus cereus sensu stricto isolates originating from the same but also from different sites displayed identical PFGE patterns, virulence gene content and enterotoxicity, indicating strong genetic and genomic relationships. The sow-bug Bacillus mycoides/Bacillus pseudomycoides strains displayed a higher diversity. The isopod-B. cereus s.l. relationship was also evaluated using antibiotic-resistant derivatives of B. cereus s.s., B. mycoides/B. pseudomycoides and Bacillus thuringiensis reintroduced into sow bugs. Both spores and vegetative cells of B. cereus s.l. were recovered from sow bugs over a 30-day period, strongly suggesting that these bacteria are natural residents of terrestrial isopods. PMID:16542411

  16. Whole-Genome Sequences of 94 Environmental Isolates of Bacillus cereus Sensu Lato.

    PubMed

    Van der Auwera, Géraldine A; Feldgarden, Michael; Kolter, Roberto; Mahillon, Jacques

    2013-01-01

    Bacillus cereus sensu lato is a species complex that includes the anthrax pathogen Bacillus anthracis and other bacterial species of medical, industrial, and ecological importance. Their phenotypes of interest are typically linked to large plasmids that are closely related to the anthrax plasmids pXO1 and pXO2. Here, we present the draft genome sequences of 94 isolates of B. cereus sensu lato, which were chosen for their plasmid content and environmental origins.

  17. 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. PMID:27372197

  18. Phylogeny and classification of Prunus sensu lato (Rosaceae).

    PubMed

    Shi, Shuo; Li, Jinlu; Sun, Jiahui; Yu, Jing; Zhou, Shiliang

    2013-11-01

    The classification of the economically important genus Prunus L. sensu lato (s.l.) is controversial due to the high levels of convergent or the parallel evolution of morphological characters. In the present study, phylogenetic analyses of fifteen main segregates of Prunus s.l. represented by eighty-four species were conducted with maximum parsimony and Bayesian approaches using twelve chloroplast regions (atpB-rbcL, matK, ndhF, psbA-trnH, rbcL, rpL16, rpoC1, rps16, trnS-G, trnL, trnL-F and ycf1) and three nuclear genes (ITS, s6pdh and SbeI) to explore their infrageneric relationships. The results of these analyses were used to develop a new, phylogeny-based classification of Prunus s.l. Our phylogenetic reconstructions resolved three main clades of Prunus s.l. with strong supports. We adopted a broad-sensed genus, Prunus, and recognised three subgenera corresponding to the three main clades: subgenus Padus, subgenus Cerasus and subgenus Prunus. Seven sections of subgenus Prunus were recognised. The dwarf cherries, which were previously assigned to subgenus Cerasus, were included in this subgenus Prunus. One new section name, Prunus L. subgenus Prunus section Persicae (T. T. Yü & L. T. Lu) S. L. Zhou and one new species name, Prunus tianshanica (Pojarkov) S. Shi, were proposed. PMID:23945216

  19. A proposal for the classification of biological weapons sensu lato.

    PubMed

    Rozsa, Lajos

    2014-12-01

    Due to historical and legislation reasons, the category of bioweapons is rather poorly defined. Authors often disagree on involving or excluding agents like hormones, psychochemicals, certain plants and animals (such as weeds or pests) or synthetic organisms. Applying a wide definition apparently threatens by eroding the regime of international legislation, while narrow definitions abandon several important issues. Therefore, I propose a category of 'biological weapons sensu lato' (BWsl) that is defined here as any tool of human aggression whose acting principle is based on disciplines of biology including particularly microbiology, epidemiology, medical biology, physiology, psychology, pharmacology and ecology, but excluding those based on inorganic agents. Synthetically produced equivalents (not necessarily exact copies) and mock weapons are also included. This definition does not involve any claim to subject all these weapons to international legislation but serves a purely scholarly purpose. BWsl may be properly categorized on the base of the magnitude of the human population potentially targeted (4 levels: individuals, towns, countries, global) and the biological nature of the weapons' intended effects (4 levels: agricultural-ecological agents, and non-pathogenic, pathogenic, or lethal agents against humans).

  20. A proposal for the classification of biological weapons sensu lato.

    PubMed

    Rozsa, Lajos

    2014-12-01

    Due to historical and legislation reasons, the category of bioweapons is rather poorly defined. Authors often disagree on involving or excluding agents like hormones, psychochemicals, certain plants and animals (such as weeds or pests) or synthetic organisms. Applying a wide definition apparently threatens by eroding the regime of international legislation, while narrow definitions abandon several important issues. Therefore, I propose a category of 'biological weapons sensu lato' (BWsl) that is defined here as any tool of human aggression whose acting principle is based on disciplines of biology including particularly microbiology, epidemiology, medical biology, physiology, psychology, pharmacology and ecology, but excluding those based on inorganic agents. Synthetically produced equivalents (not necessarily exact copies) and mock weapons are also included. This definition does not involve any claim to subject all these weapons to international legislation but serves a purely scholarly purpose. BWsl may be properly categorized on the base of the magnitude of the human population potentially targeted (4 levels: individuals, towns, countries, global) and the biological nature of the weapons' intended effects (4 levels: agricultural-ecological agents, and non-pathogenic, pathogenic, or lethal agents against humans). PMID:24992886

  1. 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.

  2. Taxonomy and molecular epidemiology of Echinococcus granulosus sensu lato.

    PubMed

    Romig, T; Ebi, D; Wassermann, M

    2015-10-30

    Echinococcus granulosus, formerly regarded as a single species with a high genotypic and phenotypic diversity, is now recognised as an assemblage of cryptic species, which differ considerably in morphology, development, host specificity (including infectivity/pathogenicity for humans) and other aspects. This diversity is reflected in the mitochondrial and nuclear genomes and has led to the construction of phylogenetic trees and hypotheses on the origin and geographic dispersal of various taxa. Based on phenotypic characters and gene sequences, E. granulosus (sensu lato) has by now been subdivided into E. granulosus sensu stricto (including the formerly identified genotypic variants G1-3), Echinococcus felidis (the former 'lion strain'), Echinococcus equinus (the 'horse strain', genotype G4), Echinococcus ortleppi (the 'cattle strain', genotype G5) and Echinococcus canadensis. The latter species, as recognised here, shows the highest diversity and is composed of the 'camel strain', genotype G6, the 'pig strain', genotype G7, and two 'cervid strains', genotypes G8 and G10. There is debate whether the closely related G6 and G7 should be placed in a separate species, but more morphological and biological data are needed to support or reject this view. In this classification, the application of rules for zoological nomenclature led to the resurrection of old species names, which had before been synonymised with E. granulosus. This nomenclatural subdivision of the agents of cystic echinococcosis (CE) may appear inconvenient for practical applications, especially because molecular tools are needed for identification of the cyst stage, and because retrospective data on 'E. granulosus' are now difficult to interpret without examination of voucher specimens. However, the increased awareness for the diversity of CE agents - now emphasised by species names rather than genotype numbers - has led to a large number of recent studies on this issue and a rapid increase of knowledge

  3. Taxonomy and molecular epidemiology of Echinococcus granulosus sensu lato.

    PubMed

    Romig, T; Ebi, D; Wassermann, M

    2015-10-30

    Echinococcus granulosus, formerly regarded as a single species with a high genotypic and phenotypic diversity, is now recognised as an assemblage of cryptic species, which differ considerably in morphology, development, host specificity (including infectivity/pathogenicity for humans) and other aspects. This diversity is reflected in the mitochondrial and nuclear genomes and has led to the construction of phylogenetic trees and hypotheses on the origin and geographic dispersal of various taxa. Based on phenotypic characters and gene sequences, E. granulosus (sensu lato) has by now been subdivided into E. granulosus sensu stricto (including the formerly identified genotypic variants G1-3), Echinococcus felidis (the former 'lion strain'), Echinococcus equinus (the 'horse strain', genotype G4), Echinococcus ortleppi (the 'cattle strain', genotype G5) and Echinococcus canadensis. The latter species, as recognised here, shows the highest diversity and is composed of the 'camel strain', genotype G6, the 'pig strain', genotype G7, and two 'cervid strains', genotypes G8 and G10. There is debate whether the closely related G6 and G7 should be placed in a separate species, but more morphological and biological data are needed to support or reject this view. In this classification, the application of rules for zoological nomenclature led to the resurrection of old species names, which had before been synonymised with E. granulosus. This nomenclatural subdivision of the agents of cystic echinococcosis (CE) may appear inconvenient for practical applications, especially because molecular tools are needed for identification of the cyst stage, and because retrospective data on 'E. granulosus' are now difficult to interpret without examination of voucher specimens. However, the increased awareness for the diversity of CE agents - now emphasised by species names rather than genotype numbers - has led to a large number of recent studies on this issue and a rapid increase of knowledge

  4. 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

  5. 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.

  6. 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. PMID:25304515

  7. 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...

  8. 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...

  9. First molecular evidence of [i]Borrelia burgdorferi[/i] sensu lato in goats, sheep, cattle and camels in Tunisia.

    PubMed

    Ben Said, Mourad; Belkahia, Hanène; Alberti, Alberto; Abdi, Khaoula; Zhioua, Manel; Daaloul-Jedidi, Monia; Messadi, Lilia

    2016-09-01

    Borrelia burgdorferi sensu lato (s.l.) are tick-transmitted spirochaetes of veterinary and human importance. Molecular epidemiology data on ruminants are still lacking in most countries of the world. Therefore, the aim of this study was to estimate the rate of B. burgdorferi s.l. infection in ruminants from Tunisia. A total of 1,021 ruminants (303 goats, 260 sheep, 232 cattle and 226 camels) from different bioclimatic areas in Tunisia were investigated for the presence of B. burgdorferi s.l. DNA in blood by real time PCR. Prevalence rates were 30.4% (92/303) in goats, 6.2% (16/260) in sheep, 1.3% (3/232) in cattle, and 1.8% (4/226) in camels. Only tick species belonging to Rhipicephalus and Hyalomma genera were found on the investigated animals. In small ruminants, the prevalence of B. burgdorferi s.l. varied significantly according to localities and farms. Goats located in humid areas were statistically more infected than those located in sub-humid areas. Prevalence rates varied significantly according to age and breed in sheep, and age and tick infestation in goats. This study provides the first insight into the presence of B. burgdorferi s.l. DNA in ruminants in Tunisia, and demonstrates that host species such as goats and sheep may play an important role in natural Lyme disease cycles in this country.

  10. First molecular evidence of [i]Borrelia burgdorferi[/i] sensu lato in goats, sheep, cattle and camels in Tunisia.

    PubMed

    Ben Said, Mourad; Belkahia, Hanène; Alberti, Alberto; Abdi, Khaoula; Zhioua, Manel; Daaloul-Jedidi, Monia; Messadi, Lilia

    2016-07-15

    Borrelia burgdorferi sensu lato (s.l.) are tick-transmitted spirochaetes of veterinary and human importance. Molecular epidemiology data on ruminants are still lacking in most countries of the world. Therefore, the aim of this study was to estimate the rate of B. burgdorferi s.l. infection in ruminants from Tunisia. A total of 1,021 ruminants (303 goats, 260 sheep, 232 cattle and 226 camels) from different bioclimatic areas in Tunisia were investigated for the presence of B. burgdorferi s.l. DNA in blood by real time PCR. Prevalence rates were 30.4% (92/303) in goats, 6.2% (16/260) in sheep, 1.3% (3/232) in cattle, and 1.8% (4/226) in camels. Only tick species belonging to Rhipicephalus and Hyalomma genera were found on the investigated animals. In small ruminants, the prevalence of B. burgdorferi s.l. varied significantly according to localities and farms. Goats located in humid areas were statistically more infected than those located in sub-humid areas. Prevalence rates varied significantly according to age and breed in sheep, and age and tick infestation in goats. This study provides the first insight into the presence of B. burgdorferi s.l. DNA in ruminants in Tunisia, and demonstrates that host species such as goats and sheep may play an important role in natural Lyme disease cycles in this country. PMID:27660865

  11. Ixodes ricinus as a vector of Borrelia burgdorferi sensu lato, Anaplasma phagocytophilum and Babesia microti in urban and suburban forests.

    PubMed

    Stańczak, Joanna; Gabre, Refaat Mohammed; Kruminis-Łozowska, Wiesława; Racewicz, Maria; Kubica-Biernat, Beata

    2004-01-01

    In the suburban and urban forests in the cities of Gdansk, Sopot and Gdynia (northern Poland), Ixodes ricinus ticks should be considered as the vector of pathogenic microorganisms that may cause significant diseases in wild and domestic animals and humans. These microorganisms include etiologic agents of Lyme disease, human anaplasmosis (HA) and babesiosis: Borrelia burgdorferi sensu lato, Anaplasma phagocytophilum and Babesia microti, respectively. DNA extracts from 701 ticks collected in 15 localities were examined by PCR for the simultaneous detection of these 3 pathogens. Overall, 14 % were infected with A. phagocytophilum followed by 12.4 % with B. burgdorferi s.l. and 2.3 % with B. microti. In total, the percentage of infected females (32.9 %) was 2.4 times higher than in males (13.7 %) and 3.2 times higher than in nymphs (10.3 %). Among adult ticks (n = 303), 8.3 % were dually infected with A. phagocytophilum and B. burgdorferi s.l., 2.0 % with the agent of human anaplasmosis and B. microti and 0.3 % with borreliae and B. microti.

  12. 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

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

    PubMed

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

    2016-02-01

    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.

  14. Seroprevalence of Rickettsia spp. in Equids and Molecular Detection of 'Candidatus Rickettsia amblyommii' in Amblyomma cajennense Sensu Lato Ticks From the Pantanal Region of Mato Grosso, Brazil.

    PubMed

    Alves, Alvair Da S; Melo, Andréia L T; Amorim, Marcus V; Borges, Alice M C M; Gaíva E Silva, Lucas; Martins, Thiago F; Labruna, Marcelo B; Aguiar, Daniel M; Pacheco, Richard C

    2014-11-01

    The aim of the study was to evaluate exposure of equids to rickettsial agents (Rickettsia rickettsii, Rickettsia parkeri, 'Candidatus Rickettsia amblyommii', Rickettsia rhipicephali, and Rickettsia bellii) and rickettsial infection in ticks of a Pantanal region of Brazil. Sera of 547 equids (500 horses and 47 donkeys) were evaluated by indirect immunofluorescence assay. In total, 665 adults and 106 nymphal pools of Amblyomma cajennense F. sensu lato, 10 Dermacentor nitens Neumann ticks, and 88 larval pools of Amblyomma sp. were tested by polymerase chain reaction (PCR). Overall, 337 (61.6%) equids were reactive (titer ≥64) to at least one antigen of Rickettsia spp. The prevalence values for Rickettsia were 66%, and the highest endpoint titers were observed for 'Ca. R. amblyommii'. By PCR 3 (0.45%) A. cajennense s.l. females were positive for 'Ca. R. amblyommii'. Minimum infection rates of 0.75% for nymphs and 0.34% for larvae were calculated. Positive samples of ticks have had a fragment of the 16S mitochondrial rRNA gene sequenced and sequences showed 99% identity to Amblyomma sculptum Berlese. This study reports a wide exposure of equids to Rickettsia agents, and PCR evidence of infection with 'Ca. R. amblyommii', for the first time, in A. sculptum.

  15. Patient isolates of Borrelia burgdorferi sensu lato with genotypic and phenotypic similarities of strain 25015.

    PubMed

    Picken, R N; Cheng, Y; Strle, F; Picken, M M

    1996-11-01

    Strain 25015 is an atypical tick isolate that belongs to a distinct genomic group (DN127) within the general taxon Borrelia burgdorferi sensu lato. Similarities between this strain and a white-footed mouse isolate from Illinois, strain CT39, have been reported. In the course of isolating B. burgdorferi sensu lato in culture from Slovenian patients, 9 isolates were identified with the same genetic profiles as strains 25015 and CT39, as evidenced by restriction enzyme MluI digestion patterns of genomic DNA. The aim of the present study was to molecularly characterize all 11 isolates to examine the extent of their genotypic and phenotypic similarity. The results of molecular studies suggest a close relationship between the patient isolates and strains 25015 and CT39. However, CT39 and several patient isolates possessed unique characteristics that reflect their discrete ontogeny. PMID:8896519

  16. 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

  17. Heterogeneity of BmpA (P39) among European isolates of Borrelia burgdorferi sensu lato and influence of interspecies variability on serodiagnosis.

    PubMed Central

    Roessler, D; Hauser, U; Wilske, B

    1997-01-01

    The molecular and antigenic variabilities of BmpA (P39) among European isolates of Borrelia burgdorferi were analyzed. The bmpA sequences of 12 isolates representing all three species of B. burgdorferi sensu lato pathogenic for humans were amplified by PCR, cloned, and sequenced. The BmpA protein of Borrelia garinii is heterogeneous, with an amino acid sequence identity ranging from 91 to 97%, whereas the BmpA proteins of Borrelia afzelii and B. burgdorferi sensu stricto strains appear to be highly conserved (>98.5% intraspecies identity). The interspecies identities ranged from 86 to 92%. Cluster analysis of BmpA reflected the subdivision of B. burgdorferi sensu lato isolates into the three species as well as a considerable heterogeneity among B. garinii strains. The BmpA protein of each species of B. burgdorferi sensu lato was recombinantly expressed in Escherichia coli, purified, and used to generate monoclonal antibodies. Seven BmpA-specific antibodies were identified; six of them recognized conserved epitopes of all three species, whereas one was specific for BmpA of B. afzelii and B. garinii. A monoclonal antibody (H1141) recommended by the Centers for Disease Control and Prevention for use in the standardization of immunoblots showed strong reactivity with BmpA of B. burgdorferi sensu stricto but no or only weak reactivity with BmpA of B. garinii and B. afzelii, respectively. Sera from 86 European patients with Lyme borreliosis in different stages and 73 controls were tested in immunoglobulin G (IgG) and IgM immunoblots with the recombinant BmpA proteins of the three species, revealing specificities of 98.6 to 100%. IgM antibodies against recombinant BmpA were only rarely detected (1.1 to 8.1%). With the BmpA proteins of B. afzelii and B. garinii, sensitivities for the IgG test (sera from stages I to III) were 36.0 and 34.9%, respectively, in contrast to 13.9% with BmpA of B. burgdorferi sensu stricto. Therefore, we recommend that recombinant BmpA of B

  18. 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.'

  19. 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.

  20. Mating type markers reveal high levels of heterothallism in Leptographium sensu lato.

    PubMed

    Duong, Tuan A; de Beer, Z Wilhelm; Wingfield, Brenda D; Wingfield, Michael J

    2016-04-01

    Species of Leptographium sensu lato are sap-stain fungi vectored by bark beetles and some species cause or are associated with tree diseases. Sexual states have been reported for more than 30 species in this group and these have been treated in the sexual genus Grosmannia. No sexual state is known for at least 59 additional species and these reside in the genus Leptographium. The discovery of sexual states for species of Leptographium relies mainly on the presence of fruiting bodies on host tissue at the time of isolation and/or intensive laboratory mating studies, which commonly have low levels of success. We developed mating-type markers to study sexual compatibility of species in Leptographium sensu lato. Using these markers, it was possible to identify mating types for 42 species and to determine thallism in many species for the first time. Surprisingly, the results showed that heterothallic and putatively heterothallic species are abundant (39 out of 42 species) in Leptographium sensu lato, and only three species were confirmed to be homothallic. The mating type markers developed in this study will be useful for future studies concerning mating type and sexual compatibility of species in this genus. PMID:27020155

  1. Selection of Beauveria bassiana sensu lato and Metarhizium anisopliae sensu lato isolates as microbial control agents against the boll weevil (Anthonomus grandis) in Argentina.

    PubMed

    Nussenbaum, A L; Lecuona, R E

    2012-05-01

    The boll weevil (Anthonomus grandis) is the main pest of cotton in the Americas. The aim of this work was to evaluate isolates of the entomopathogenic fungi Beauveria bassiana sensu lato and Metarhizium anisopliae sensu lato virulent against A. grandis. Screening was performed to evaluate the pathogenicity of 28 isolates of M. anisopliae s.l. and 66 isolates of B. bassiana s.l. against boll weevil adults. To select the isolates, LC(50) values of the most virulent isolates were calculated, and compatibility between the fungi and insecticides was studied. In addition, the effects of these isolates on the feeding behavior of the adults were evaluated. Isolates Ma 50 and Ma 20 were the most virulent against A. grandis and their LC(50) values were 1.13×10(7) and 1.20×10(7) conidia/ml, respectively. In addition, these isolates were compatible with pyrethroid insecticides, but none with endosulfan. On the other hand, infected females reduced the damage caused by feeding on the cotton squares and their weight gain. This shows that entomopathogenic fungi cause mortality in the insects, but also these fungi could influence the feeding behavior of the females. In summary, these results indicate the possibility of the use of M. anisopliae s.l. as a microbiological control agent against boll weevils. Also, this species could be included in an Integrated Pest Management program.

  2. 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.

  3. Molecular and immunological characterization of the p83/100 protein of various Borrelia burgdorferi sensu lato strains.

    PubMed

    Rössler, D; Eiffert, H; Jauris-Heipke, S; Lehnert, G; Preac-Mursic, V; Teepe, J; Schlott, T; Soutschek, E; Wilske, B

    1995-05-01

    The complete coding regions of the chromosomally encoded p83/100 protein of four Borrelia garinii strains and one Borrelia burgdorferi sensu stricto strain have been amplified by the polymerase chain reaction (PCR), cloned and sequenced. From alignment studies with the deduced amino acid sequences presented here, and five other published p83/100 sequences, the most heterologous region of the p83/100 molecule was identified to be located between amino acid position 390-540. To study the structure of this heterogeneous region, and internal fragment of the p83/100 genes from 11 additional B. burgdorferi sensu lato strains was amplified by PCR. The PCR products were analyzed by DNA sequencing and restriction enzyme analysis. These internal p83/100 fragments varied in size and sequence. Cluster analysis of internal p83/100 fragments, as well as restriction enzyme analysis, revealed three major groups in accordance with grouping into the three species causing Lyme disease. Strains within the same species (six B. burgdorferi sensu stricto and six B. afzelii strains) showed similar p83/100 partial structures. Nevertheless, nine B. garinii strains showed more sequence variations and could be further divided into two major subgroups. One group is represented by OspA serotype 4 strains, the other more heterogeneous group is represented by OspA serotypes 3, 5, 6 and 7 strains. Phenotypic analysis with four p83/100-specific monoclonal antibodies revealed four distinct reactivity patterns. Antibody L100 1B4 recognized a common epitope of B. burgdorferi sensu stricto and B. afzelii. Antibodies L100 17D3 and L100 18B4 were reactive with an epitope shared by strains of all three species. The broadest reactivity was shown by L100 18B4 which, in contrast to L100 17D3, additionally recognized the relapsing fever borreliae B. turicatae and B. hermsii. L100 8B8 detected a subgroup of the B. burgdorferi sensu stricto strains. Since comparison of the p83/100 molecule with sequences from

  4. Genetic Heterogeneity of Borrelia burgdorferi Sensu Lato in the Southern United States Based on Restriction Fragment Length Polymorphism and Sequence Analysis

    PubMed Central

    Lin, T.; Oliver, J. H.; Gao, L.; Kollars, T. M.; Clark, K. L.

    2001-01-01

    Fifty-six strains of Borrelia burgdorferi sensu lato, isolated from ticks and vertebrate animals in Missouri, South Carolina, Georgia, Florida, and Texas, were identified and characterized by PCR-restriction fragment length polymorphism (RFLP) analysis of rrf (5S)-rrl (23S) intergenic spacer amplicons. A total of 241 to 258 bp of intergenic spacers between tandemly duplicated rrf (5S) and rrl (23S) was amplified by PCR. MseI and DraI restriction fragment polymorphisms were used to analyze these strains. PCR-RFLP analysis results indicated that the strains represented at least three genospecies and 10 different restriction patterns. Most of the strains isolated from the tick Ixodes dentatus in Missouri and Georgia belonged to the genospecies Borrelia andersonii. Excluding the I. dentatus strains, most southern strains, isolated from the ticks Ixodes scapularis and Ixodes affinis, the cotton rat (Sigmodon hispidus), and cotton mouse (Peromyscus gossypinus) in Georgia and Florida, belonged to Borrelia burgdorferi sensu stricto. Seven strains, isolated from Ixodes minor, the wood rat (Neotoma floridana), the cotton rat, and the cotton mouse in South Carolina and Florida, belonged to Borrelia bissettii. Two strains, MI-8 from Florida and TXW-1 from Texas, exhibited MseI and DraI restriction patterns different from those of previously reported genospecies. Eight Missouri tick strains (MOK-3a group) had MseI patterns similar to that of B. andersonii reference strain 21038 but had a DraI restriction site in the spacer. Strain SCGT-8a had DraI restriction patterns identical to that of strain 25015 (B. bissettii) but differed from strain 25015 in its MseI restriction pattern. Strain AI-1 had the same DraI pattern as other southern strains in the B. bissettii genospecies but had a distinct MseI profile. The taxonomic status of these atypical strains needs to be further evaluated. To clarify the taxonomic positions of these atypical Borrelia strains, the complete sequences of

  5. 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

  6. The role of Rhipicephalus sanguineus sensu lato saliva in the dissemination of Rickettsia conorii in C3H/HeJ mice.

    PubMed

    Milhano, N; Saito, T B; Bechelli, J; Fang, R; Vilhena, M; DE Sousa, R; Walker, D H

    2015-09-01

    Animal models have been developed for the study of rickettsial pathogenesis. However, to understand what occurs during the natural route of rickettsial transmission via the tick bite, the role of tick saliva should be considered in these models. To address this, we analysed the role of tick saliva in the transmission of Rickettsia conorii (Rickettsiales: Rickettsiaceae) in a murine host by intradermally (i.d.) inoculating two groups of susceptible C3H/HeJ mice with this Rickettsia, and infesting one group with nymphal Rhipicephalus sanguineus sensu lato (Ixodida: Ixodidae) ticks. Quantification of bacterial loads and mRNA levels of interleukin-1β (IL-1β), IL-10 and NF-κB was performed in C3H/HeJ lung samples by real-time quantitative polymerase chain reaction (PCR) and real-time reverse transcriptase PCR, respectively. Lung histology was examined to evaluate the pathological manifestations of infection. No statistically significant difference in bacterial load in the lungs of mice was observed between these two groups; however, a statistically significant difference was observed in levels of IL-1β and NF-κB, both of which were higher in the group inoculated with rickettsiae but not infected with ticks. Lung histology in both groups of animals revealed infiltration of inflammatory cells. Overall, this study showed that i.d. inoculation of R. conorii caused infection in the lungs of C3H/HeJ mice and tick saliva inhibited proinflammatory effects.

  7. 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.

  8. 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.

  9. Draft Genome Sequences of Human Pathogenic Fungus Geomyces pannorum Sensu Lato and Bat White Nose Syndrome Pathogen Geomyces (Pseudogymnoascus) destructans

    PubMed Central

    Crabtree, Jonathan; Nagaraj, Sushma; Chaturvedi, Sudha

    2013-01-01

    We report the draft genome sequences of Geomyces pannorum sensu lato and Geomyces (Pseudogymnoascus) destructans. G. pannorum has a larger proteome than G. destructans, containing more proteins with ascribed enzymatic functions. This dichotomy in the genomes of related psychrophilic fungi is a valuable target for defining their distinct saprobic and pathogenic attributes. PMID:24356829

  10. Draft Genome Sequences of Human Pathogenic Fungus Geomyces pannorum Sensu Lato and Bat White Nose Syndrome Pathogen Geomyces (Pseudogymnoascus) destructans.

    PubMed

    Chibucos, Marcus C; Crabtree, Jonathan; Nagaraj, Sushma; Chaturvedi, Sudha; Chaturvedi, Vishnu

    2013-01-01

    We report the draft genome sequences of Geomyces pannorum sensu lato and Geomyces (Pseudogymnoascus) destructans. G. pannorum has a larger proteome than G. destructans, containing more proteins with ascribed enzymatic functions. This dichotomy in the genomes of related psychrophilic fungi is a valuable target for defining their distinct saprobic and pathogenic attributes. PMID:24356829

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

    PubMed Central

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

    2015-01-01

    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. PMID:25888168

  12. 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

  13. 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. PMID:26100492

  14. 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. PMID:25661649

  15. Seroepidemiology of Borrelia burgdorferi sensu lato and Anaplasma phagocytophilum in wild mice captured in northern Turkey.

    PubMed

    Güner, E S; Watanabe, M; Kadosaka, T; Polat, E; Gargili, A; Gulanber, A; Ohashi, N; Kaneda, K; Imai, Y; Masuzawa, T

    2005-04-01

    An expedition across the Asian part of the Black Sea coast and national parks of Northern Turkey was organized in the summer of 2001 to investigate the presence of Borrelia burgdorferi sensu lato (s.l.), Lyme borreliosis agent, and Anaplasma phagocytophilum, human granulocytic ehrlichiosis, agent, in wild mice. A total of 65 Apodemus flavicollis, Apodemus sylvaticus, Microtus epiroticus, Crocidura suaveolens and Mus macedonicus, were captured. Two out of 22 Apodemus sylvaticus specimens were seropositive for B. afzelii by enzyme-linked immunosorbent assay as confirmed by Western blotting, however cultures of skin and bladder samples from all small mammals in Barbour-Stoenner-Kelly's medium-II remained negative for B. burgdorferi s.l. All sera tested were negative for Anaplasma phagocytophilum by indirect immunofluorescent assay. The prevalence of B. burgdorferi s.l. and Anaplasma phagocytophilum is low in wild mice of the Asian part of Northern Turkey.

  16. A taxonomic revision of the Neoserica (sensu lato) calva group (Coleoptera, Scarabaeidae, Sericini)

    PubMed Central

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

    2014-01-01

    Abstract The species of the Neoserica (sensu lato) calva group are revised. Neoserica calva Frey, 1972, comb. n. is redescribed. Thirteen new species are described from China and South Korea: Neoserica ailaoshanica sp. n., Neoserica anonyma sp. n., Neoserica calvoides sp. n., Neoserica gulinqingensis sp. n., Neoserica koelkebecki sp. n., Neoserica liangi sp. n., Neoserica luxiensis sp. n., Neoserica menghaiensis sp. n., Neoserica mengi sp. n., Neoserica taipingensis sp. n., Neoserica zheijangensis sp. n., Neoserica zhibenshanica sp. n., and Neoserica zongyuani sp. n. A key to Sericini genera with multilamellate antenna and species groups of Neoserica of mainland Asia as well as a key to species of the Neoserica calva group are provided. A map of species distribution is given, habitus and male genitalia are illustrated. PMID:25408610

  17. 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". PMID:26790741

  18. 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

  19. Immunochemical analysis of lipopolysaccharide-like component extracted from Borrelia burgdorferi sensu lato.

    PubMed

    Schwarzová, K; Ciznár, I

    2004-01-01

    Immunoelectrophoresis and its modifications were applied to analysis of a lipopolysaccharide-like component (LPS-LC) extracted from Borrelia garinii strains K24 and K48 isolated from Ixodes ricinus and Borrelia burgdorferi sensu stricto strain B31. A modification of the hot phenol-water method was used for isolation of LPS. Immunoelectrophoresis (IE) and crossed immunoelectrophoresis (CIE) of LPS-LC with polyclonal rabbit antisera revealed a pattern and properties partially similar to LPS from other Gram-negative bacteria. B. garinii LPS-LC formed in CIE a diffuse band extending from the start to the anode. Similarly, the shape and position of the band in IE did not show major differences from LPS of other Gram-negative bacteria. The LPS-LC extracted from the three genomic groups of B. burgdorferi sensu lato were found to have similar immunochemical properties irrespective of their genotype origin.

  20. 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".

  1. [Heterogeneity of the gene P83/100 of Borrelia borrelia burgdorferi sensu lato complex].

    PubMed

    Fomenko, N V; Sabitova, Iu B; Khasnatinov, M A; Gol'tsova, N A; Danchinova, G A; Bataa, Zh; Ambed, D; Stronin, O V

    2007-01-01

    The 35 full-length Borrelia burgdorferi sensu lato complex a83/100 gene nucleotide sequences were determined. High level of homology was observed in the nucleotide sequences corresponding to the strains and isolates of Borrelia fzelii. The analysis of the nucleotide sequences revealed two groups of Borrelia garinii. The most variable p83/100 gene region containing species-typical insertions and deletions was demonstrated to be included into the region where the antigenic determinants of protein were encoded. According to the data obtained in this work, the modification of the P83/100 protein structure and immunological properties could be suggested to exist even within species. The results of this work could be used for receiving recombinant P83/100 proteins useful for diagnostic applications.

  2. Discrimination of Mycobacterium abscessus subsp. massiliense from Mycobacterium abscessus subsp. abscessus in clinical isolates by multiplex PCR.

    PubMed

    Nakanaga, Kazue; Sekizuka, Tsuyoshi; Fukano, Hanako; Sakakibara, Yumi; Takeuchi, Fumihiko; Wada, Shinpei; Ishii, Norihisa; Makino, Masahiko; Kuroda, Makoto; Hoshino, Yoshihiko

    2014-01-01

    The rapidly growing mycobacterium M. abscessus sensu lato is the causative agent of emerging pulmonary and skin diseases and of infections following cosmetic surgery and postsurgical procedures. M. abscessus sensu lato can be divided into at least three subspecies: M. abscessus subsp. abscessus, M. abscessus subsp. massiliense, and M. abscessus subsp. bolletii. Clinical isolates of rapidly growing mycobacteria were previously identified as M. abscessus by DNA-DNA hybridization. More than 30% of these 117 clinical isolates were differentiated as M. abscessus subsp. massiliense using combinations of multilocus genotyping analyses. A much more cost-effective technique to distinguish M. abscessus subsp. massiliense from M. abscessus subsp. abscessus, a multiplex PCR assay, was developed using the whole-genome sequence of M. abscessus subsp. massiliense JCM15300 as a reference. Several primer sets were designed for single PCR to discriminate between the strains based on amplicons of different sizes. Two of these single-PCR target sites were chosen for development of the multiplex PCR assay. Multiplex PCR was successful in distinguishing clinical isolates of M. abscessus subsp. massiliense from samples previously identified as M. abscessus. This approach, which spans whole-genome sequencing and clinical diagnosis, will facilitate the acquisition of more-precise information about bacterial genomes, aid in the choice of more relevant therapies, and promote the advancement of novel discrimination and differential diagnostic assays. PMID:24197885

  3. Occurrence of Bartonella henselae and Borrelia burgdorferi sensu lato co-infections in ticks collected from humans in Germany.

    PubMed

    Mietze, A; Strube, C; Beyerbach, M; Schnieder, T; Goethe, R

    2011-06-01

    Bartonella (B.) henselae is the zoonotic agent of cat scratch disease. B. henselae has been associated with therapy-resistant Lyme disease in humans suggesting that B. henselae and Borrelia burgdorferi sensu lato might be transmitted concurrently by ticks. In the present study we found that 16 (6.9%) of 230 Ixodes ricinus collected from humans harboured DNA of Bartonella spp. Fifteen positive ticks were infected with B. henselae and one tick with B. clarridgeiae. Twenty-five percent of the 16 Bartonella positive ticks were co-infected with Borrelia burgdorferi sensu lato. Our data show that B. henselae is present in Ixodes ricinus and that ticks may serve as source of infection for humans.

  4. Reverse-transcription PCR (RT-PCR).

    PubMed

    Bachman, Julia

    2013-01-01

    RT-PCR is commonly used to test for genetic diseases and to characterize gene expression in various tissue types, cell types, and over developmental time courses. This serves as a form of expression profiling, but typically as a candidate approach. RT-PCR is also commonly used to clone cDNAs for further use with other molecular biology techniques (e.g., see Oligo(dT)-primed RT-PCR isolation of polyadenylated RNA degradation intermediates and Circularized RT-PCR (cRT-PCR): analysis of RNA 5' ends, 3' ends, and poly(A) tails).

  5. 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.

  6. 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.

  7. 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.

  8. First detection of Borrelia burgdorferi sensu lato DNA in king penguins (Aptenodytes patagonicus halli).

    PubMed

    Schramm, Frédéric; Gauthier-Clerc, Michel; Fournier, Jean-Charles; McCoy, Karen D; Barthel, Cathy; Postic, Danièle; Handrich, Yves; Le Maho, Yvon; Jaulhac, Benoît

    2014-10-01

    The hard tick Ixodes uriae parasitises a wide range of seabird species in the circumpolar areas of both Northern and Southern hemispheres and has been shown to be infected with Borrelia burgdorferi sensu lato, the bacterial agents of Lyme borreliosis. Although it is assumed that seabirds represent viable reservoir hosts, direct demonstrations of infection are limited to a single study from the Northern hemisphere. Here, the blood of 50 tick-infested adult king penguins (Aptenodytes patagonicus halli) breeding in the Crozet Archipelago (Southern Indian Ocean) was examined for B. burgdorferi sl exposure by serology and for spirochetemia by in vitro DNA amplification. Four birds were found positive by serology, whereas B. burgdorferi sl DNA was detected in two other birds. Our data therefore provide the first direct proof of Borrelia burgdorferi sl spirochetes in seabirds of the Southern hemisphere and indicate a possible reservoir role for king penguins in the natural maintenance of this bacterium. Although the bacterial genetic diversity present in these hosts and the infectious period for tick vectors remain to be elucidated, our results add to a growing body of knowledge on the contribution of seabirds to the complex epizootiology of Lyme disease and the global dissemination of B. burgdorferi sl spirochetes. PMID:25150726

  9. Comparative exine ontogeny in some members of the family Zygophyllaceae sensu lato.

    PubMed

    Nasri-Ayachi, M Ben; Nabli, M A

    2006-08-01

    Exine ontogeny is studied in five taxa of the family Zygophyllaceae sensu lato (Peganum harmala L., Zygophyllum album L., Fagonia cretica L., Tribulus terrestris L., and Nitraria retusa [Forsk.] Asch.). In the beginning of the tetrad stage, the plasmalemma is attached to the callose wall, except in T. terrestris, where it describes crests and hollows. The primexine matrix is fibrillar, bilayered in T. terrestris and unilayered in the other taxa. In all species except P. harmala, the procolumellae are heterogeneous with clear zones and they become compact later. In T. terrestris, they are built on the crests. In Z. album and T. terrestris, a primordial nexinic lamella is set up. It is tripartite with a white line seen at some levels; on its external leaflet, the foot layer is observed, and on its internal leaflet, there is the endexine with numerous lamellae. This white line disappears often in the mature exine. In T. terrestris, there is a thick nexine that is coarsely lamellate inside. In the aperture zone, the columellae are lacking, the tectum and the foot layer get thinner; they unite and form the apertural membrane with the external part of the endexine. There is a granulolamellar endexinic zone well developed in P. harmala, whereas it is threelayered and weakly developed in T. terrestris. PMID:16937054

  10. Quest to elucidate the life cycle of Puccinia psidii sensu lato.

    PubMed

    Morin, Louise; Talbot, Mark J; Glen, Morag

    2014-02-01

    There is controversy surrounding the described life cycle of the rust fungus Puccinia psidii sensu lato, which causes disease on several plant species in the family Myrtaceae. The objective of this study was to determine whether P. psidii s.l. is autoecious by performing basidiospore inoculations, and microscopically examining the fate of basidiospores on the leaf surface and nuclear condition at different stages of rust development. No spermogonia developed on leaves of Agonis flexuosa inoculated either with a teliospore suspension or basidiospores naturally discharged from telia. Uredinial sori that developed in all three inoculations with teliospore suspensions and in one of the five inoculations with naturally-discharged basidiospores from telia were most likely the result of urediniospore infections. Microsatellite analysis revealed that isolates made from these uredinial sori had the same multilocus genotype as that of the original isolate. No signs of penetration of plant cells by basidiospores were observed on A. flexuosa and Syzygium jambos. The nuclear condition of mycelia of uredinial sori, urediniospores, teliospores, and four-celled metabasidia was typical of that in many rust fungi. Our study could not provide unequivocal proof that P. psidii s.l. is autoecious. While it is possible that it could be heteroecious, with an unknown alternate aecial host, it is also possible that basidiospores have lost the ability to infect Myrtaceae or are infrequently operational. PMID:24528646

  11. Seroprevalence of Leptospira spp. and Borrelia burgdorferi sensu lato in Italian horses.

    PubMed

    Ebani, Valentina V; Bertelloni, Fabrizio; Pinzauti, Paolo; Cerri, Domenico

    2012-01-01

    The aim of the study was to determine the seroprevalence of Leptospira spp. and Borrelia burgdorferi sensu lato in healthy horses living in 7 provinces of central Italy. In the period 2007-2009, sera from 386 horses were tested by microagglutination test (MAT) to detect antibodies to Leptospira spp., employing the following serovars as antigens: Bratislava, Ballum, Canicola, Icterohaemorrhagiae, Grippotyphosa, Hardjo, Pomona, Tarassovi. 3 animals were positive for the serovars Icterohaemorrhagiae, 2 to Bratislava, and 1 to Pomona, for a total 1.5% seroprevalence. All sera were examined by immunofluorence antibody test (IFAT) to reveal anti-B. burgdorferi s.l. antibodies. 94 (24.3%) horses were positive with antibody titres ranging from 1:64 to 1:1,024. The seroprevalence was significantly higher in >10 year-old horses compared to younger subjects. No significant differences in the mean seroprevalence were observed in the respective years. The total mean seroprevalence were strictly related to the environmental conditions of the areas in which the horses lived. No cross-reactions between Leptospira and Borrelia were observed. This is the first serological survey on antibodies to B. burgdorferi s.l. in Italian horses. PMID:22742794

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

    PubMed

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

    2014-01-01

    The present paper revises the species belonging to the Neoserica (sensu lato) abnormis group, so far known only with two nominal species. Twenty new species are herein described from Indochina and southern China: N. abnormoides sp. n. (Vietnam, China), N. allolaotica sp. n., N. namthaensis sp. n., N. simplicissima sp. n. (Laos), N. thailandensis sp. n. (Thailand), N. alloputaoana sp. n., N. kanphantensis sp. n., N. natmatoungensis sp. n., N. putaoana sp. n., N. taunggyiana sp. n. (Myanmar), N. lamellosa sp. n., N. tonkinea sp. n. (Vietnam), N. bairailingshanica sp. n., N. euyunnanica sp. n., N. huangi sp. n., N. jiangxiensis sp. n., N. trifida sp. n., N. yaoi sp. n., N. yingjiangensis sp. n. (China), N. cardamomensis sp. n. (Indochina and southern China). One new combination is established: Neoserica ponderosa Arrow, 1946, comb. n. The lectotypes of Neoserica abnormis Moser, 1908 and the taxonomically uncertain N. inclinata Brenske, 1898, which very likely also belongs to this species group, are designated herein. A key to the species and to species groups is given, the genitalia of all species including their habitus are illustrated. Maps of species distribution are included. PMID:25317056

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

    PubMed Central

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

    2014-01-01

    Abstract The present paper revises the species belonging to the Neoserica (sensu lato) abnormis group, so far known only with two nominal species. Twenty new species are herein described from Indochina and southern China: N. abnormoides sp. n. (Vietnam, China), N. allolaotica sp. n., N. namthaensis sp. n., N. simplicissima sp. n. (Laos), N. thailandensis sp. n. (Thailand), N. alloputaoana sp. n., N. kanphantensis sp. n., N. natmatoungensis sp. n., N. putaoana sp. n., N. taunggyiana sp. n. (Myanmar), N. lamellosa sp. n., N. tonkinea sp. n. (Vietnam), N. bairailingshanica sp. n., N. euyunnanica sp. n., N. huangi sp. n., N. jiangxiensis sp. n., N. trifida sp. n., N. yaoi sp. n., N. yingjiangensis sp. n. (China), N. cardamomensis sp. n. (Indochina and southern China). One new combination is established: Neoserica ponderosa Arrow, 1946, comb. n. The lectotypes of Neoserica abnormis Moser, 1908 and the taxonomically uncertain N. inclinata Brenske, 1898, which very likely also belongs to this species group, are designated herein. A key to the species and to species groups is given, the genitalia of all species including their habitus are illustrated. Maps of species distribution are included. PMID:25317056

  14. 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.

  15. Spatial and seasonal variation in the prevalence of Anaplasma phagocytophilum and Borrelia burgdorferi sensu lato in questing Ixodes ricinus ticks in Norway

    PubMed Central

    2013-01-01

    Background Understanding the variation in prevalence of Borrelia burgdorferi sensu lato (Lyme Borreliosis Spirochaetes, LBS) and Anaplasma phagocytophilum (causing tick-borne fever in ruminants and human granulocytic ehrlichiosis) in ticks is vital from both a human and an animal disease perspective to target the most effective mitigation measures. From the host competence hypothesis, we predicted that prevalence of LBS would decrease with red deer density, while prevalence of A. phagocytophilum would increase. Methods Based on a sample of 112 adult and 686 nymphal Ixodes ricinus ticks collected with flagging during questing from 31 transects (4–500 m long) corresponding to individual seasonal home ranges of 41 red deer along the west coast of Norway, we tested whether there were spatial and seasonal variations in prevalence with a special emphasis on the population density of the most common large host in this area, the red deer (Cervus elaphus). We used a multiplex real-time PCR assay for detection of A. phagocytophilum and LBS. Results Prevalence of LBS was higher in adult female ticks (21.6%) compared to adult male ticks (11.5%) and nymphs (10.9%), while prevalence was similar among stages for prevalence of A. phagocytophilum (8.8%). Only partly consistent with predictions, we found a lower prevalence of LBS in areas of high red deer density, while there was no relationship between red deer density and prevalence of A. phagocytophilum in ticks. Prevalence of both bacteria was much higher in ticks questing in May compared to August. Conclusions Our study provides support to the notion that spatial variation in host composition forms a role for prevalence of LBS in ticks also in a northern European ecosystem, while no such association was found for A. phagocytophilum. Further studies are needed to fully understand the similar seasonal pattern of prevalence of the two pathogens. PMID:23786850

  16. 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…

  17. 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.

  18. Risk indicators for the tick Ixodes ricinus and Borrelia burgdorferi sensu lato in Sweden.

    PubMed

    Jaenson, T G T; Eisen, L; Comstedt, P; Mejlon, H A; Lindgren, E; Bergström, S; Olsen, B

    2009-09-01

    The distributional area of the tick Ixodes ricinus (L.), the primary European vector to humans of Lyme borreliosis spirochaetes (Borrelia burgdorferi sensu lato) and tick-borne encephalitis virus, appears to be increasing in Sweden. It is therefore important to determine which environmental factors are most useful to assess risk of human exposure to this tick and its associated pathogens. The geographical distribution of I. ricinus in Sweden was analysed with respect to vegetation zones and climate. The northern limit of I. ricinus and B. burgdorferi s.l. in Sweden corresponds roughly to the northern limit of the southern boreal vegetation zone, and is characterized climatically by snow cover for a mean duration of 150 days and a vegetation period averaging 170 days. The zoogeographical distribution of I. ricinus in Sweden can be classified as southerly-central, with the centre of the distribution south of the Limes Norrlandicus. Ixodes ricinus nymphs from 13 localities in different parts of Sweden were examined for the presence of B. burgdorferi s.l. and found to be infected with Borrelia afzelii and Borrelia garinii. Tick sampling localities were characterized on the basis of the density of Borrelia-infected I. ricinus nymphs, presence of specific mammals, dominant vegetation and climate. Densities of I. ricinus nymphs and Borrelia-infected nymphs were significantly correlated, and nymphal density can thus serve as a general indicator of risk for exposure to Lyme borreliosis spirochaetes. Analysis of data from this and other studies suggests that high densities of Borrelia-infected nymphs typically occur in coastal, broadleaf vegetation and in mixed deciduous/spruce vegetation in southern Sweden. Ixodes ricinus populations consistently infected with B. burgdorferi s.l. can occur in: (a) biotopes with shrews, rodents, hares and birds; (b) biotopes with shrews, rodents, hares, deer and birds, and (c) island locations where the varying hare (Lepus timidus) is the

  19. Genome size variation and incidence of polyploidy in Scrophulariaceae sensu lato from the Iberian Peninsula

    PubMed Central

    Castro, Mariana; Castro, Sílvia; Loureiro, João

    2012-01-01

    Background and aims In the last decade, genomic studies using DNA markers have strongly influenced the current phylogeny of angiosperms. Genome size and ploidy level have contributed to this discussion, being considered important characters in biosystematics, ecology and population biology. Despite the recent increase in studies related to genome size evolution and polyploidy incidence, only a few are available for Scrophulariaceae. In this context, we assessed the value of genome size, mostly as a taxonomic marker, and the role of polyploidy as a process of genesis and maintenance of plant diversity in Scrophulariaceae sensu lato in the Iberian Peninsula. Methodology Large-scale analyses of genome size and ploidy-level variation across the Iberian Peninsula were performed using flow cytometry. One hundred and sixty-two populations of 59 distinct taxa were analysed. A bibliographic review on chromosome counts was also performed. Principal results From the 59 sampled taxa, 51 represent first estimates of genome size. The majority of the Scrophulariaceae species presented very small to small genome sizes (2C ≤ 7.0 pg). Furthermore, in most of the analysed genera it was possible to use this character to separate several taxa, independently if these genera were homoploid or heteroploid. Also, some genome-related phenomena were detected, such as intraspecific variation of genome size in some genera and the possible occurrence of dysploidy in Verbascum spp. With respect to polyploidy, despite a few new DNA ploidy levels having been detected in Veronica, no multiple cytotypes have been found in any taxa. Conclusions This work contributed with important basic scientific knowledge on genome size and polyploid incidence in the Scrophulariaceae, providing important background information for subsequent studies, with several perspectives for future studies being opened. PMID:23240073

  20. Phylogeography of Rhipicephalus sanguineus sensu lato and its relationships with climatic factors.

    PubMed

    Zemtsova, Galina E; Apanaskevich, Dmitry A; Reeves, Will K; Hahn, Micah; Snellgrove, Alyssa; Levin, Michael L

    2016-06-01

    Brown dog ticks morphologically identifiable as Rhipicephalus sanguineus sensu lato, are distributed world-wide and their systematics is controversial. Results of genetic and reproductive compatibility studies of geographically distinct populations of R. sanguineus s.l. indicate that the R. sanguineus complex is paraphyletic. To further elucidate systematic relationships within R. sanguineus s.l. and geographic boundaries of its lineages, we conducted a phylogeographical study of 136 tick specimens from 23 countries. Voucher specimens were morphologically identified. A phylogenetic tree was constructed using concatenated partial mitochondrial 12S and 16S rDNA gene sequences and analyzed by the Neighbor-Joining method. A set of 19 bioclimatic variables within the WorldClim dataset were extracted and analyzed to assess correlations between distribution of R. sanguineus s.l. lineages and climatic variables. The following four branches are clearly recognized on the phylogenetic tree: R. sanguineus s.l.-tropical and temperate clades, R. leporis, and R. turanicus. DNA sequences of Rhipicephalus ticks from Israel differ from those of other groups. Strong association between geographical locations of major clades of R. sanguineus s.l. and temperature was identified. The tropical clade of R. sanguineus s.l. occupies areas with the annual mean temperature >20 °C, whereas the temperate clade is present in areas with the annual mean temperature <20 °C. Our results indicate that ticks in two closely related phylogenetic clades are adapted to different environmental conditions and support proposals for re-classification of R. sanguineus complex. Differences in R. sanguineus s.l. ecology and human/animal pathogens transmitted by different taxa of brown dog tick need to be studied. PMID:27003273

  1. Borrelia burgdorferi sensu lato infection pressure shapes innate immune gene evolution in natural rodent populations across Europe

    PubMed Central

    Tschirren, Barbara

    2015-01-01

    Although parasite-mediated selection is assumed to be the main driver of immune gene evolution, empirical evidence that parasites induce allele frequency changes at host immune genes in time and/or space remains scarce. Here, I show that the frequency of a protective gene variant of the innate immune receptor Toll-like receptor 2 in natural bank vole (Myodes glareolus) populations is positively associated with the strength of Borrelia burgdorferi sensu lato infection risk across the European continent. Thereby, this study provides rare evidence for the role of spatially variable infection pressures in moulding the vertebrate immune system. PMID:26018834

  2. Comparison of different strains of Borrelia burgdorferi sensu lato used as antigens in enzyme-linked immunosorbent assays.

    PubMed Central

    Magnarelli, L A; Anderson, J F; Johnson, R C; Nadelman, R B; Wormser, G P

    1994-01-01

    Eight strains of Borrelia burgdorferi sensu lato were tested with serum samples from persons who had Lyme borreliosis or syphilis in class-specific enzyme-linked immunosorbent assays (ELISAs). Antigens of B. burgdorferi sensu stricto, of Borrelia garinii, and of Borrelia spirochetes in group VS461 were prepared from cultured bacteria isolated from ticks, a white-footed mouse (Peromyscus leucopus), or human tissues in North America, the former Soviet Union, and Japan. Nearly all of the serum specimens that contained immunoglobulins to strain 2591, a Connecticut isolate, were also positive in antibody tests with the other seven strains. In general, all eight strains reacted similarly and were suitable as coating antigens in class-specific ELISAs. Assay sensitivities ranged from 82.6 to 100% in analyses for immunoglobulin M and G antibodies. Compared with reference antigen strain 2591, strains 231 (a tick isolate from Canada) and NCH-1 (a human skin isolate from Wisconsin) resulted in higher antibody titers in an ELISA. Syphilitic sera cross-reacted in all tests regardless of the antigen used. Key immunodominant proteins are shared among the closely related strains of B. burgdorferi sensu lato tested, but it is suspected that variations in antigen compositions among these spirochetes may sometimes affect assay performance for detecting serum antibodies. PMID:8051239

  3. 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.

  4. Echinococcus granulosus sensu lato genotypes infecting humans--review of current knowledge.

    PubMed

    Alvarez Rojas, Cristian A; Romig, Thomas; Lightowlers, Marshall W

    2014-01-01

    Genetic variability in the species group Echinococcus granulosus sensu lato is well recognised as affecting intermediate host susceptibility and other biological features of the parasites. Molecular methods have allowed discrimination of different genotypes (G1-10 and the 'lion strain'), some of which are now considered separate species. An accumulation of genotypic analyses undertaken on parasite isolates from human cases of cystic echinococcosis provides the basis upon which an assessment is made here of the relative contribution of the different genotypes to human disease. The allocation of samples to G-numbers becomes increasingly difficult, because much more variability than previously recognised exists in the genotypic clusters G1-3 (=E. granulosus sensu stricto) and G6-10 (Echinococcus canadensis). To accommodate the heterogeneous criteria used for genotyping in the literature, we restrict ourselves to differentiate between E. granulosus sensu stricto (G1-3), Echinococcus equinus (G4), Echinococcus ortleppi (G5) and E. canadensis (G6-7, G8, G10). The genotype G1 is responsible for the great majority of human cystic echinococcosis worldwide (88.44%), has the most cosmopolitan distribution and is often associated with transmission via sheep as intermediate hosts. The closely related genotypes G6 and G7 cause a significant number of human infections (11.07%). The genotype G6 was found to be responsible for 7.34% of infections worldwide. This strain is known from Africa and Asia, where it is transmitted mainly by camels (and goats), and South America, where it appears to be mainly transmitted by goats. The G7 genotype has been responsible for 3.73% of human cases of cystic echinococcosis in eastern European countries, where the parasite is transmitted by pigs. Some of the samples (11) could not be identified with a single specific genotype belonging to E. canadensis (G6/10). Rare cases of human cystic echinococcosis have been identified as having been caused by

  5. 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

  6. 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

    2015-08-01

    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. PMID:26185097

  7. 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 Central

    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

    2015-01-01

    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. PMID:26185097

  8. 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.

  9. Wild turkey (Meleagris gallopavo) as a host of ixodid ticks, lice, and Lyme disease spirochetes (Borrelia burgdorferi sensu lato) in California state parks.

    PubMed

    Lane, Robert S; Kucera, Thomas F; Barrett, Reginald H; Mun, Jeomhee; Wu, Chunling; Smith, Vincent S

    2006-10-01

    Rio Grande wild turkeys (Meleagris gallopavo intermedia) were evaluated as potential hosts of ixodid ticks, lice, and Lyme disease spirochetes (Borrelia burgdorferi sensu lato [s.l.]) in three state parks in Sonoma County, California, USA, during 2003 and 2004. In total, 113 birds were collected, 50 (44.2%) of which were found to be infested by 361 ixodid ticks representing three species: the western black-legged tick (Ixodes pacificus, n=248), the rabbit tick (Haemaphysalis leporispalustris, n=112), and one American dog tick (Dermacentor variabilis). Year-round the prevalence of all ticks combined was unrelated to the age or sex of turkeys, and the prevalence of infestation by I. pacificus (35.4%) was significantly higher than it was for either H. leporispalustris (14.2%) or D. variabilis (0.9%). The proportion of the two prevalent tick species differed significantly by life stage with 86.3% of the I. pacificus and 82.1% of the H. leporispalustris enumerated being nymphs and larvae, respectively. Three species of lice were collected, including the chicken body louse Menacanthus stramineus (12.5% of total), Chelopistes meleagridis (37.5% of total), and Oxylipeurus polytrapezius (50% of total). The records for all three ticks are the first ever from wild turkeys, and those for the lice are the first from this host in the far-western United States. Wild turkeys potentially were exposed to the feeding activities of I. pacificus nymphs infected with B. burgdorferi s.l. as 15% of host-seeking nymphs (n=200) collected in woodlands used by turkeys as roosting or foraging areas were infected mainly with B. burgdorferi sensu stricto (s.s.). However, only one (1%) of 90 turkey blood specimens tested by PCR contained B. burgdorferi s.s., and four in vitro, complement-protein assays demonstrated that domestic turkey serum is moderately bacteriolytic for this spirochete. Taken together, these findings indicate that wild turkeys are important avian hosts of I. pacificus nymphs

  10. Isolation and identification of Pseudomonas syringae facilitated by a PCR targeting the whole P. syringae group.

    PubMed

    Guilbaud, Caroline; Morris, Cindy E; Barakat, Mohamed; Ortet, Philippe; Berge, Odile

    2016-01-01

    We present a reliable PCR-based method to avoid the biases related to identification based on the conventional phenotypes currently used in the identification of Pseudomonas syringae sensu lato, a ubiquitous environmental bacterium including plant pathogens. We identified a DNA target suitable for this purpose by applying a comparative genomic pipeline to Pseudomonas genomes. We designed primers and developed PCR conditions that led to a clean and strong PCR product from 97% of the 185 strains of P. syringae strains tested and gave a clear negative result for the 31 non-P. syringae strains tested. The sensitivity of standard PCR was determined with pure strains to be 10(6) bacteria mL(-1) or 0.4 ng of DNA μL(-1). Sensitivity could be improved with the touchdown method. The new PCR-assisted isolation of P. syringae was efficient when deployed on an environmental sample of river water as compared to the isolation based on phenotypes. This innovation eliminates the need for extensive expertise in isolating P. syringae colonies, was simpler, faster and very reliable. It will facilitate discovery of more diversity of P. syringae and research on emergence, dispersion and evolution to understand the varied functions of this environmental bacterium. PMID:26610434

  11. 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. PMID:24794201

  12. 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.

  13. Predators of Anopheles gambiae sensu lato (Diptera: Culicidae) Larvae in Wetlands, Western Kenya: Confirmation by Polymerase Chain Reaction Method

    PubMed Central

    OHBA, SHIN-YA; KAWADA, HITOSHI; DIDA, GABRIEL O.; JUMA, DUNCAN; SONYE, GORGE; MINAKAWA, NOBORU; TAKAGI, MASAHIRO

    2010-01-01

    Polymerase chain reaction analysis was performed to determine whether mosquito predators in wetland habitats feed on Anopheles gambiae sensu lato (s.l.) larvae. Aquatic mosquito predators were collected from six wetlands near Lake Victoria in Mbita, Western Kenya. This study revealed that the whole positive rate of An. gambiae s.l. from 330 predators was 54.2%. The order of positive rate was the highest in Odonata (70.2%), followed by Hemiptera (62.8%), Amphibia (41.7%), and Coleoptera (18%). This study demonstrates that the polymerase chain reaction method can determine whether aquatic mosquito predators feed on An. gambiae s.l. larvae if the predators have undigested An. gambiae s.l. in their midgut or stomach. PMID:20939371

  14. 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

  15. Sex determination using PCR.

    PubMed

    Kima, Peter E; Rasche, Madeline E

    2004-03-01

    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 distinguish between male and female samples and determine the gender of an unknown DNA donor. The exercise is presented for upper division undergraduate majors in microbiology, biochemistry, and molecular biology, but can be adapted to different academic levels and disciplines. The use of student samples in the exercise can enhance learning of these techniques by making PCR and agarose gel electrophoresis directly relevant to the students.

  16. In silico PCR analysis.

    PubMed

    Yu, Bing; Zhang, Changbin

    2011-01-01

    In silico PCR analysis is a useful and efficient complementary method to ensure primer specificity for an extensive range of PCR applications from gene discovery, molecular diagnosis, and pathogen detection to forensic DNA typing. In silico PCR, SNPCheck, and Primer-BLAST are commonly used web-based in silico PCR tools. Their applications are discussed here in stepwise detail along with several examples, which aim to make it easier for the intended users to apply the tools. This virtual PCR method can assist in the selection of newly designed primers, identify potential mismatches in the primer binding sites due to known SNPs, and avoid the amplification of unwanted amplicons so that potential problems can be prevented before any "wet bench" experiment.

  17. The Historical Speciation of Mauremys Sensu Lato: Ancestral Area Reconstruction and Interspecific Gene Flow Level Assessment Provide New Insights

    PubMed Central

    Zhou, Huaxing; Jiang, Yuan; Nie, Liuwang; Yin, Huazong; Li, Haifeng; Dong, Xianmei; Zhao, Feifei; Zhang, Huanhuan; Pu, Youguang; Huang, Zhenfeng; Song, Jiaolian; Sun, Entao

    2015-01-01

    Mauremys sensu lato was divided into Mauremys, Chinemys, Ocadia, and Annamemys based on earlier research on morphology. Phylogenetic research on this group has been controversial because of disagreements regarding taxonomy, and the historical speciation is still poorly understood. In this study, 32 individuals of eight species that are widely distributed in Eurasia were collected. The complete mitochondrial (mt) sequences of 14 individuals of eight species were sequenced. Phylogenetic relationships, interspecific divergence times, and ancestral area reconstructions were explored using mt genome data (10,854 bp). Subsequent interspecific gene flow level assessment was performed using five unlinked polymorphic microsatellite loci. The Bayesian and maximum likelihood analyses revealed a paraphyletic relationship among four old genera (Mauremys, Annamemys, Chinemys, and Ocadia) and suggested the four old genera should be merged into the genus (Mauremys). Ancestral area reconstruction and divergence time estimation suggested Southeast Asia may be the area of origin for the common ancestral species of this genus and genetic drift may have played a decisive role in species divergence due to the isolated event of a glacial age. However, M. japonica may have been speciated due to the creation of the island of Japan. The detection of extensive gene flow suggested no vicariance occurred between Asia and Southeast Asia. Inconsistent results between gene flow assessment and phylogenetic analysis revealed the hybrid origin of M. mutica (Southeast Asian). Here ancestral area reconstruction and interspecific gene flow level assessment were first used to explore species origins and evolution of Mauremys sensu lato, which provided new insights on this genus. PMID:26657158

  18. Correlation of Borrelia burgdorferi sensu lato prevalence in questing Ixodes ricinus ticks with specific abiotic traits in the western palearctic.

    PubMed

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

    2011-06-01

    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. Real-Time PCR

    NASA Astrophysics Data System (ADS)

    Evrard, A.; Boulle, N.; Lutfalla, G. S.

    Over the past few years there has been a considerable development of DNA amplification by polymerase chain reaction (PCR), and real-time PCR has now superseded conventional PCR techniques in many areas, e.g., the quantification of nucleic acids and genotyping. This new approach is based on the detection and quantification of a fluorescent signal proportional to the amount of amplicons generated by PCR. Real-time detection is achieved by coupling a thermocycler with a fluorimeter. This chapter discusses the general principles of quantitative real-time PCR, the different steps involved in implementing the technique, and some examples of applications in medicine. The polymerase chain reaction (PCR) provides a way of obtaining a large number of copies of a double-stranded DNA fragment of known sequence. This DNA amplification technique, developed in 1985 by K. Mullis (Cetus Corporation), saw a spectacular development over the space of a few years, revolutionising the methods used up to then in molecular biology. Indeed, PCR has many applications, such as the detection of small amounts of DNA, cloning, and quantitative analysis (assaying), each of which will be discussed further below.

  20. Prevalence of Borrelia burgdorferi Sensu Lato in Ticks from Eastern China

    PubMed Central

    Hou, Juan; Ling, Feng; Chai, Chengliang; Lu, Ye; Yu, Xianghua; Lin, Junfen; Sun, Jimin; Chang, Yue; Ye, Xiaodong; Gu, Shiping; Pang, Weilong; Wang, Chengwei; Zheng, Xiaohua; Jiang, Jianmin; Chen, Zhiping; Gong, Zhenyu

    2015-01-01

    To explore the tick distribution and prevalence of Borrelia in Zhejiang Province, we performed a survey in nine sites. A total of 447 adult ticks of 11 species were captured and the dominant tick species were Haemaphysalis longicornis and Ixodes sinensis and the abundance of tick species in different areas varied significantly. Overall, 4.70% of the ticks were polymerase chain reaction (PCR) positive for Borrelia. The average PCR positive rates were 5.19% for H. longicornis, 3.45% for Amblyomma testudinarium, 1.06% for I. sinensis, 5.00% for Rhipicephalus (Boophilus) microplus, and 19.44% for Ixodes granulatus, respectively. No Borrelia DNA was detected in Rhiphicephalus haemaphysaloides, Haemaphysalis yeni, Dermacentor taiwanensis, Haemaphysalis hystricis, Hyalomna asiaticum, and Ixodes ovatus. The prevalence of Borrelia was significantly different among tick species and the prevalence in I. granulatus was significantly higher than that in other tick species. Of note, experimentally confirmed vectors for B. burgdorferi s.l. including I. sinensis and I. granulatus were found in Zhejiang Province. Two species of B. burgdorferi s.l. exist in Zhejiang Province of which 12 sequences were most similar to the sequence of Borrelia garinii and nine sequences were most similar to the sequence of Borrelia valaisiana or Borrelia yangtze sp. nov. PMID:25548382

  1. 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

  2. Molecular characterization of Echinococcus granulosus sensu lato from farm animals in Egypt.

    PubMed

    Amer, Said; Helal, Ibrahim B; Kamau, Evelyne; Feng, Yaoyu; Xiao, Lihua

    2015-01-01

    Little is known on the diversity and public health significance of Echinococcus species in livestock in Egypt. In this study, 37 individual hydatid cysts were collected from dromedary camels (n=28), sheep (n=7) and buffalos (n=2). DNA was extracted from protoscoleces/germinal layer of individual cysts and amplified by PCR targeting nuclear (actin II) and mitochondrial (COX1 and NAD1) genes. Direct sequencing of amplicons indicated the presence of Echinococcus canadenesis (G6 genotype) in 26 of 28 camel cysts, 3 of 7 sheep cysts and the 2 buffalo derived cysts. In contrast, Echinococcus granulosus sensu stricto (G1 genotype) was detected in one cyst from a camel and 4 of 7 cysts from sheep, whereas Echinococcus ortleppi (G5 genotype) was detected in one cyst from a camel. This is the first identification of E. ortleppi in Egypt.

  3. Molecular Characterization of Echinococcus granulosus Sensu Lato from Farm Animals in Egypt

    PubMed Central

    Amer, Said; Helal, Ibrahim B.; Kamau, Evelyne; Feng, Yaoyu; Xiao, Lihua

    2015-01-01

    Little is known on the diversity and public health significance of Echinococcus species in livestock in Egypt. In this study, 37 individual hydatid cysts were collected from dromedary camels (n=28), sheep (n=7) and buffalos (n=2). DNA was extracted from protoscoleces/germinal layer of individual cysts and amplified by PCR targeting nuclear (actin II) and mitochondrial (COX1 and NAD1) genes. Direct sequencing of amplicons indicated the presence of Echinococcus canadenesis (G6 genotype) in 26 of 28 camel cysts, 3 of 7 sheep cysts and the 2 buffalo derived cysts. In contrast, Echinococcus granulosus sensu stricto (G1 genotype) was detected in one cyst from a camel and 4 of 7 cysts from sheep, whereas Echinococcus ortleppi (G5 genotype) was detected in one cyst from a camel. This is the first identification of E. ortleppi in Egypt. PMID:25760944

  4. Overlap extension PCR cloning.

    PubMed

    Bryksin, Anton; Matsumura, Ichiro

    2013-01-01

    Rising demand for recombinant proteins has motivated the development of efficient and reliable cloning methods. Here we show how a beginner can clone virtually any DNA insert into a plasmid of choice without the use of restriction endonucleases or T4 DNA ligase. Chimeric primers encoding plasmid sequence at the 5' ends and insert sequence at the 3' ends are designed and synthesized. Phusion(®) DNA polymerase is utilized to amplify the desired insert by PCR. The double-stranded product is subsequently employed as a pair of mega-primers in a PCR-like reaction with circular plasmids. The original plasmids are then destroyed in restriction digests with Dpn I. The product of the overlap extension PCR is used to transform competent Escherichia coli cells. Phusion(®) DNA polymerase is used for both the amplification and fusion reactions, so both steps can be monitored and optimized in the same way. PMID:23996437

  5. 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. PMID:25918210

  6. 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

  7. 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. PMID:26619925

  8. 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.

  9. Dental microwear in anubis and hybrid baboons (Papio hamadryas, sensu lato) living in Awash National Park, Ethiopia.

    PubMed

    Nystrom, Pia; Phillips-Conroy, Jane E; Jolly, Clifford J

    2004-11-01

    We describe dental microwear in baboons (Papio hamadryas sensu lato) from the anubis-hamadryas hybrid zone of Awash National Park, Ethiopia, outline its variation with sex and age, and attempt to relate the observed microwear pattern to environment and diet. Casts of the maxillary second molar of 52 adult and subadult individuals of both sexes were examined with a scanning electron microscope at x 500. Digitized micrographs were taken at a consistent location on facet 9, and microwear was recorded with an image analysis software package. Univariate and multivariate statistics were used to investigate the shape, size, and density of microwear features. The overall pattern of microwear exhibits an unusual combination of high feature density, with numerous small pits and relatively wide striations, and a high correlation between width of pits and striations across individuals. We interpret this pattern as predominantly the consequence of abrasion by relatively small-caliber environmental grit when accidentally ingested with tough foods such as dried seeds and fruits, as expected in a terrestrial omnivore living in a dusty habitat. Statistical analysis revealed no significant differences between groups defined by sex, age, or troop membership, a result consistent with qualitative observations of feeding habits in this population, and which lends no support to the hypothesis that the longer jaws of adult males should result in longer striations. A trend towards greater feature density in females, however, might be due to limited sexual dinichism, and merits further investigation.

  10. 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.

  11. Explanatory chapter: PCR primer design.

    PubMed

    Álvarez-Fernández, Rubén

    2013-01-01

    This chapter is intended as a guide on polymerase chain reaction (PCR) primer design (for information on PCR, see General PCR and Explanatory Chapter: Troubleshooting PCR). In the next section, general guidelines will be provided, followed by a discussion on primer design for specific applications. A list of recommended software tools is shown at the end.

  12. Mapping human risk of infection with Borrelia burgdorferi sensu lato, the agent of Lyme borreliosis, in a periurban forest in France.

    PubMed

    Vourc'h, G; Abrial, D; Bord, S; Jacquot, M; Masséglia, S; Poux, V; Pisanu, B; Bailly, X; Chapuis, J-L

    2016-07-01

    Lyme borreliosis is a major zoonosis in Europe, with estimates of over 26,000 cases per year in France alone. The etiological agents are spirochete bacteria that belong to the Borrelia burgdorferi sensu lato (s. l.) complex and are transmitted by hard ticks among a large range of vertebrate hosts. In Europe, the tick Ixodes ricinus is the main vector. In the absence of a vaccine and given the current difficulties to diagnose and treat chronic Lyme syndromes, there is urgent need for prevention. In this context, accurate information on the spatial patterns of risk of exposure to ticks is of prime importance for public health. The objective of our study was to provide a snapshot map of the risk of human infection with B. burgdorferi s. l. pathogens in a periurban forest at a high resolution, and to analyze the factors that contribute to variation in this risk. Field monitoring took place over three weeks in May 2011 in the suburban Sénart forest (3,200ha; southeast of Paris), which receives over 3 million people annually. We sampled ticks over the entire forest area (from 220 forest stands with a total area of 35,200m(2)) and quantified the density of questing nymphs (DON), the prevalence of infection among nymphs (NIP), and the density of infected nymphs (DIN), which is the most important predictor of the human risk of Lyme borreliosis. For each of these response variables, we explored the relative roles of weather (saturation deficit), hosts (abundance indices of ungulates and Tamias sibiricus, an introduced rodent species), vegetation and forest cover, superficial soil composition, and the distance to forest roads. In total, 19,546 questing nymphs were collected and the presence of B. burgdorferi s. l. was tested in 3,903 nymphs by qPCR. The mean DON was 5.6 nymphs per 10m(2) (standard deviation=10.4) with an average NIP of 10.1% (standard deviation=0.11). The highest DIN was 8.9 infected nymphs per 10m(2), with a mean of 0.59 (standard deviation=0.6). Our

  13. Mapping human risk of infection with Borrelia burgdorferi sensu lato, the agent of Lyme borreliosis, in a periurban forest in France.

    PubMed

    Vourc'h, G; Abrial, D; Bord, S; Jacquot, M; Masséglia, S; Poux, V; Pisanu, B; Bailly, X; Chapuis, J-L

    2016-07-01

    Lyme borreliosis is a major zoonosis in Europe, with estimates of over 26,000 cases per year in France alone. The etiological agents are spirochete bacteria that belong to the Borrelia burgdorferi sensu lato (s. l.) complex and are transmitted by hard ticks among a large range of vertebrate hosts. In Europe, the tick Ixodes ricinus is the main vector. In the absence of a vaccine and given the current difficulties to diagnose and treat chronic Lyme syndromes, there is urgent need for prevention. In this context, accurate information on the spatial patterns of risk of exposure to ticks is of prime importance for public health. The objective of our study was to provide a snapshot map of the risk of human infection with B. burgdorferi s. l. pathogens in a periurban forest at a high resolution, and to analyze the factors that contribute to variation in this risk. Field monitoring took place over three weeks in May 2011 in the suburban Sénart forest (3,200ha; southeast of Paris), which receives over 3 million people annually. We sampled ticks over the entire forest area (from 220 forest stands with a total area of 35,200m(2)) and quantified the density of questing nymphs (DON), the prevalence of infection among nymphs (NIP), and the density of infected nymphs (DIN), which is the most important predictor of the human risk of Lyme borreliosis. For each of these response variables, we explored the relative roles of weather (saturation deficit), hosts (abundance indices of ungulates and Tamias sibiricus, an introduced rodent species), vegetation and forest cover, superficial soil composition, and the distance to forest roads. In total, 19,546 questing nymphs were collected and the presence of B. burgdorferi s. l. was tested in 3,903 nymphs by qPCR. The mean DON was 5.6 nymphs per 10m(2) (standard deviation=10.4) with an average NIP of 10.1% (standard deviation=0.11). The highest DIN was 8.9 infected nymphs per 10m(2), with a mean of 0.59 (standard deviation=0.6). Our

  14. 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...

  15. 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...

  16. Echinococcus granulosus sensu lato GENOTYPES IN DOMESTIC LIVESTOCK AND HUMANS IN GOLESTAN PROVINCE, IRAN.

    PubMed

    Sharbatkhori, Mitra; Tanzifi, Asal; Rostami, Sima; Rostami, Masoomeh; Fasihi Harandi, Majid

    2016-01-01

    Cystic echinococcosis (CE) is a globally parasitic zoonosis caused by larval stages of Echinococcus granulosus. This study investigated E. granulosus genotypes isolated from livestock and humans in the Golestan province, northern Iran, southeast of the Caspian sea, using partial sequencing data of the cytochrome c oxidase subunit 1 (cox1) and NADH dehydrogenase 1 (nad1) mitochondrial genes. Seventy E. granulosus isolates were collected from animals in slaughterhouses: 18 isolates from sheep, 40 from cattle, nine from camels, two from buffaloes and one from a goat, along with four human isolates (formalin-fixed, paraffin-embedded tissues) from CE patients of provincial hospitals. All isolates were successfully analysed by PCR amplification and sequencing. The sequence analysis found four E. granulosus genotypes among the 74 CE isolates: G1 (78.3%), G2 (2.7%), G3 (15%) and G6 (4%). The G1-G3 complex genotype was found in all of the sheep, goat, cattle and buffalo isolates. Among the nine camel isolates, the frequency of G1-G3 and G6 genotypes were 66.7% and 33.3%, respectively. All four human CE isolates belonged to E. granulosus sensu stricto. This study reports the first occurrence of the G2 genotype in cattle from Iran and confirms the previously reported G3 genotype in camels in the same country. PMID:27253740

  17. Echinococcus granulosus sensu lato GENOTYPES IN DOMESTIC LIVESTOCK AND HUMANS IN GOLESTAN PROVINCE, IRAN

    PubMed Central

    SHARBATKHORI, Mitra; TANZIFI, Asal; ROSTAMI, Sima; ROSTAMI, Masoomeh; HARANDI, Majid FASIHI

    2016-01-01

    Cystic echinococcosis (CE) is a globally parasitic zoonosis caused by larval stages of Echinococcus granulosus. This study investigated E. granulosus genotypes isolated from livestock and humans in the Golestan province, northern Iran, southeast of the Caspian sea, using partial sequencing data of the cytochrome c oxidase subunit 1 (cox1) and NADH dehydrogenase 1 (nad1) mitochondrial genes. Seventy E. granulosus isolates were collected from animals in slaughterhouses: 18 isolates from sheep, 40 from cattle, nine from camels, two from buffaloes and one from a goat, along with four human isolates (formalin-fixed, paraffin-embedded tissues) from CE patients of provincial hospitals. All isolates were successfully analysed by PCR amplification and sequencing. The sequence analysis found four E. granulosus genotypes among the 74 CE isolates: G1 (78.3%), G2 (2.7%), G3 (15%) and G6 (4%). The G1-G3 complex genotype was found in all of the sheep, goat, cattle and buffalo isolates. Among the nine camel isolates, the frequency of G1-G3 and G6 genotypes were 66.7% and 33.3%, respectively. All four human CE isolates belonged to E. granulosus sensu stricto. This study reports the first occurrence of the G2 genotype in cattle from Iran and confirms the previously reported G3 genotype in camels in the same country. PMID:27253740

  18. Echinococcus granulosus sensu lato GENOTYPES IN DOMESTIC LIVESTOCK AND HUMANS IN GOLESTAN PROVINCE, IRAN.

    PubMed

    Sharbatkhori, Mitra; Tanzifi, Asal; Rostami, Sima; Rostami, Masoomeh; Fasihi Harandi, Majid

    2016-01-01

    Cystic echinococcosis (CE) is a globally parasitic zoonosis caused by larval stages of Echinococcus granulosus. This study investigated E. granulosus genotypes isolated from livestock and humans in the Golestan province, northern Iran, southeast of the Caspian sea, using partial sequencing data of the cytochrome c oxidase subunit 1 (cox1) and NADH dehydrogenase 1 (nad1) mitochondrial genes. Seventy E. granulosus isolates were collected from animals in slaughterhouses: 18 isolates from sheep, 40 from cattle, nine from camels, two from buffaloes and one from a goat, along with four human isolates (formalin-fixed, paraffin-embedded tissues) from CE patients of provincial hospitals. All isolates were successfully analysed by PCR amplification and sequencing. The sequence analysis found four E. granulosus genotypes among the 74 CE isolates: G1 (78.3%), G2 (2.7%), G3 (15%) and G6 (4%). The G1-G3 complex genotype was found in all of the sheep, goat, cattle and buffalo isolates. Among the nine camel isolates, the frequency of G1-G3 and G6 genotypes were 66.7% and 33.3%, respectively. All four human CE isolates belonged to E. granulosus sensu stricto. This study reports the first occurrence of the G2 genotype in cattle from Iran and confirms the previously reported G3 genotype in camels in the same country.

  19. 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

  20. The Heterogeneity, Distribution, and Environmental Associations of Borrelia burgdorferi Sensu Lato, the Agent of Lyme Borreliosis, in Scotland

    PubMed Central

    James, Marianne C.; Gilbert, Lucy; Bowman, Alan S.; Forbes, Ken J.

    2014-01-01

    Lyme borreliosis is an emerging infectious human disease caused by the Borrelia burgdorferi sensu lato complex of bacteria with reported cases increasing in many areas of Europe and North America. To understand the drivers of disease risk and the distribution of symptoms, which may improve mitigation and diagnostics, here we characterize the genetics, distribution, and environmental associations of B. burgdorferi s.l. genospecies across Scotland. In Scotland, reported Lyme borreliosis cases have increased almost 10-fold since 2000 but the distribution of B. burgdorferi s.l. is so far unstudied. Using a large survey of over 2200 Ixodes ricinus tick samples collected from birds, mammals, and vegetation across 25 sites we identified four genospecies: Borrelia afzelii (48%), Borrelia garinii (36%), Borrelia valaisiana (8%), and B. burgdorferi sensu stricto (7%), and one mixed genospecies infection. Surprisingly, 90% of the sequence types were novel and, importantly, up to 14% of samples were mixed intra-genospecies co-infections, suggesting tick co-feeding, feeding on multiple hosts, or multiple infections in hosts. B. garinii (hosted by birds) was considerably more genetically diverse than B. afzelii (hosted by small mammals), as predicted since there are more species of birds than small mammals and birds can import strains from mainland Europe. Higher proportions of samples contained B. garinii and B. valaisiana in the west, while B. afzelii and B. garinii were significantly more associated with mixed/deciduous than with coniferous woodlands. This may relate to the abundance of transmission hosts in different regions and habitats. These data on the genetic heterogeneity within and between Borrelia genospecies are a first step to understand pathogen spread and could help explain the distribution of patient symptoms, which may aid local diagnosis. Understanding the environmental associations of the pathogens is critical for rational policy making for disease risk

  1. Mitochondrial DNA analysis of Rhipicephalus sanguineus sensu lato (Acari: Ixodidae) in the Southern Cone of South America.

    PubMed

    Nava, Santiago; Mastropaolo, Mariano; Venzal, José M; Mangold, Atilio J; Guglielmone, Alberto A

    2012-12-21

    A genetic analysis of partial sequences of the mitochondrial 16S and 12S rDNA genes of Rhipicephalus sanguineus sensu lato collected in the Southern Cone of South America was carried out. Also, sequences of ticks belonging to this taxon from Europe, Africa and other South American localities were included. TCS networks constructed with 16S rDNA sequences showed two clusters of haplotypes, namely, Southern lineage (ST) and Northern lineage (NT). Haplotypes representing the specimens coming from localities of Argentina, Uruguay and Chile were included in the ST lineage, while haplotypes from Brazil, Paraguay, Colombia, South Africa, Mozambique and from two localities of Northern Argentina were grouped in the NT lineage. The phylogenetic trees obtained with both 16S and 12S sequences showed two distinct clades, one containing R. sanguineus s.l from Argentina, Uruguay, Chile (ST lineage) and Western Europe (Italy and France), and a second clade including R. sanguineus s.l from Argentina, Paraguay, Brazil, Colombia (NT lineage), South Africa and Mozambique. The results herein reported revealed that the taxon R. sanguineus s.l is represented by two lineages in the Southern Cone of South America. According with the genetic comparative analysis, NT lineage and the ticks from Mozambique and South Africa represent a species that is not R. sanguineus s.s, while R. sanguineus s.l ticks from Western Europe and Southern South America (ST lineage) probably represent true R. sanguineus, because the type locality of R. sanguineus s.s is located in France. The taxonomic issue described for R. sanguineus s.l in the South America has epidemiological implications. Difference in the vectorial competence for Ehrlichia canis between the two lineages of R. sanguineus s.l was found in previous works. Further investigations are needed in order to verify a possible different vectorial competence for the other pathogens transmitted by these ticks.

  2. Inter- and intra-specific pan-genomes of Borrelia burgdorferi sensu lato: genome stability and adaptive radiation

    PubMed Central

    2013-01-01

    Background Lyme disease is caused by spirochete bacteria from the Borrelia burgdorferi sensu lato (B. burgdorferi s.l.) species complex. To reconstruct the evolution of B. burgdorferi s.l. and identify the genomic basis of its human virulence, we compared the genomes of 23 B. burgdorferi s.l. isolates from Europe and the United States, including B. burgdorferi sensu stricto (B. burgdorferi s.s., 14 isolates), B. afzelii (2), B. garinii (2), B. “bavariensis” (1), B. spielmanii (1), B. valaisiana (1), B. bissettii (1), and B. “finlandensis” (1). Results Robust B. burgdorferi s.s. and B. burgdorferi s.l. phylogenies were obtained using genome-wide single-nucleotide polymorphisms, despite recombination. Phylogeny-based pan-genome analysis showed that the rate of gene acquisition was higher between species than within species, suggesting adaptive speciation. Strong positive natural selection drives the sequence evolution of lipoproteins, including chromosomally-encoded genes 0102 and 0404, cp26-encoded ospC and b08, and lp54-encoded dbpA, a07, a22, a33, a53, a65. Computer simulations predicted rapid adaptive radiation of genomic groups as population size increases. Conclusions Intra- and inter-specific pan-genome sizes of B. burgdorferi s.l. expand linearly with phylogenetic diversity. Yet gene-acquisition rates in B. burgdorferi s.l. are among the lowest in bacterial pathogens, resulting in high genome stability and few lineage-specific genes. Genome adaptation of B. burgdorferi s.l. is driven predominantly by copy-number and sequence variations of lipoprotein genes. New genomic groups are likely to emerge if the current trend of B. burgdorferi s.l. population expansion continues. PMID:24112474

  3. 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

  4. 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.

  5. 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...

  6. Prevalence of granulocytic Ehrlichia and Borrelia burgdorferi sensu lato in Ixodes ricinus ticks collected from Southwestern Finland and from Vormsi Island in Estonia.

    PubMed

    Mäkinen, Johanna; Vuorinen, Ilppo; Oksi, Jarmo; Peltomaa, Miikka; He, Qiushui; Marjamäki, Merja; Viljanen, Matti K

    2003-02-01

    Altogether, 343 adult and 111 nymphal Ixodes ricinus ticks collected from parks in Turku and suburban and rural islands of the Turku archipelago, Finland, and 100 adult I. ricinus ticks collected from Vormsi Island, Estonia, were included in this study. Using the polymerase chain reaction the ticks were examined for 16S rDNA of the Ehrlichia phagocytophila genogroup and for Borrelia burgdorferi sensu lato recA and flagellin genes. None of the Finnish ticks was found to be infected with E. phagocytophila, whereas 3% of the Estonian ticks were positive for this organism. The rate of Finnish ticks infected with B. burgdorferi sensu lato varied from 0% to 11.6% (mean 5%; 9% for adult and 4% for nymphal ticks). The corresponding rate for Estonian ticks was 15%. Borrelia afzelii was the most common genospecies in both Finnish (2.6%) and Estonian (12%) ticks. B. burgdorferi sensu stricto was detected in 2.0% of the Finnish ticks, but in none of the Estonian ticks. These results suggest that the E. phagocytophila genogroup is very rare in Finnish ticks, although the ticks were collected from an area endemic for Lyme borreliosis. In Estonia, E. phagocytophila is found in ticks and may cause disease.

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

    PubMed

    Kathirithamby, Jeyaraney; Johnston, J Spencer

    2004-02-01

    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

  8. Digital droplet PCR on disk.

    PubMed

    Schuler, Friedrich; Trotter, Martin; Geltman, Marcel; Schwemmer, Frank; Wadle, Simon; Domínguez-Garrido, Elena; López, María; Cervera-Acedo, Cristina; Santibáñez, Paula; von Stetten, Felix; Zengerle, Roland; Paust, Nils

    2016-01-01

    Existing systems for digital droplet PCR (ddPCR) either suffer from low integration or are difficult to introduce to mass fabrication. Here we present an integrated system that is compatible to mass fabrication and combines emulsification, PCR, and fluorescence readout in a single chamber within a disposable cartridge (disk). Droplets are generated by injecting the sample into fluorinated oil via centrifugal step emulsification. The resulting emulsion is aligned in the PCR and readout zone by capillary action. During thermocycling, gas bubbles generated by degassing are removed by capillary driven transport through tapered regions in the PCR chamber. Thereby, the positioning of the emulsion within the readout zone of the PCR chamber is ensured at any time and no bubbles are present during readout. Manual handling of the disk solely requires pipetting of oil and PCR mix into the inlet structures, placing the disk into the thermocycler and subsequently into a microarray scanner. The functionality of the ddPCR process chain is demonstrated by quantitative detection of the cystic fibrosis causing mutation p.Phe508del, which is of interest for non-invasive prenatal testing (NIPT). The mutation was detected in a concentration range spanning four orders of magnitude. We envision that this work will lay the base for the development of highly integrated sample-to-digital-answer PCR systems that can be employed in routine clinical diagnosis. PMID:26610263

  9. Molecular survey of Dirofilaria immitis and Dirofilaria repens by direct PCR for wild caught mosquitoes in the Republic of Korea.

    PubMed

    Lee, Sang-Eun; Kim, Heung-Chul; Chong, Sung-Tae; Klein, Terry A; Lee, Won-Ja

    2007-09-01

    Adult mosquito collections using New Jersey light traps and Black-hole light traps were conducted to determine the potential vectors and the relative mosquito infection rates of Dirofilaria immitis and Dirofilaria repens in Gyeonggi and Gangwon Provinces, Republic of Korea, 2005. Dirofilaria spp. were confirmed by polymerase chain reaction (PCR) using species-specific primers for D. immitis and D. repens. Minimum field infection rates (MFIR) [MFIR = (number infected pools/total number of mosquitoes) x 1000] of 12 pools/2059 total number mosquitoes (5.8) and 21 pools (10.1) of the wild caught mosquitoes were positive by PCR for D. immitis and D. repens, respectively. Dual infections of both D. immitis and D. repens were detected by PCR in eight pools (3.9). Anopheles sinensis sensu lato (includes An. sinensis s.s., An. pullus, An. kleini, An. belenrae, and An. lesteri), An. sineroides, Aedes vexans nipponi, Culex pipiens and Armigeres subalbatus were found to be infected and are potential vectors of D. immitis and D. repens in Korea. Infections observed in mosquitoes represent potential health risks for domestic animal and human populations.

  10. 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

  11. 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

  12. Review of the genus Mansonella Faust, 1929 sensu lato (Nematoda: Onchocercidae), with descriptions of a new subgenus and a new subspecies.

    PubMed

    Bain, Odile; Mutafchiev, Yasen; Junker, Kerstin; Guerrero, Ricardo; Martin, Coralie; Lefoulon, Emilie; Uni, Shigehiko

    2015-01-01

    Based on material deposited in museum collections, twelve species within Mansonella sensu lato were examined and their descriptions amended. Based on additional morphological details, the erection of the new monotypic subgenus Filyamagutia Bain & Uni for M. (F.) akitensis (Uni, 1983), and the new combination M. (Pseudolitomosa) musasabi (Yamaguti, 1941) Bain & Uni are proposed. A new subspecies, M. (Tetrapetalonema) atelensis amazonae Bain & Guerrero is described and a key to the seven subgenera of Mansonella is provided. Furthermore, the elevation of Sandnema to full genus rank comprising the two species S. digitatum (Chandler, 1929) n. comb. and S. sunci (Sandground, 1933) n. comb., is proposed. Host and geographic records for the species of Mansonella and Sandnema are included. PMID:25781087

  13. Further studies on Contracaecum spasskii Mozgovoi, 1950 and C. rudolphii Hartwich, 1964 (sensu lato) (Ascaridida: Anisakidae) from piscivorous birds in China.

    PubMed

    Li, Liang; Xu, Zhen; Zhang, Lu-Ping

    2013-03-01

    Contracaecum spasskii Mozgovoi, 1950, collected from the great crested grebe Podiceps cristatus (Linnaeus) (Podicipediformes: Podicipedidae), is redescribed using both light and, for the first time, scanning electron microscopy. Contracaecum spasskii differs from its congeners by having marked transverse cuticular annulations, the length of the oesophagus and spicules, the ratio between the intestinal caecum and the ventricular appendix, the number and arrangement of male caudal papillae, and especially by the particular morphology of the lips and interlabia. Some previously unreported morphological features of C. spasskii are also revealed and others corrected. Contracaecum rudolphii Hartwich, 1964 (sensu lato) is also redescribed based on the specimens collected from the great cormorant Phalacrocorax carbo sinensis (Blumenbach) (Pelecaniformes: Phalacrocoracidae) from China. Based on the geographical perspective, the present Chinese material may represent the species C. rudolphii B. PMID:23404759

  14. 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.

  15. Occurrence of Borrelia burgdorferi Sensu Lato in Ixodes ricinus Ticks with First Identification of Borrelia miyamotoi in Vojvodina, Serbia.

    PubMed

    Potkonjak, Aleksandar; Kleinerman, Gabriela; Gutiérrez, Ricardo; Savić, Sara; Vračar, Vuk; Nachum-Biala, Yaarit; Jurišić, Aleksandar; Rojas, Alicia; Petrović, Aleksandra; Ivanović, Ivana; Harrus, Shimon; Baneth, Gad

    2016-10-01

    Lyme borreliosis is the most common tick-borne infectious disease in Eurasia. Borrelia miyamotoi is the only known relapsing fever Borrelia group spirochete transmitted by Ixodes species. The aim of this study was to investigate the presence of Lyme Borrelia spp. and relapsing fever Borrelia spp. in Ixodes ricinus ticks collected from dogs and the vegetation from different parts of Vojvodina, Serbia. A total of 71 Ixodes ricinus ticks were collected and screened for the presence of Lyme Borrelia spp. group and relapsing fever Borrelia spp. by real-time PCR for the Borrelia flagellin B (flaB) gene followed by DNA sequencing of PCR products. Species identification was verified by PCR of the outer surface protein A (ospA) gene for Lyme Disease Borrelia spp. and by PCR of the glycerophosphodiester phosphodiesterase (glpQ) gene for relapsing fever Borrelia spp. Lyme Borrelia spp. were found in 15/71 (21.13%) of the ticks evaluated and included B. luisitaniae (11.3%), B. afzelii (7%), B. valaisiana (1.4%), and B. garinii (1.4%). Borrelia miyamotoi, from the relapsing fever Borrelia complex, was found, for the first time in Serbia, in one (1.4%) nymph collected from the environment. Co-infections between Borrelia species in ticks were not detected. These results suggest that the dominance of species within B. burgdorferi s.l. complex in I. ricinus ticks may vary over time and in different geographic regions. Further systematic studies of Borrelia species in vectors and reservoir hosts are needed to understand eco-epidemiology of these zoonotic infections and how to prevent human infection in the best way.

  16. Occurrence of Borrelia burgdorferi Sensu Lato in Ixodes ricinus Ticks with First Identification of Borrelia miyamotoi in Vojvodina, Serbia.

    PubMed

    Potkonjak, Aleksandar; Kleinerman, Gabriela; Gutiérrez, Ricardo; Savić, Sara; Vračar, Vuk; Nachum-Biala, Yaarit; Jurišić, Aleksandar; Rojas, Alicia; Petrović, Aleksandra; Ivanović, Ivana; Harrus, Shimon; Baneth, Gad

    2016-10-01

    Lyme borreliosis is the most common tick-borne infectious disease in Eurasia. Borrelia miyamotoi is the only known relapsing fever Borrelia group spirochete transmitted by Ixodes species. The aim of this study was to investigate the presence of Lyme Borrelia spp. and relapsing fever Borrelia spp. in Ixodes ricinus ticks collected from dogs and the vegetation from different parts of Vojvodina, Serbia. A total of 71 Ixodes ricinus ticks were collected and screened for the presence of Lyme Borrelia spp. group and relapsing fever Borrelia spp. by real-time PCR for the Borrelia flagellin B (flaB) gene followed by DNA sequencing of PCR products. Species identification was verified by PCR of the outer surface protein A (ospA) gene for Lyme Disease Borrelia spp. and by PCR of the glycerophosphodiester phosphodiesterase (glpQ) gene for relapsing fever Borrelia spp. Lyme Borrelia spp. were found in 15/71 (21.13%) of the ticks evaluated and included B. luisitaniae (11.3%), B. afzelii (7%), B. valaisiana (1.4%), and B. garinii (1.4%). Borrelia miyamotoi, from the relapsing fever Borrelia complex, was found, for the first time in Serbia, in one (1.4%) nymph collected from the environment. Co-infections between Borrelia species in ticks were not detected. These results suggest that the dominance of species within B. burgdorferi s.l. complex in I. ricinus ticks may vary over time and in different geographic regions. Further systematic studies of Borrelia species in vectors and reservoir hosts are needed to understand eco-epidemiology of these zoonotic infections and how to prevent human infection in the best way. PMID:27574731

  17. Nanoliter high throughput quantitative PCR

    PubMed Central

    Morrison, Tom; Hurley, James; Garcia, Javier; Yoder, Karl; Katz, Arrin; Roberts, Douglas; Cho, Jamie; Kanigan, Tanya; Ilyin, Sergey E.; Horowitz, Daniel; Dixon, James M.; Brenan, Colin J.H.

    2006-01-01

    Understanding biological complexity arising from patterns of gene expression requires accurate and precise measurement of RNA levels across large numbers of genes simultaneously. Real time PCR (RT-PCR) in a microtiter plate is the preferred method for quantitative transcriptional analysis but scaling RT-PCR to higher throughputs in this fluidic format is intrinsically limited by cost and logistic considerations. Hybridization microarrays measure the transcription of many thousands of genes simultaneously yet are limited by low sensitivity, dynamic range, accuracy and sample throughput. The hybrid approach described here combines the superior accuracy, precision and dynamic range of RT-PCR with the parallelism of a microarray in an array of 3072 real time, 33 nl polymerase chain reactions (RT-PCRs) the size of a microscope slide. RT-PCR is demonstrated with an accuracy and precision equivalent to the same assay in a 384-well microplate but in a 64-fold smaller reaction volume, a 24-fold higher analytical throughput and a workflow compatible with standard microplate protocols. PMID:17000636

  18. 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

  19. 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. PMID:26986669

  20. 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.

  1. First molecular evidence of the simultaneous human infection with two species of Echinococcus granulosus sensu lato: Echinococcus granulosus sensu stricto and Echinococcus canadensis.

    PubMed

    Oudni-M'rad, Myriam; M'rad, Selim; Ksia, Amine; Lamiri, Rachida; Mekki, Mongi; Nouri, Abdellatif; Mezhoud, Habib; Babba, Hamouda

    2016-03-01

    Cystic echinococcosis is a widespread zoonotic parasitic disease especially in Tunisia which is one of the most endemic countries in the Mediterranean area. The etiological agent, Echinococcus granulosus sensu lato, implies dogs and other canids as definitive hosts and different herbivore species as intermediate hosts. Human contamination occurs during the consumption of parasite eggs passed in the environment through canid feces. Hydatid cysts coming from a child operated for multiple echinococcosis were collected and analyzed in order to genotype and to obtain some epidemiological molecular information. Three targets, ribosomal DNA ITS1 fragment, NADH dehydrogenase subunit 1 (nad1), and mitochondrial cytochrome c oxydase subunit 1 (CO1) genes, were amplified and analyzed by RFLP and sequencing approach. This study presents the first worldwide report in human of a simultaneous infection with Echinococcus granulosus sensu stricto (genotype G1) and Echinococcus canadensis (genotype G6) species. This is also the first report of the presence of E. canadensis in the Tunisian population which argues in favor of a greater importance of this species in human infestation in Tunisia than previously believed.

  2. 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

  3. Phylogenetic relationships and Y genome origin in Elymus L. sensu lato (Triticeae; Poaceae) based on single-copy nuclear Acc1 and Pgk1 gene sequences.

    PubMed

    Fan, Xing; Sha, Li-Na; Dong, Zhen-Zhen; Zhang, Hai-Qin; Kang, Hou-Yang; Wang, Yi; Wang, Xiao-Li; Zhang, Li; Ding, Chun-Bang; Yang, Rui-Wu; Zheng, You-Liang; Zhou, Yong-Hong

    2013-12-01

    To estimate the origin and genomic relationships of the polyploid species within Elymus L. sensu lato, two unlinked single-copy nuclear gene (Acc1 and Pgk1) sequences of eighteen tetraploids (StH and StY genomes) and fourteen hexaploids (StStH, StYP, StYH, and StYW genomes) were analyzed with those of 35 diploid taxa representing 18 basic genomes in Triticeae. Sequence and phylogenetic analysis suggested that: (1) the St, H, W, and P genomes were donated by Pseudoroegneria, Hordeum, Australopyrum, and Agropyron, respectively, while the Y genome is closely related to the Xp genome in Peridictyon sanctum; (2) different hexaploid Elymus s.l. species may derived their StY genome from different StY genome tetraploid species via independent origins; (3) due to incomplete lineage sorting and/or hybridization events, the genealogical conflict between the two gene trees suggest introgression involving some Elymus s.l. species, Pseudoroegneria, Agropyron and Aegilops/Triticum; (4) it is reasonable to recognize the StH genome species as Elymus sensu stricto, the StY genome species as Roegneria, the StYW genome species as Anthosachne, the StYH genome species as Campeiostachys, and the StYP genome species as Kengyilia. The occurrence of multiple origin and introgression could account for the rich diversity and ecological adaptation of Elymus s.l. species. PMID:23816902

  4. Environmental determinants of Ixodes ricinus ticks and the incidence of Borrelia burgdorferi sensu lato, the agent of Lyme borreliosis, in Scotland.

    PubMed

    James, M C; Bowman, A S; Forbes, K J; Lewis, F; McLeod, J E; Gilbert, L

    2013-02-01

    Lyme borreliosis (LB) is the most common arthropod-borne disease of humans in the Northern hemisphere. In Europe, the causative agent, Borrelia burgdorferi sensu lato complex, is principally vectored by Ixodes ricinus ticks. The aim of this study was to identify environmental factors influencing questing I. ricinus nymph abundance and B. burgdorferi s.l. infection in questing nymphs using a large-scale survey across Scotland. Ticks, host dung and vegetation were surveyed at 25 woodland sites, and climatic variables from a Geographical Information System (GIS) were extracted for each site. A total of 2397 10 m2 transect surveys were conducted and 13 250 I. ricinus nymphs counted. Questing nymphs were assayed for B. burgdorferi s.l. and the average infection prevalence was 5·6% (range 0·8-13·9%). More questing nymphs and higher incidence of B. burgdorferi s.l. infection were found in areas with higher deer abundance and in mixed/deciduous compared to coniferous forests, as well as weaker correlations with season, altitude, rainfall and ground vegetation. No correlation was found between nymph abundance and infection prevalence within the ranges encountered. An understanding of the environmental conditions associated with tick abundance and pathogen prevalence may be used to reduce risk of exposure and to predict future pathogen prevalence and distributions under environmental changes.

  5. High resolution melting technique for molecular epidemiological studies of cystic echinococcosis: differentiating G1, G3, and G6 genotypes of Echinococcus granulosus sensu lato.

    PubMed

    Rostami, Sima; Talebi, Saeed; Babaei, Zahra; Sharbatkhori, Mitra; Ziaali, Naser; Rostami, Habib; Harandi, Majid Fasihi

    2013-10-01

    Reliable and rapid genotyping of large number of Echinococcus granulosus sensu lato isolates is crucial for understanding the epidemiology and transmission of cystic echinococcosis. We have developed a method for distinguishing and discriminating common genotypes of E. granulosus s.l. (G1, G3, and G6) in Iran. This method is based on polymerase chain reaction coupled with high resolution melting curve (HRM), ramping from 70 to 86 °C with fluorescence data acquisition set at 0.1 °C increments and continuous fluorescence monitoring. Consistency of this technique was assessed by inter- and intra-assays. Assessment of intra- and inter-assay variability showed low and acceptable coefficient of variations ranging from 0.09 to 0.17 %. Two hundred and eighty E. granulosus s.l. isolates from sheep, cattle, and camel were used to evaluate the applicability and accuracy of the method. The isolates were categorized as G1 (93, 94, and 25%), G3 (7, 4, and 4%), and G6 (0, 2, and 71%) for sheep, cattle, and camel, respectively. HRM results were completely compatible with those obtained from sequencing and rostellar hook measurement. This method proved to be a valuable screening tool for large-scale molecular epidemiological studies. PMID:23832641

  6. Design and experiment of silicon PCR chips

    NASA Astrophysics Data System (ADS)

    Cui, Zheng; Zhao, Zhan; Xia, Shanhong

    2002-04-01

    There are considerable interests in integrating Polymerase chain reaction (PCR) on a microchip can have much fast heating and cooling rate, the delicacy in its structure makes the PCR experiment difficult and cracks often occur particularly for the thin membrane type of PCR chips. Design study and experiment of silicon PCR chips are presented with the aim of identifying the problems encountered in experiment and finding an optimum chip structure. Heating characteristics of four different heater designs have been compared, so have the PCR chambers with fixed frame and with suspended frame. The thermal stress analysis has shown that the structure and heater design can make a significant difference in heating characteristics and in reducing the failure of PCR chips. Different solutions to reduce PCR chip failure have been proposed. One of the solutions was implemented in the experiment, confirming the design study results. Silicon PCR chips have been fabricated. Thermal cycling and initial DNA amplification results are presented.

  7. Investigating the host-range of the rust fungus Puccinia psidii sensu lato across tribes of the family Myrtaceae present in Australia.

    PubMed

    Morin, Louise; Aveyard, Ruth; Lidbetter, Jonathan R; Wilson, Peter G

    2012-01-01

    The exotic rust fungus Puccinia psidii sensu lato was first detected in Australia in April 2010. This study aimed to determine the host-range potential of this accession of the rust by testing its pathogenicity on plants of 122 taxa, representative of the 15 tribes of the subfamily Myrtoideae in the family Myrtaceae. Each taxon was tested in two separate trials (unless indicated otherwise) that comprised up to five replicates per taxon and six replicates of a positive control (Syzygium jambos). No visible symptoms were observed on the following four taxa in either trial: Eucalyptus grandis×camaldulensis, E. moluccana, Lophostemon confertus and Sannantha angusta. Only small chlorotic or necrotic flecks without any uredinia (rust fruiting bodies) were observed on inoculated leaves of seven other taxa (Acca sellowiana, Corymbia calophylla 'Rosea', Lophostemon suaveolens, Psidium cattleyanum, P. guajava 'Hawaiian' and 'Indian', Syzygium unipunctatum). Fully-developed uredinia were observed on all replicates across both trials of 28 taxa from 8 tribes belonging to the following 17 genera: Agonis, Austromyrtus, Beaufortia, Callistemon, Calothamnus, Chamelaucium, Darwinia, Eucalyptus, Gossia, Kunzea, Leptospermum, Melaleuca, Metrosideros, Syzygium, Thryptomene, Tristania, Verticordia. In contrast, the remaining 83 taxa inoculated, including the majority of Corymbia and Eucalyptus species, developed a broad range of symptoms, often across the full spectrum, from fully-developed uredinia to no visible symptoms. These results were encouraging as they indicate that some levels of genetic resistance to the rust possibly exist in these taxa. Overall, our results indicated no apparent association between the presence or absence of disease symptoms and the phylogenetic relatedness of taxa. It is most likely that the majority of the thousands of Myrtaceae species found in Australia have the potential to become infected to some degree by the rust, although this wide host range may

  8. Investigating the Host-Range of the Rust Fungus Puccinia psidii sensu lato across Tribes of the Family Myrtaceae Present in Australia

    PubMed Central

    Morin, Louise; Aveyard, Ruth; Lidbetter, Jonathan R.; Wilson, Peter G.

    2012-01-01

    The exotic rust fungus Puccinia psidii sensu lato was first detected in Australia in April 2010. This study aimed to determine the host-range potential of this accession of the rust by testing its pathogenicity on plants of 122 taxa, representative of the 15 tribes of the subfamily Myrtoideae in the family Myrtaceae. Each taxon was tested in two separate trials (unless indicated otherwise) that comprised up to five replicates per taxon and six replicates of a positive control (Syzygium jambos). No visible symptoms were observed on the following four taxa in either trial: Eucalyptus grandis×camaldulensis, E. moluccana, Lophostemon confertus and Sannantha angusta. Only small chlorotic or necrotic flecks without any uredinia (rust fruiting bodies) were observed on inoculated leaves of seven other taxa (Acca sellowiana, Corymbia calophylla ‘Rosea’, Lophostemon suaveolens, Psidium cattleyanum, P. guajava ‘Hawaiian’ and ‘Indian’, Syzygium unipunctatum). Fully-developed uredinia were observed on all replicates across both trials of 28 taxa from 8 tribes belonging to the following 17 genera: Agonis, Austromyrtus, Beaufortia, Callistemon, Calothamnus, Chamelaucium, Darwinia, Eucalyptus, Gossia, Kunzea, Leptospermum, Melaleuca, Metrosideros, Syzygium, Thryptomene, Tristania, Verticordia. In contrast, the remaining 83 taxa inoculated, including the majority of Corymbia and Eucalyptus species, developed a broad range of symptoms, often across the full spectrum, from fully-developed uredinia to no visible symptoms. These results were encouraging as they indicate that some levels of genetic resistance to the rust possibly exist in these taxa. Overall, our results indicated no apparent association between the presence or absence of disease symptoms and the phylogenetic relatedness of taxa. It is most likely that the majority of the thousands of Myrtaceae species found in Australia have the potential to become infected to some degree by the rust, although this wide

  9. 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.

  10. Repeated landmass reformation limits diversification in the widespread littoral zone mosquito Anopheles sundaicus sensu lato in the Indo-Oriental Region.

    PubMed

    Zarowiecki, Magdalena; Linton, Yvonne-Marie; Post, Rory J; Bangs, Michael J; Htun, Pe Than; Hlaing, Thaung; Seng, Chang Moh; Baimai, Visut; Ding, Trung Ho; Sochantha, Tho; Walton, Catherine

    2014-05-01

    Southeast Asia harbours abundant biodiversity, hypothesized to have been generated by Pliocene and Pleistocene climatic and environmental change. Vicariance between the island of Borneo, the remaining Indonesian archipelago and mainland Southeast Asia caused by elevated sea levels during interglacial periods has been proposed to lead to diversification in the littoral zone mosquito Anopheles (Cellia) sundaicus (Rodenwaldt) sensu lato. To test this biogeographical hypothesis, we inferred the population history and assessed gene flow of A. sundaicus s.l. sampled from 18 populations across its pan-Asian species range, using sequences from mitochondrial cytochrome c oxidase subunit 1 (CO1), the internal transcribed spacer 2 (ITS2) and the mannose phosphate isomerase (Mpi) gene. A hypothesis of ecological speciation for A. sundaicus involving divergent adaptation to brackish and freshwater larval habitats was also previously proposed, based on a deficiency of heterozygotes for Mpi allozyme alleles in sympatry. This hypothesis was not supported by Mpi sequence data, which exhibited no fixed differences between brackish and freshwater larval habitats. Mpi and CO1 supported the presence of up to eight genetically distinct population groupings. Counter to the hypothesis of three allopatric species, divergence was often no greater between Borneo, Sumatra/Java and the Southeast Asian mainland than it was between genetic groupings within these landmasses. An isolation-with-migration (IM) model indicates recurrent gene flow between the current major landmasses. Such gene flow would have been possible during glacial periods when the current landmasses merged, presenting opportunities for dispersal along expanding and contracting coastlines. Consequently, Pleistocene climatic variation has proved a homogenizing, rather than diversifying, force for A. sundaicus diversity. PMID:24750501

  11. 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

  12. 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

  13. 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...

  14. 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. PMID:23513039

  15. 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.

  16. 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

  17. 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…

  18. Electrothermal modeling of silicon PCR chips

    NASA Astrophysics Data System (ADS)

    Cui, Zheng; Zhao, Zhan; Xia, Shanhong

    2001-04-01

    Polymerase chain reaction (PCR) on a microchip has drawn considerable attention in recent years. Although a microchip can have must fast heating and cooling rate, the delicacy in its structure makes the PCR experiment difficult and cracks often occurs particularly for the thin membrane type of PCR chips. Electrothermal modeling of PCR chips is presented using commercial MEMS software tool IntelliSuiteTM, with the aim of identifying the problems encountered in experiment and finding an optimum chip structure. Heating characteristics of four different heater designs have been compared, so have the PCR chambers with fixed frame and with suspended frame. The thermal stress analysis has shown that the structure and heater design can make significant difference in heating characteristics and in reducing the failure of PCR chips. The computer simulation has confirmed what has been found in experiment the reason of membrane cracks. Improvement in PCR chip design has been proposed.

  19. 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.

  20. Multiplexed Primer Prediction for PCR

    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 sequencesmore » 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.« less

  1. 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.

  2. Identification of non-host semiochemicals for the brown dog tick, Rhipicephalus sanguineus sensu lato (Acari: Ixodidae), from tick-resistant beagles, Canis lupus familiaris.

    PubMed

    Borges, Lígia Miranda Ferreira; de Oliveira Filho, Jaires Gomes; Ferreira, Lorena Lopes; Louly, Carla Cristina Braz; Pickett, John A; Birkett, Michael A

    2015-07-01

    Studies have shown that the brown dog tick, Rhipicephalus sanguineus sensu lato, when fed on the beagle breed of dog, Canis lupus familiaris, development negatively affected in comparison with tick development after feeding on the English cocker spaniel breed. Thus leading to the suggestion that beagle dogs are be tick-resistant dogs. Behavioural studies have demonstrated that more ticks are attracted by extracts from cocker spaniels than from beagles and that the odour of beagles is a repellent. To test the hypothesis that resistant hosts produce repellent compounds, we undertook comparative chemical analysis on beagle odour and cocker spaniel extracts using coupled high-resolution gas chromatography-mass spectrometry (GC-MS) and also used Petri-dish and olfactometer behavioural assays to assess the response of ticks to identified non-host compounds. The beagle odour extracts contained almost three times as many chemical compounds as cocker spaniel samples. Several non-host compounds were identified, i.e. 2-hexanone, benzaldehyde, nonane, decane and undecane. In Petri-dish assays, 2-hexanone was repellent at 30 min at concentrations of 0.200 and 0.050 mg cm(-2), whilst at 10 min, the 0.100 mg cm(-2) concentration was repellent. Benzaldehyde repelled ticks at 30 min (0.200 mg cm(-2)) and at 5 min (0.050 mg cm(-2)). Undecane was repellent for R. sanguineus s.l. ticks for the first 5 min at the highest concentration tested. Nonane and decane did not show any significant repellency at any concentration or time evaluated. When 2-hexanone and benzaldehyde were combined, an increase in the repellency rate was observed, with activity comparable or better than N,N-diethyl-3-methylbenzamide (DEET). In olfactometer bioassays, a 1:1 mixture of 2-hexanone:benzaldehyde and DEET were repellent for R. sanguineus s.l. adults at the concentration of 0.200 mg cm(-2). This study identified non-host semiochemicals that mediate avoidance of the beagle dog breed by R. sanguineus s

  3. Identification of non-host semiochemicals for the brown dog tick, Rhipicephalus sanguineus sensu lato (Acari: Ixodidae), from tick-resistant beagles, Canis lupus familiaris.

    PubMed

    Borges, Lígia Miranda Ferreira; de Oliveira Filho, Jaires Gomes; Ferreira, Lorena Lopes; Louly, Carla Cristina Braz; Pickett, John A; Birkett, Michael A

    2015-07-01

    Studies have shown that the brown dog tick, Rhipicephalus sanguineus sensu lato, when fed on the beagle breed of dog, Canis lupus familiaris, development negatively affected in comparison with tick development after feeding on the English cocker spaniel breed. Thus leading to the suggestion that beagle dogs are be tick-resistant dogs. Behavioural studies have demonstrated that more ticks are attracted by extracts from cocker spaniels than from beagles and that the odour of beagles is a repellent. To test the hypothesis that resistant hosts produce repellent compounds, we undertook comparative chemical analysis on beagle odour and cocker spaniel extracts using coupled high-resolution gas chromatography-mass spectrometry (GC-MS) and also used Petri-dish and olfactometer behavioural assays to assess the response of ticks to identified non-host compounds. The beagle odour extracts contained almost three times as many chemical compounds as cocker spaniel samples. Several non-host compounds were identified, i.e. 2-hexanone, benzaldehyde, nonane, decane and undecane. In Petri-dish assays, 2-hexanone was repellent at 30 min at concentrations of 0.200 and 0.050 mg cm(-2), whilst at 10 min, the 0.100 mg cm(-2) concentration was repellent. Benzaldehyde repelled ticks at 30 min (0.200 mg cm(-2)) and at 5 min (0.050 mg cm(-2)). Undecane was repellent for R. sanguineus s.l. ticks for the first 5 min at the highest concentration tested. Nonane and decane did not show any significant repellency at any concentration or time evaluated. When 2-hexanone and benzaldehyde were combined, an increase in the repellency rate was observed, with activity comparable or better than N,N-diethyl-3-methylbenzamide (DEET). In olfactometer bioassays, a 1:1 mixture of 2-hexanone:benzaldehyde and DEET were repellent for R. sanguineus s.l. adults at the concentration of 0.200 mg cm(-2). This study identified non-host semiochemicals that mediate avoidance of the beagle dog breed by R. sanguineus s

  4. 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

  5. Application of Reverse Transcription-PCR and Real-Time PCR in Nanotoxicity Research

    PubMed Central

    Mo, Yiqun; Wan, Rong; Zhang, Qunwei

    2016-01-01

    Reverse transcription-polymerase chain reaction (RT-PCR) is a relatively simple and inexpensive technique to determine the expression level of target genes and is widely used in biomedical science research including nanotoxicology studies for semiquantitative analysis. Real-time PCR allows for the detection of PCR amplification in the exponential growth phase of the reaction and is much more quantitative than traditional RT-PCR. Although a number of kits and reagents for RT-PCR and real-time PCR are commercially available, the basic principles are the same. Here, we describe the procedures for total RNA isolation by using TRI Reagent, for reverse transcription (RT) by M-MLV reverse transcriptase, and for PCR by GoTaq® DNA Polymerase. And real-time PCR will be performed on an iQ5 multicolor real-time PCR detection system by using iQ™ SYBR Green Supermix. PMID:22975959

  6. The polymerase chain reaction (PCR): general methods.

    PubMed

    Waters, Daniel L E; Shapter, Frances M

    2014-01-01

    The polymerase chain reaction (PCR) converts very low quantities of DNA into very high quantities and is the foundation of many specialized techniques of molecular biology. PCR utilizes components of the cellular machinery of mitotic cell division in vitro which respond predictably to user inputs. This chapter introduces the principles of PCR and discusses practical considerations from target sequence definition through to optimization and application.

  7. Pitfalls in PCR troubleshooting: Expect the unexpected?

    PubMed

    Schrick, Livia; Nitsche, Andreas

    2016-01-01

    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.

  8. 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

  9. Propidium monoazide reverse transcriptase PCR and RT-qPCR for detecting infectious enterovirus and norovirus.

    PubMed

    Karim, Mohammad R; Fout, G Shay; Johnson, Clifford H; White, Karen M; Parshionikar, Sandhya U

    2015-07-01

    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 public health significance of positive findings are limited. In this study, PMA RT-PCR and RT-qPCR assays were evaluated for selective detection of infectious poliovirus, murine norovirus (MNV-1), and Norwalk virus. Viruses were inactivated using heat, chlorine, and ultraviolet light (UV). Infectious and non-infectious viruses were treated with PMA before RT-PCR and RT-qPCR. PMA RT-PCR was able to differentiate selectively between infectious and heat and chlorine inactivated poliovirus. PMA RT-PCR was able to differentiate selectively between infectious and noninfectious murine norovirus only when inactivated by chlorine. However, PMA RT-PCR could not differentiate infectious Norwalk virus from virus particles rendered non-infectious by any treatment. PMA RT-PCR assay was not able to differentiate between infectious and UV inactivated viruses suggesting that viral capsid damage may be necessary for PMA to enter and bind to the viral genome. PMA RT-PCR on naked MNV-1 and Norwalk virus RNA suggest that PMA RT-PCR can be used to detect intact, potentially infectious MNV-1 and Norwalk viruses and can be used to exclude the detection of free viral RNA by PCR assay. PMID:25796356

  10. 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…

  11. 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...

  12. COLD-PCR: Applications and Advantages.

    PubMed

    Zuo, Zhuang; Jabbar, Kausar J

    2016-01-01

    Co-amplification at lower denaturation temperature-based polymerase chain reaction (COLD-PCR) is a single-step amplification method that results in the enhancement of both known and unknown minority alleles during PCR, irrespective of mutation type and position. This method is based on exploitation of the critical temperature, Tc, at which mutation-containing DNA is preferentially melted over wild type. COLD-PCR can be a good strategy for mutation detection in specimens with high nonneoplastic cell content, small specimens in which neoplastic cells are difficult to micro-dissect and therefore enrich, and whenever a mutation is suspected to be present but is undetectable using conventional PCR and sequencing methods. We describe in this chapter our COLD-PCR-based pyrosequencing method for KRAS mutation detection in various clinical samples using DNA extracted from either fresh or fixed paraffin-embedded tissue specimens.

  13. 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.

  14. Mechanisms of karyotype differentiation in Cassidinae sensu lato (Coleoptera, Polyphaga, Chrysomelidae) based on seven species of the Brazilian fauna and an overview of the cytogenetic data.

    PubMed

    de Julio, Milena; Fernandes, Flávia Rodrigues; Costa, Cleide; Almeida, Mara Cristina; Cella, Doralice Maria

    2010-01-01

    Among the subfamilies of Chrysomelidae, Cassidinae sensu lato (s.l.) includes 6000 species distributed in 43 tribes. Approximately 100 of these species were cytogenetically analyzed and most of them presented 2n=18=16+Xy(p), which was smaller than 2n=20=18+Xy(p) considered basal for Polyphaga. However, some groups of species presented maintenance of the basal diploid number and others showed increase in this number. Certain species of the latter group also exhibited variation in the type of sex chromosome system (SCS). Considering the recent taxonomic revision accomplished for the Cassidinae s.l. species, the existence of phylogenetic relationship for some species of this subfamily, the high diversity of species of this group in the Neotropical region, and the low number of Cassidinae s.l. species karyotyped so far, the aim of the present work was to establish the main mechanisms involved in the karyotype evolution of this subfamily through the study of seven species of the Brazilian fauna and overview of the cytogenetic data. The individuals were collected in southeast and south of Brazil. The chromosomal preparations obtained from embryo and testes of adult males were stained with Giemsa solution. The species Agroiconota inedita (2n=42=40+Xy(p)), Charidotella (s.str.) immaculata (2n=22=20+Xy(p)), Charidotella (s.str.) sexpunctata (2n=22=20+Xy(p)), and Stolas chalybaea (2n=24=22+Xy(p)) revealed diploid number higher than that established as basal for Polyphaga and biarmed chromosomes. The karyotype of Cteisella confusa, Deloyala cruciata, and Metriona elatior showed the chromosomal formulae 2n=18=16+Xy(p) considered modal for Cassidinae s.l. and biarmed chromosomes. The seven species exhibited easily identified sex chromosomes due to their size and/or morphology. The analysis of meiotic cells of all the species showed pachytenes with a positively heteropycnotic block probably corresponding to the sex chromosomes; diplotenes with a high number of bivalents with two

  15. Single-molecule PCR: an artifact-free PCR approach for the analysis of somatic mutations.

    PubMed

    Kraytsberg, Yevgenya; Khrapko, Konstantin

    2005-09-01

    A critical review of the clone-by-clone approach to the analysis of complex spectra of somatic mutations is presented. The study of a priori unknown somatic mutations requires painstaking analysis of complex mixtures of multiple mutant and non-mutant DNA molecules. If mutant fractions are sufficiently high, these mixtures can be dissected by the cloning of individual DNA molecules and scanning of the individual clones for mutations (e.g., by sequencing). Currently, the majority of such cloning is performed using PCR fragments. However, post-PCR cloning may result in various PCR artifacts - PCR errors and jumping PCR - and preferential amplification of certain mutations. This review argues that single-molecule PCR is a simple alternative that promises to evade the disadvantages inherent to post-PCR cloning and enhance mutational analysis in the future. PMID:16149882

  16. Real-time PCR detection chemistry.

    PubMed

    Navarro, E; Serrano-Heras, G; Castaño, M J; Solera, J

    2015-01-15

    Real-time PCR is the method of choice in many laboratories for diagnostic and food applications. This technology merges the polymerase chain reaction chemistry with the use of fluorescent reporter molecules in order to monitor the production of amplification products during each cycle of the PCR reaction. Thus, the combination of excellent sensitivity and specificity, reproducible data, low contamination risk and reduced hand-on time, which make it a post-PCR analysis unnecessary, has made real-time PCR technology an appealing alternative to conventional PCR. The present paper attempts to provide a rigorous overview of fluorescent-based methods for nucleic acid analysis in real-time PCR described in the literature so far. Herein, different real-time PCR chemistries have been classified into two main groups; the first group comprises double-stranded DNA intercalating molecules, such as SYBR Green I and EvaGreen, whereas the second includes fluorophore-labeled oligonucleotides. The latter, in turn, has been divided into three subgroups according to the type of fluorescent molecules used in the PCR reaction: (i) primer-probes (Scorpions, Amplifluor, LUX, Cyclicons, Angler); (ii) probes; hydrolysis (TaqMan, MGB-TaqMan, Snake assay) and hybridization (Hybprobe or FRET, Molecular Beacons, HyBeacon, MGB-Pleiades, MGB-Eclipse, ResonSense, Yin-Yang or displacing); and (iii) analogues of nucleic acids (PNA, LNA, ZNA, non-natural bases: Plexor primer, Tiny-Molecular Beacon). In addition, structures, mechanisms of action, advantages and applications of such real-time PCR probes and analogues are depicted in this review.

  17. Gene amplification and qRT-PCR.

    PubMed

    Jones, Cerith; Filloux, Alain

    2014-01-01

    This chapter includes methods for the use of the polymerase chain reaction (PCR) with Pseudomonas, and several specific tips for their successful application in this organism. The first part of the chapter includes methods for purifying genomic DNA from, and amplifying genes from, Pseudomonas, in addition to methods which describe how to prepare a cell lysate from Pseudomonas species for colony PCR reactions. The chapter continues with a switch in focus from DNA to RNA, describing methods for RNA isolation from Pseudomonas, cDNA generation, and finally q-RT-PCR to investigate relative changes in gene expression. PMID:24818925

  18. 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.

  19. Detection of Onchocerca volvulus in Latin American black flies for pool screening PCR using high-throughput automated DNA isolation for transmission surveillance.

    PubMed

    Rodríguez-Pérez, Mario A; Gopal, Hemavathi; Adeleke, Monsuru Adebayo; De Luna-Santillana, Erick Jesús; Gurrola-Reyes, J Natividad; Guo, Xianwu

    2013-11-01

    The posttreatment entomological surveillance (ES) of onchocerciasis in Latin America requires quite large numbers of flies to be examined for parasite infection to prove that the control strategies have worked and that the infection is on the path of elimination. Here, we report a high-throughput automated DNA isolation of Onchocerca volvulus for PCR using a major Latin American black fly vector of onchocerciasis. The sensitivity and relative effectiveness of silica-coated paramagnetic beads was evaluated in comparison with phenol chloroform (PC) method which is known as the gold standard of DNA extraction for ES in Latin America. The automated method was optimized in the laboratory and validated in the field to detect parasite DNA in Simulium ochraceum sensu lato flies in comparison with PC. The optimization of the automated method showed that it is sensitive to detect O. volvulus with a pool size of 100 flies as compared with PC which utilizes 50 flies pool size. The validation of the automated method in comparison with PC in an endemic community showed that 5/67 and 3/134 heads pools were positive for the two methods, respectively. There was no statistical variation (P < 0.05) in the estimation of transmission indices generated by automated method when compared with PC method. The fact that the automated method is sensitive to pool size up to 100 confers advantage over PC method and can, therefore, be employed in large-scale ES of onchocerciasis transmission in endemic areas of Latin America. PMID:24030195

  20. 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.

  1. A systematic analysis of PCR contamination.

    PubMed

    Scherczinger, C A; Ladd, C; Bourke, M T; Adamowicz, M S; Johannes, P M; Scherczinger, R; Beesley, T; Lee, H C

    1999-09-01

    In light of the strict legal scrutiny surrounding DNA typing at this time, it has become necessary to systematically address the issue of PCR contamination. To precisely define the parameters affecting PCR contamination under casework analysis conditions, PCR amplification reactions were intentionally compromised by employing sub-standard laboratory technique and by introducing secondary sources of DNA. The PCR parameters considered for potential sources of contamination include amplification set-up, amplification product handling, aerosol DNA and storage. In addition, analyst technique was evaluated by modifying or eliminating standard safeguards. Under the circumstances normally encountered during casework analysis, PCR contamination was never noted. Significantly, using the dot blot detection method, contamination was never observed when nanogram quantities of genomic DNA were mishandled or aerosolized. Contamination occurred only when amplification product was carelessly manipulated or purposefully sprayed near or directly into open tubes containing water or genomic DNA. Although standard precautions should be employed during PCR-based DNA typing, our data indicates that contamination during amplification procedures is not prevalent when detected by dot blot analysis. PMID:10486955

  2. Specific PCR and real-time PCR assays for detection and quantitation of 'Candidatus Phytoplasma phoenicium'.

    PubMed

    Jawhari, Maan; Abrahamian, Peter; Sater, Ali Abdel; Sobh, Hana; Tawidian, Patil; Abou-Jawdah, Yusuf

    2015-02-01

    Almond witches' broom (AlmWB) is a fast-spreading lethal disease of almond, peach and nectarine associated with 'Candidatus Phytoplasma phoenicium'. The development of PCR and quantitative real-time PCR (qPCR) assays for the sensitive and specific detection of the phytoplasma is of prime importance for early detection of 'Ca. P. phoenicium' and for epidemiological studies. The developed qPCR assay herein uses a TaqMan(®) probe labeled with Black Hole Quencher Plus. The specificity of the PCR and that of the qPCR detection protocols were tested on 17 phytoplasma isolates belonging to 11 phytoplasma 16S rRNA groups, on samples of almond, peach, nectarine, native plants and insects infected or uninfected with the phytoplasma. The developed assays showed high specificity against 'Ca. P. phoenicium' and no cross-reactivity against any other phytoplasma, plant or insect tested. The sensitivity of the developed PCR and qPCR assays was similar to the conventional nested PCR protocol using universal primers. The qPCR assay was further validated by quantitating AlmWB phytoplasma in different hosts, plant parts and potential insect vectors. The highest titers of 'Ca. P. phoenicium' were detected in the phloem tissues of stems and roots of almond and nectarine trees, where they averaged from 10(5) to 10(6) genomic units per nanogram of host DNA (GU/ng of DNA). The newly developed PCR and qPCR protocols are reliable, specific and sensitive methods that are easily applicable to high-throughput diagnosis of AlmWB in plants and insects and can be used for surveys of potential vectors and alternative hosts.

  3. 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. PMID:27393656

  4. Rational primer design greatly improves differential display-PCR (DD-PCR).

    PubMed Central

    Graf, D; Fisher, A G; Merkenschlager, M

    1997-01-01

    Since its conception in 1992, differential display PCR (DD-PCR) has attracted widespread interest. Theoretically an attractive cloning approach, it combines the comparative analysis of several samples with the sensitivity of PCR. Although a large number of studies embracing this technology have been initiated, few novel genes of interest have been identified, suggesting that the method has not realised its potential. The present report shows that by modifying primer design, sampling of differentially expressed genes can be greatly enhanced and relevant genes can be isolated. Using our modified conditions DD-PCR efficiently screens a wide range of gene expression levels, in which differences are represented on a linear scale. PMID:9153330

  5. 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.

  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. PCR-based diagnostics for anaerobic infections.

    PubMed

    Song, Yuli

    2005-01-01

    Conventional methods to identify anaerobic bacteria have often relied on unique clinical findings, isolation of organisms, and laboratory identification by morphology and biochemical tests (phenotypic tests). Although these methods are still fundamental, there is an increasing move toward molecular diagnostics of anaerobes. In this review, some of the molecular approaches to anaerobic diagnostics based on the polymerase chain reaction (PCR) are discussed. This includes several technological advances in PCR-based methods for the detection, identification, and quantitation of anaerobes including real-time PCR which has been successfully used to provide rapid, quantitative data on anaerobic species on clinical samples. Since its introduction in the mid-1980s, PCR has provided many molecular diagnostic tools, some of which are discussed within this review. With the advances in micro-array technology and real-time PCR methods, the future is bright for the development of accurate, quantitative diagnostic tools that can provide information not only on individual anaerobic species but also on whole communities.

  8. Direct chromatin PCR (DC-PCR): hypotonic conditions allow differentiation of chromatin states during thermal cycling.

    PubMed

    Vatolin, Sergei; Khan, Shahper N; Reu, Frederic J

    2012-01-01

    Current methods to study chromatin configuration are not well suited for high throughput drug screening since they require large cell numbers and multiple experimental steps that include centrifugation for isolation of nuclei or DNA. Here we show that site specific chromatin analysis can be achieved in one step by simply performing direct chromatin PCR (DC-PCR) on cells. The basic underlying observation was that standard hypotonic PCR buffers prevent global cellular chromatin solubilization during thermal cycling while more loosely organized chromatin can be amplified. Despite repeated heating to >90 °C, 41 of 61 tested 5' sequences of silenced genes (CDKN2A, PU.1, IRF4, FOSB, CD34) were not amplifiable while 47 could be amplified from expressing cells. Two gene regions (IRF4, FOSB) even required pre-heating of cells in isotonic media to allow this differentiation; otherwise none of 19 assayed sequences yielded PCR products. Cells with baseline expression or epigenetic reactivation gave similar DC-PCR results. Silencing during differentiation of CD34 positive cord blood cells closed respective chromatin while treatment of myeloma cells with an IRF4 transcriptional inhibitor opened a site to DC-PCR that was occupied by RNA polymerase II and NFκB as determined by ChIP. Translation into real-time PCR can not be achieved with commercial real-time PCR buffers which potently open chromatin, but even with simple ethidium bromide addition to standard PCR mastermix we were able to identify hits in small molecules screens that suppressed IRF4 expression or reactivated CDKN2A in myeloma cells using densitometry or visual inspection of PCR plates under UV light. While need in drug development inspired this work, application to genome-wide analysis appears feasible using phi29 for selective amplification of open cellular chromatin followed by library construction from supernatants since such supernatants yielded similar results as gene specific DC-PCR.

  9. [DNA amplification using PCR with abutting primers].

    PubMed

    Garafutdinov, R R; Galimova, A A; Sakhabutdinova, A R; Vakhitov, V A; Chemeris, A V

    2015-01-01

    DNA analysis of ñîmplex biological objects (wastewater, soil, archaeological and forensic samples, etc.) is currently of great interest. DNA of these objects is characterized by low suitability for research due to the violation of its integrity and chemical structure; thus, the detection of specific nucleic acid fragments can be achieved by PCR with contiguous primers. In this paper, we present the results that clarify the specific characteristics of PCR with abutting primers. The 3'-ends of these primers are annealed at adjacent nucleotides of complementary chains of DNA target. It has been shown that the proximity of primers enables the formation of specific reaction products with a higher sensitivity and less reaction time. Using artificially damaged DNA and DNA from the soil we demonstrated that the abutting primers provide assured detection of specific DNA fragments. The results of this work may be taken into account in PCR with degraded (fragmented) DNA.

  10. 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...

  11. [A novel quantitative PCR with fluorogenic probe].

    PubMed

    Isono, K

    1997-03-01

    The polymerase chain reaction(PCR) is a powerful tool to amplify small amounts of DNA or RNA for various molecular analysis. However, in these analyses, PCR only provides qualitative results. The availability of quantitative PCR provides valuable additional information in various applications. It is difficult to establish absolute quantitation, because PCR amplification is a complicated reaction process of exponential growth. To trace the amplification process, the initial amount of template and the efficiency of amplification in each cycle, has to be determined. Conventional methods have not achieved absolute quantitative analysis. The ABI PRISM 7700 Sequence Detection System has solved these problems with real-time monitoring of the PCR process. The real-time detection system provides essential information to quantify the initial target copy number, because it can draw an amplification curve. Using the 5' nuclease assay, a specific fluorescent signal is generated and measured at every cycle during a run. This system can perform a variety of applications including, quantitation, allele discrimination, PCR optimization and viral screening. Using the ABI PRISM 7700 Sequence Detection System, the rice genome has been quantitatively analyzed. To monitor maturation of the chloroplast genome from proplastid during germ development, 5' nuclease assay set up for Cab and rbcL genes which are located in the nuclear genome and chloroplast genome, respectively. Cab was used as an internal standard for normalization of cell numbers. The maturation process of chloroplast was estimated using the ratio of gene dosage, [rbcL]/[Cab]. After development of cotyledon, a significant increase in copy numbers of the chloroplast was observed. These results indicate that a light-induced chloroplast maturation process is coupled with an increase in chloroplast genome copy numbers.

  12. 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...

  13. 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…

  14. Modeling PCR in Natural Convection Systems

    NASA Astrophysics Data System (ADS)

    Dorfman, Kevin; Yariv, Ehud; Ben Dov, Guy

    2007-03-01

    Polymerase chain reaction (PCR) is a biochemical protocol for making many copies of a DNA template by thermal cycling between a hot temperature (where the strands are separated) and a cool temperature (where primers are annealed). In natural convection PCR, the requisite thermal cycling is provided by a buoyancy-driven circulating flow of the carrying buffer between a lower hot plate (at the denaturing temperature) and an upper cold plate (at the annealing temperature). We present a multi-component convection-diffusion-reaction model for natural convection-driven PCR when both primers and PCR enzyme are in excess. The evolution of the DNA population achieves a stationary state, wherein the problem is recast as an eigenvalue problem for computing the exponential amplification rate. With a realistic choice of parameters, the model predicts a doubling time on the order of two minutes, in agreement with experiments and much slower than the fluid cycling time. In contrast to what might be expected, the doubling time increases monotonically with the diffusion coefficient.

  15. [Mutation detection by PCR-TGGE].

    PubMed

    Shirakawa, T; Nishiyama, K; Matsuo, M

    1995-07-01

    The variants in enterotoxigenic Escherichia coli (ETEC) heat-labile toxin (LT) gene were detected by temperature gradient gel electrophoresis (TGGE). 15 clinical strains isolated from patients and a wild type strain (B2C) were analyzed after the conventional PCR. Although all PCR products (707bp) corresponded to A-subunit of LT, three strains were the different electrophoretic patterns after silver staining as compared to the wild type. For further electrophoretic analyses, the 707bp region was divided into 4 parts. The different ones were localized in the downstream part (183bp), but each those DNA bands was not so clear than DNA bands in 707bp. The clearer patterns were obtained by using a primer attached GC-clamp. The hetero-duplex assays in TGGE were proceeded by a series of procedures in mixing with the equal quantity of PCR products derived from a variant and a wild type, heat-denaturation and then annealing. TGGE of those mixed samples had 4 bands that were 2 front bands as homo-duplex and 2 slower migration bands as hetero-duplex. To Confirm the site of the mutations, the nucleotide sequences in each 183bp PCR products were decided by dideoxynucleotide-fluorescent dye method. Indeed, two variants were recognized four one-base substitutions without deletions and the one was five. Thus, the difference of migration in TGGE depended on the number and the localization in mutation sites.

  16. DNA profiles from fingernails using direct PCR.

    PubMed

    Ottens, Renée; Taylor, Duncan; Linacre, Adrian

    2015-03-01

    We report on the successful routine amplification of DNA profiles from small sections of fingernails using direct PCR. The data are from 40 nail clippings from eight donors where approximately 4 mm(2) of nail is added directly to the PCR. The NGM™ kit was used that amplifies 15 STR loci plus amelogenin. No increase in cycle number was used and no enrichment of the PCR products was performed. Full DNA profiles were observed in 17 of the 40 profiles with 21 generating partial DNA profiles. The process omits the DNA extraction process, and hence there is no opportunity to quantify the DNA prior to amplifying the STRs, but by not performing a DNA extraction step, the amount of DNA available for PCR is maximized. Single source DNA profiles were observed in 29 of the 38 profiles obtained. The source of the DNA is assumed to be adhering to the underside of the nail. This simple method offers a significant reduction in time to generate DNA profiles from nail clippings, such as those taken from victims of mass disasters, and should be included into a forensic process relatively easily as it requires no change to manufacturer's instructions for amplification.

  17. 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.

  18. 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.

  19. Multiplex digital PCR: breaking the one target per color barrier of quantitative PCR.

    PubMed

    Zhong, Qun; Bhattacharya, Smiti; Kotsopoulos, Steven; Olson, Jeff; Taly, Valérie; Griffiths, Andrew D; Link, Darren R; Larson, Jonathan W

    2011-07-01

    Quantitative polymerase chain reactions (qPCR) based on real-time PCR constitute a powerful and sensitive method for the analysis of nucleic acids. However, in qPCR, the ability to multiplex targets using differently colored fluorescent probes is typically limited to 4-fold by the spectral overlap of the fluorophores. Furthermore, multiplexing qPCR assays requires expensive instrumentation and most often lengthy assay development cycles. Digital PCR (dPCR), which is based on the amplification of single target DNA molecules in many separate reactions, is an attractive alternative to qPCR. Here we report a novel and easy method for multiplexing dPCR in picolitre droplets within emulsions-generated and read out in microfluidic devices-that takes advantage of both the very high numbers of reactions possible within emulsions (>10(6)) as well as the high likelihood that the amplification of only a single target DNA molecule will initiate within each droplet. By varying the concentration of different fluorogenic probes of the same color, it is possible to identify the different probes on the basis of fluorescence intensity. Adding multiple colors increases the number of possible reactions geometrically, rather than linearly as with qPCR. Accurate and precise copy numbers of up to sixteen per cell were measured using a model system. A 5-plex assay for spinal muscular atrophy was demonstrated with just two fluorophores to simultaneously measure the copy number of two genes (SMN1 and SMN2) and to genotype a single nucleotide polymorphism (c.815A>G, SMN1). Results of a pilot study with SMA patients are presented.

  20. 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 ...

  1. 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%.

  2. The Next-Generation PCR-Based Quantification Method for Ambient Waters: Digital PCR.

    PubMed

    Cao, Yiping; Griffith, John F; Weisberg, Stephen B

    2016-01-01

    Real-time quantitative PCR (qPCR) is increasingly being used for ambient water monitoring, but development of digital polymerase chain reaction (digital PCR) has the potential to further advance the use of molecular techniques in such applications. Digital PCR refines qPCR by partitioning the sample into thousands to millions of miniature reactions that are examined individually for binary endpoint results, with DNA density calculated from the fraction of positives using Poisson statistics. This direct quantification removes the need for standard curves, eliminating the labor and materials associated with creating and running standards with each batch, and removing biases associated with standard variability and mismatching amplification efficiency between standards and samples. Confining reactions and binary endpoint measurements to small partitions also leads to other performance advantages, including reduced susceptibility to inhibition, increased repeatability and reproducibility, and increased capacity to measure multiple targets in one analysis. As such, digital PCR is well suited for ambient water monitoring applications and is particularly advantageous as molecular methods move toward autonomous field application. PMID:27460373

  3. 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.

  4. 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

  5. [Indications for PCR in travel medicine].

    PubMed

    Chappuis, F

    2011-05-11

    The use of PCR-based molecular diagnosis in travel medicine remains limited to specific indications such as clinical suspicion of some of the viral hemorrhagic fevers (e.g. Ebola, Marburg), differential diagnosis between Entamoeba histolytica (pathogen) and E. dispar (non pathogen) in the stools, and parasitological diagnosis of cutaneous leishmaniasis. The scope of indications is likely to expand in the coming years with the development of techniques (e.g. multiplex PCR) able to identify several pathogens from a single sample. Simplification and cost-reduction of molecular techniques, which would allow for more equitable access to these diagnostic tools in countries where the targeted diseases are highly prevalent, pose major technological and ethical challenges.

  6. 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

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

    PubMed

    Zhao, Yun; Xia, Qingyan; 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

  8. 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. PMID:25828047

  9. Clostridium difficile PCR Ribotypes in Calves, Canada

    PubMed Central

    Stämpfli, Henry R.; Duffield, Todd; Peregrine, Andrew S.; Trotz-Williams, Lise A.; Arroyo, Luis G.; Brazier, Jon S.; Weese, J. Scott

    2006-01-01

    We investigated Clostridium difficile in calves and the similarity between bovine and human C. difficile PCR ribotypes by conducting a case-control study of calves from 102 dairy farms in Canada. Fecal samples from 144 calves with diarrhea and 134 control calves were cultured for C. difficile and tested with an ELISA for C. difficile toxins A and B. C. difficile was isolated from 31 of 278 calves: 11 (7.6%) of 144 with diarrhea and 20 (14.9%) of 134 controls (p = 0.009). Toxins were detected in calf feces from 58 (56.8%) of 102 farms, 57 (39.6%) of 144 calves with diarrhea, and 28 (20.9%) of 134 controls (p = 0.0002). PCR ribotyping of 31 isolates showed 8 distinct patterns; 7 have been identified in humans, 2 of which have been associated with outbreaks of severe disease (PCR types 017 and 027). C. difficile may be associated with calf diarrhea, and cattle may be reservoirs of C. difficile for humans. PMID:17283624

  10. A PCR amplification method without DNA extraction.

    PubMed

    Li, Hongwei; Xu, Haiyue; Zhao, Chunjiang; Sulaiman, Yiming; Wu, Changxin

    2011-02-01

    To develop a simple and inexpensive method for direct PCR amplification of animal DNA from tissues, we optimized different components and their concentration in lysis buffer systems. Finally, we acquired the optimized buffer system composed of 10 mmol tris(hydroxymethyl)aminomethane (Tris)-Cl (pH 8.0), 2 mmol ethylene diamine tetraacetic (EDTA) (pH 8.0), 0.2 mol NaCl and 200 μg/mL Proteinase K. Interestingly, the optimized buffer is also very effective when working with common human sample types, including blood, buccal cells and hair. The direct PCR method requires fewer reagents (Tris-Cl, EDTA, Protease K and NaCl) and less incubation time (only 35 min). The cost of treating every sample is less than $0.02, and all steps can be completed on a thermal cycler in a 96-well format. So, the proposed method will significantly improve high-throughput PCR-based molecular assays in animal systems and in common human sample types.

  11. Recent Developments in Miniaturized PCR-Microchips, Microarrays and Microdroplets

    PubMed Central

    Pollak, Eleanor S; Fortina, Paolo

    2012-01-01

    Microminiaturization of assays and lab-on-a-chip devices hold considerable promise for the future of analysis, especially in point-of-care testing. This article focuses on developments that have occurred during the last five years in the specific area of microchip PCR and miniaturized PCR in arrays of reaction vessels and droplets. Although, this area continues to be an active focus of research and development and the variety and ingenuity of microchip PCR and integrated microchip PCR devices continue to increase, commercialization lags behind the progress being made in digital PCR and arrays for real-time PCR.

  12. 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

  13. 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.

  14. Comparative detection of rabies RNA by NASBA, real-time PCR and conventional PCR.

    PubMed

    Wacharapluesadee, Supaporn; Phumesin, Patta; Supavonwong, Pornpun; Khawplod, Pakamatz; Intarut, Nirun; Hemachudha, Thiravat

    2011-08-01

    Five methods for the RNA detection of rabies virus were directly compared in this study. These included conventional nucleic acid sequence-based amplification with electrochemiluminescence (NASBA-ECL) assay, reverse transcription (RT)-heminested (hn) polymerase chain reaction (PCR) and TaqMan real-time RT-PCR using protocols as described previously. The first two methods have been routinely utilised for ante-mortem diagnosis of human rabies in Thailand and other rabies-endemic Asian and African countries. In addition, two real-time NASBA assays based on the use of a NucliSens EasyQ analyser (NASBA-Beacon-EQ) and LightCycler real-time PCR machine (NASBA-Beacon-LC) were studied in parallel. All methods target the N gene, whereas the L gene is used for RT-hnPCR. Using serial dilutions of purified RNA from rabies-infected dog brain tissue to assess sensitivity, all five methods had comparable degrees of sensitivities of detection. However, both real-time NASBA assays had slightly lower sensitivities by 10-fold than the other three assays. This finding was also true (except for TaqMan real-time RT-PCR due to a mismatch between the target and probe sequences) when laboratory-adapted (challenge virus standard-11) virus was used in the assays. Testing on previously NASBA-ECL positive clinical samples from 10 rabies patients (saliva [6] and brain [4]) and 10 rabies-infected dog brain tissues, similar results were obtained among the five methods; real-time NASBA assays yielded false-negative results on 2 saliva samples. None of the assays showed positive results on cerebrospinal fluid specimens of 10 patients without rabies encephalitis. Due to the unavailability of the NASBA-ECL assay, the results show that TaqMan real-time RT-PCR and RT-hnPCR can be useful for ante- and post-mortem diagnosis of rabies.

  15. 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

  16. Species identification and quantification in meat and meat products using droplet digital PCR (ddPCR).

    PubMed

    Floren, C; Wiedemann, I; Brenig, B; Schütz, E; Beck, J

    2015-04-15

    Species fraud and product mislabelling in processed food, albeit not being a direct health issue, often results in consumer distrust. Therefore methods for quantification of undeclared species are needed. Targeting mitochondrial DNA, e.g. CYTB gene, for species quantification is unsuitable, due to a fivefold inter-tissue variation in mtDNA content per cell resulting in either an under- (-70%) or overestimation (+160%) of species DNA contents. Here, we describe a reliable two-step droplet digital PCR (ddPCR) assay targeting the nuclear F2 gene for precise quantification of cattle, horse, and pig in processed meat products. The ddPCR assay is advantageous over qPCR showing a limit of quantification (LOQ) and detection (LOD) in different meat products of 0.01% and 0.001%, respectively. The specificity was verified in 14 different species. Hence, determining F2 in food by ddPCR can be recommended for quality assurance and control in production systems.

  17. 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.

  18. mRNA profiling for body fluid identification by reverse transcription endpoint PCR and realtime PCR.

    PubMed

    Haas, C; Klesser, B; Maake, C; Bär, W; Kratzer, A

    2009-03-01

    mRNA profiling is a promising new method for the identification of body fluids from biological stains. Major advantages of mRNA profiling are the possibility of detecting several body fluids in one multiplex reaction and of simultaneously isolating DNA without loss of material. A reverse transcription endpoint polymerase chain reaction (PCR) method and a realtime PCR assay were established for the identification of blood, saliva, semen, vaginal secretions and menstrual blood, and were compared to conventional enzymatic and immunologic tests. The results for specificity, sensitivity and suitability to biological stains were satisfying and mRNA stability was demonstrated for up to 2-year-old stains. Two novel multiplex assays were created with the endpoint PCR primers: multiplex 1 amplifies two markers for each of the above mentioned body fluids and is suited for screening; multiplex 2 was designed for the detection of blood, vaginal secretions and menstrual blood. The results demonstrate that both endpoint PCR and realtime PCR are suitable for the identification of body fluids in forensic stains and represent an effective alternative to conventional enzymatic and immunologic tests.

  19. Optimized PCR-based detection of mycoplasma.

    PubMed

    Dobrovolny, Paige L; Bess, Dan

    2011-06-20

    The maintenance of contamination-free cell lines is essential to cell-based research. Among the biggest contaminant concerns are mycoplasma contamination. Although mycoplasma do not usually kill contaminated cells, they are difficult to detect and can cause a variety of effects on cultured cells, including altered metabolism, slowed proliferation and chromosomal aberrations. In short, mycoplasma contamination compromises the value of those cell lines in providing accurate data for life science research. The sources of mycoplasma contamination in the laboratory are very challenging to completely control. As certain mycoplasma species are found on human skin, they can be introduced through poor aseptic technique. Additionally, they can come from contaminated supplements such as fetal bovine serum, and most importantly from other contaminated cell cultures. Once mycoplasma contaminates a culture, it can quickly spread to contaminate other areas of the lab. Strict adherence to good laboratory practices such as good aseptic technique are key, and routine testing for mycoplasma is highly recommended for successful control of mycoplasma contamination. PCR-based detection of mycoplasma has become a very popular method for routine cell line maintenance. PCR-based detection methods are highly sensitive and can provide rapid results, which allows researchers to respond quickly to isolate and eliminate contamination once it is detected in comparison to the time required using microbiological techniques. The LookOut Mycoplasma PCR Detection Kit is highly sensitive, with a detection limit of only 2 genomes per μl. Taking advantage of the highly specific JumpStart Taq DNA Polymerase and a proprietary primer design, false positives are greatly reduced. The convenient 8-tube format, strips pre-coated with dNTPs, and associated primers helps increase the throughput to meet the needs of customers with larger collections of cell lines. Given the extreme sensitivity of the kit, great

  20. 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 ...

  1. Multiplex PCR serogrouping of Listeria monocytogenes isolated in Japan

    PubMed Central

    SHIMOJIMA, Yukako; IDA, Miki; NISHINO, Yukari; ISHITSUKA, Rie; KURODA, Sumiyo; HIRAI, Akihiko; SADAMASU, Kenji; NAKAMA, Akiko; KAI, Akemi

    2015-01-01

    PCR serogrouping methods were used to examine strains of L. monocytogenes isolated in Japan. Among 187 strains, 99.5% were classified into 4 PCR serogroups corresponding to conventional serotypes. Only one isolate had a new PCR profile, which may be a variant of serogroup IVb. PMID:26537550

  2. An Efficient Multistrategy DNA Decontamination Procedure of PCR Reagents for Hypersensitive PCR Applications

    PubMed Central

    Pruvost, Mélanie; Bennett, E. Andrew; Grange, Thierry; Geigl, Eva-Maria

    2010-01-01

    Background PCR amplification of minute quantities of degraded DNA for ancient DNA research, forensic analyses, wildlife studies and ultrasensitive diagnostics is often hampered by contamination problems. The extent of these problems is inversely related to DNA concentration and target fragment size and concern (i) sample contamination, (ii) laboratory surface contamination, (iii) carry-over contamination, and (iv) contamination of reagents. Methodology/Principal Findings Here we performed a quantitative evaluation of current decontamination methods for these last three sources of contamination, and developed a new procedure to eliminate contaminating DNA contained in PCR reagents. We observed that most current decontamination methods are either not efficient enough to degrade short contaminating DNA molecules, rendered inefficient by the reagents themselves, or interfere with the PCR when used at doses high enough to eliminate these molecules. We also show that efficient reagent decontamination can be achieved by using a combination of treatments adapted to different reagent categories. Our procedure involves γ- and UV-irradiation and treatment with a mutant recombinant heat-labile double-strand specific DNase from the Antarctic shrimp Pandalus borealis. Optimal performance of these treatments is achieved in narrow experimental conditions that have been precisely analyzed and defined herein. Conclusions/Significance There is not a single decontamination method valid for all possible contamination sources occurring in PCR reagents and in the molecular biology laboratory and most common decontamination methods are not efficient enough to decontaminate short DNA fragments of low concentration. We developed a versatile multistrategy decontamination procedure for PCR reagents. We demonstrate that this procedure allows efficient reagent decontamination while preserving the efficiency of PCR amplification of minute quantities of DNA. PMID:20927390

  3. [Ticks Ixodes ricinus (Linnaeus, 1758) from selected municipal forests of the city Poznań and their infection with the spirochetes Borrelia burgdorferi senso lato].

    PubMed

    Nowosad, A; Jenek, J; Głazaczow, A; Wal, M

    1999-01-01

    The results of research (1997-1998) on the occurrence of the spirochetes Borrelia burgdorferi s.l. in ticks Ixodes ricinus (L.) in selected municipal forests of the city of Poznań (localities--districts of Poznań: 1 Debiec, 2 Marcelin, 3 Golecin and Wola, 4 Krzyzowniki and Smochowice, 5 Kiekrz and Strzeszynek, 6 Morasko, 7 Piatkowo, 8 Umultowo, 9 Naramowice and Rózany Młyn, 10 Malta and Antoninek) are presented. A total of 1432 ticks were collected from 10 localities (748 larvae, 590 nymphs, 47 males, and 47 females). Out of this number, 266 specimens were selected at random for further analysis (20 larvae, 160 nymphs, 44 males, and 42 females) which were then tested for the presence of the spirochetes using the PCR technique (tab. I-III, fig. 1). Spirochetes were found in 60 specimens (22.6%). Percentages of infected nymphs and males were similar: 25.6% and 27.3%, respectively. The level of infection of females was lower (14.3%) and of larvae lowest (5.0%). Infected ticks were found in all ten localities, but their proportions differed from site to site and varied from 9.5% (Piatkowo) to 34.6% (Krzyzowniki and Smochowice). Special attention was devoted to those municipal woods where ticks were abundant (Kiekrz and Strzeszynek, as well as Malta and Antoninek, and relatively numerous Debiec) and where their extensiveness of infestation was high (Krzyzowniki and Smochowice: 34.6%; Debiec: 29.6%; Naramowice and Rózany Młyn: 28.6%; Malta and Antoninek: 28.1%; and Golecin and Wola: 26.9%). After summing up all the data from the literature on Wielkopolska and the city of Poznań, the mean extensiveness of infestation in Wielkopolska turns out to be 21.8% and in Poznań slightly higher, 22.2% (tab. IV). The infection levels of the particular developmental stages are different, though: highly variable in nymphs (8.3-25.6%), and more stable in males (22.0-27.3%) and females (22.0-24.5%).

  4. Heterozygote PCR product melting curve prediction.

    PubMed

    Dwight, Zachary L; Palais, Robert; Kent, Jana; Wittwer, Carl T

    2014-03-01

    Melting curve prediction of PCR products is limited to perfectly complementary strands. Multiple domains are calculated by recursive nearest neighbor thermodynamics. However, the melting curve of an amplicon containing a heterozygous single-nucleotide variant (SNV) after PCR is the composite of four duplexes: two matched homoduplexes and two mismatched heteroduplexes. To better predict the shape of composite heterozygote melting curves, 52 experimental curves were compared with brute force in silico predictions varying two parameters simultaneously: the relative contribution of heteroduplex products and an ionic scaling factor for mismatched tetrads. Heteroduplex products contributed 25.7 ± 6.7% to the composite melting curve, varying from 23%-28% for different SNV classes. The effect of ions on mismatch tetrads scaled to 76%-96% of normal (depending on SNV class) and averaged 88 ± 16.4%. Based on uMelt (www.dna.utah.edu/umelt/umelt.html) with an expanded nearest neighbor thermodynamic set that includes mismatched base pairs, uMelt HETS calculates helicity as a function of temperature for homoduplex and heteroduplex products, as well as the composite curve expected from heterozygotes. It is an interactive Web tool for efficient genotyping design, heterozygote melting curve prediction, and quality control of melting curve experiments. The application was developed in Actionscript and can be found online at http://www.dna.utah.edu/hets/.

  5. Prediction of neonatal alloimmune thrombocytopenia using PCR.

    PubMed

    Marshall, L R; Jones, C; Munro, T E

    1994-01-01

    Neonatal alloimmune thrombocytopenia (NAIT) is a potentially fatal condition and in the majority of cases is associated with maternal antibodies to the HPA-1a (PLA1) haplotype. Early diagnosis in utero can enhance survival rates. The application of DNA genomic analysis and PCR technology for the determination of the HPA-1a/HPA-1b (PLA1/PLA2) locus is described and applied in a family study where the fetus was diagnosed to have NAIT. This rapid technique differentiated between the 3 haplotypes HPA-1a/HPA-1a, HPA-1b/HPA-1b and HPA-1a/HPA-1b using the polymorphism at base 196 of the GPIIIa gene. This is the first Australian report on the establishment of this technology for platelet genotype typing and the application in the diagnosis of NAIT. This technique can be performed on DNA extracted from any nucleated cells and avoids the difficulty of requiring fetal platelets for serological typing when NAIT is suspected. The PCR technique of genomic DNA analysis has an important application in the prediction and management of this potentially severe condition.

  6. 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. PMID:25547077

  7. 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.

  8. Genetic Characterization of Human-Derived Hydatid Cysts of Echinococcus granulosus Sensu Lato in Heilongjiang Province and the First Report of G7 Genotype of E. canadensis in Humans in China

    PubMed Central

    Zeng, Zhaolin; Zhao, Wei; Liu, Aiqin; Piao, Daxun; Jiang, Tao; Cao, Jianping; Shen, Yujuan; Liu, Hua; Zhang, Weizhe

    2014-01-01

    Cystic echinococcosis (CE) caused by the larval stage of Echinococcus granulosus sensu lato (s.l.) is one of the most important zoonotic parasitic diseases worldwide and 10 genotypes (G1–G10) have been reported. In China, almost all the epidemiological and genotyping studies of E. granulosus s.l. are from the west and northwest pasturing areas. However, in Heilongjiang Province of northeastern China, no molecular information is available on E. granulosus s.l. To understand and to speculate on possible transmission patterns of E. granulosus s.l., we molecularly identified and genotyped 10 hydatid cysts from hepatic CE patients in Heilongjiang Province based on mitochondrial cytochrome c oxidase subunit I (cox1), cytochrome b (cytb) and NADH dehydrogenase subunit 1 (nad1) genes. Two genotypes were identified, G1 genotype (n = 6) and G7 genotype (n = 4). All the six G1 genotype isolates were identical to each other at the cox1 locus; three and two different sequences were obtained at the cytb and nad1 loci, respectively, with two cytb gene sequences not being described previously. G7 genotype isolates were identical to each other at the cox1, cytb and nad1 loci; however, the cytb gene sequence was not described previously. This is the first report of G7 genotype in humans in China. Three new cytb gene sequences from G1 and G7 genotypes might reflect endemic genetic characterizations. Pigs might be the main intermediate hosts of G7 genotype in our investigated area by homology analysis. The results will aid in making more effective control strategies for the prevention of transmission of E. granulosus s.l. PMID:25329820

  9. Genetic characterization of human-derived hydatid cysts of Echinococcus granulosus sensu lato in Heilongjiang Province and the first report of G7 genotype of E. canadensis in humans in China.

    PubMed

    Zhang, Tiemin; Yang, Dong; Zeng, Zhaolin; Zhao, Wei; Liu, Aiqin; Piao, Daxun; Jiang, Tao; Cao, Jianping; Shen, Yujuan; Liu, Hua; Zhang, Weizhe

    2014-01-01

    Cystic echinococcosis (CE) caused by the larval stage of Echinococcus granulosus sensu lato (s.l.) is one of the most important zoonotic parasitic diseases worldwide and 10 genotypes (G1-G10) have been reported. In China, almost all the epidemiological and genotyping studies of E. granulosus s.l. are from the west and northwest pasturing areas. However, in Heilongjiang Province of northeastern China, no molecular information is available on E. granulosus s.l. To understand and to speculate on possible transmission patterns of E. granulosus s.l., we molecularly identified and genotyped 10 hydatid cysts from hepatic CE patients in Heilongjiang Province based on mitochondrial cytochrome c oxidase subunit I (cox1), cytochrome b (cytb) and NADH dehydrogenase subunit 1 (nad1) genes. Two genotypes were identified, G1 genotype (n = 6) and G7 genotype (n = 4). All the six G1 genotype isolates were identical to each other at the cox1 locus; three and two different sequences were obtained at the cytb and nad1 loci, respectively, with two cytb gene sequences not being described previously. G7 genotype isolates were identical to each other at the cox1, cytb and nad1 loci; however, the cytb gene sequence was not described previously. This is the first report of G7 genotype in humans in China. Three new cytb gene sequences from G1 and G7 genotypes might reflect endemic genetic characterizations. Pigs might be the main intermediate hosts of G7 genotype in our investigated area by homology analysis. The results will aid in making more effective control strategies for the prevention of transmission of E. granulosus s.l.

  10. 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. PMID:27671928

  11. 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.

  12. DNA fingerprinting of medically important microorganisms by use of PCR.

    PubMed Central

    van Belkum, A

    1994-01-01

    Selected segments of any DNA molecule can be amplified exponentially by PCR. This technique provides a powerful tool to detect and identify minimal numbers of microorganisms. PCR is applicable both in diagnosis and in epidemiology. By amplification of hypervariable DNA domains, differences can be detected even among closely related strains. PCR fingerprinting is a valuable tool for medical microbiologists, epidemiologists, and microbial taxonomists. The current state of PCR-mediated genotyping is reviewed, and a comparison with conventional molecular typing methods is included. Because of its speed and versatility, PCR fingerprinting will play an important role in microbial genetics, epidemiology, and systematics. Images PMID:8055466

  13. 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.

  14. PDMS-based micro PCR chip with Parylene coating

    NASA Astrophysics Data System (ADS)

    Shin, Young Shik; Cho, Keunchang; Lim, Sun Hee; Chung, Seok; Park, Sung-Jin; Chung, Chanil; Han, Dong-Chul; Chang, Jun Keun

    2003-09-01

    We have developed a microchip for polymerase chain reaction (PCR) with polydimethylsiloxane (PDMS). PDMS has good characteristics: it is cheap, transparent, easy to fabricate and biocompatible. But in micro PCR, the porosity of PDMS causes several critical problems such as bubble formation, sample evaporation and protein adsorption. To solve those problems, we coated the micro PCR chips with Parylene film, which has low permeability to moisture and long-term stability. We investigated the influence of low thermal conductivity of PDMS and Parylene on the thermal characteristics of the PCR chips with numerical analysis. The thermal responses of micro PCR chips were compared for three materials: silicon, glass and PDMS. From the results, we identified appropriate thermal responses of the PDMS-based micro PCR chips by heating both the top and bottom sides. We could successfully amplify the angiotensin converting enzyme gene with as small a volume as 2 mul on the PDMS-based micro PCR chips without any additives.

  15. PCR and real-time PCR assays to detect fungi of Alternaria alternata species.

    PubMed

    Kordalewska, Milena; Brillowska-Dąbrowska, Anna; Jagielski, Tomasz; Dworecka-Kaszak, Bożena

    2015-01-01

    Fungi of the Alternaria genus are mostly associated with allergic diseases. However, with a growing number of immunocompromised patients, these fungi, with A. alternata being the most prevalent one, are increasingly recognized as etiological agents of infections (phaeohyphomycoses) in humans. Nowadays, identification of Alternaria spp. requires their pure culture and is solely based on morphological criteria. Clinically, Alternaria infections may be indistinguishable from other fungal diseases. Therefore, a diagnostic result is often delayed or even not achieved at all. In this paper we present easy to perform and interpret PCR and real-time PCR assays enabling detection of A. alternata species. On the basis of alignment of β-tubulin gene sequences, A. alternata-specific primers were designed. DNA from fungal isolates, extracted in a two-step procedure, were used in PCR and real-time PCR assays followed by electrophoresis or melting temperature analysis, respectively. The assays specificity was confirmed, since positive results were obtained for all A. alternata isolates, and no positive results were obtained neither for other molds, dermatophytes, yeast-like fungi, nor human DNA. The assays developed here enable fast and unambiguous identification of A. alternata pathogens. PMID:26610309

  16. PCR and real-time PCR assays to detect fungi of Alternaria alternata species.

    PubMed

    Kordalewska, Milena; Brillowska-Dąbrowska, Anna; Jagielski, Tomasz; Dworecka-Kaszak, Bożena

    2015-01-01

    Fungi of the Alternaria genus are mostly associated with allergic diseases. However, with a growing number of immunocompromised patients, these fungi, with A. alternata being the most prevalent one, are increasingly recognized as etiological agents of infections (phaeohyphomycoses) in humans. Nowadays, identification of Alternaria spp. requires their pure culture and is solely based on morphological criteria. Clinically, Alternaria infections may be indistinguishable from other fungal diseases. Therefore, a diagnostic result is often delayed or even not achieved at all. In this paper we present easy to perform and interpret PCR and real-time PCR assays enabling detection of A. alternata species. On the basis of alignment of β-tubulin gene sequences, A. alternata-specific primers were designed. DNA from fungal isolates, extracted in a two-step procedure, were used in PCR and real-time PCR assays followed by electrophoresis or melting temperature analysis, respectively. The assays specificity was confirmed, since positive results were obtained for all A. alternata isolates, and no positive results were obtained neither for other molds, dermatophytes, yeast-like fungi, nor human DNA. The assays developed here enable fast and unambiguous identification of A. alternata pathogens.

  17. Simultaneous Detection of Ricin and Abrin DNA by Real-Time PCR (qPCR)

    PubMed Central

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

    2012-01-01

    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. PMID:23105972

  18. 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.

  19. 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.

  20. PCR detection of uncultured rumen bacteria.

    PubMed

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

    2012-07-01

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

  1. 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.

  2. IDH1 mutation detection by droplet digital PCR in glioma.

    PubMed

    Wang, Jing; Zhao, Yi-ying; Li, Jian-feng; Guo, Cheng-cheng; Chen, Fu-rong; Su, Hong-kai; Zhao, Hua-fu; Long, Ya-kang; Shao, Jian-yong; To, Shing shun Tony; Chen, Zhong-ping

    2015-11-24

    Glioma is the most frequent central nervous system tumor in adults. The overall survival of glioma patients is disappointing, mostly due to the poor prognosis of glioblastoma (Grade IV glioma). Isocitrate dehydrogenase (IDH) is a key factor in metabolism and catalyzes the oxidative decarboxylation of isocitrate. Mutations in IDH genes are observed in over 70% of low-grade gliomas and some cases of glioblastoma. As the most frequent mutation, IDH1(R132H) has been served as a predictive marker of glioma patients. The recently developed droplet digital PCR (ddPCR) technique generates a large amount of nanoliter-sized droplets, each of which carries out a PCR reaction on one template. Therefore, ddPCR provides high precision and absolute quantification of the nucleic acid target, with wide applications for both research and clinical diagnosis. In the current study, we collected 62 glioma tissue samples (Grade II to IV) and detected IDH1 mutations by Sanger direct sequencing, ddPCR, and quantitative real-time PCR (qRT-PCR). With the results from Sanger direct sequencing as the standard, the characteristics of ddPCR were compared with qRT-PCR. The data indicated that ddPCR was much more sensitive and much easier to interpret than qRT-PCR. Thus, we demonstrated that ddPCR is a reliable and sensitive method for screening the IDH mutation. Therefore, ddPCR is able to applied clinically in predicting patient prognosis and selecting effective therapeutic strategies. Our data also supported that the prognosis of Grade II and III glioma was better in patients with an IDH mutation than in those without mutation.

  3. IDH1 mutation detection by droplet digital PCR in glioma

    PubMed Central

    Wang, Jing; Zhao, Yi-ying; Li, Jian-feng; Guo, Cheng-cheng; Chen, Fu-rong; Su, Hong-kai; Zhao, Hua-fu; Long, Ya-kang; Shao, Jian-yong; Tony To, Shing-shun; Chen, Zhong-ping

    2015-01-01

    Glioma is the most frequent central nervous system tumor in adults. The overall survival of glioma patients is disappointing, mostly due to the poor prognosis of glioblastoma (Grade IV glioma). Isocitrate dehydrogenase (IDH) is a key factor in metabolism and catalyzes the oxidative decarboxylation of isocitrate. Mutations in IDH genes are observed in over 70% of low-grade gliomas and some cases of glioblastoma. As the most frequent mutation, IDH1(R132H) has been served as a predictive marker of glioma patients. The recently developed droplet digital PCR (ddPCR) technique generates a large amount of nanoliter-sized droplets, each of which carries out a PCR reaction on one template. Therefore, ddPCR provides high precision and absolute quantification of the nucleic acid target, with wide applications for both research and clinical diagnosis. In the current study, we collected 62 glioma tissue samples (Grade II to IV) and detected IDH1 mutations by Sanger direct sequencing, ddPCR, and quantitative real-time PCR (qRT-PCR). With the results from Sanger direct sequencing as the standard, the characteristics of ddPCR were compared with qRT-PCR. The data indicated that ddPCR was much more sensitive and much easier to interpret than qRT-PCR. Thus, we demonstrated that ddPCR is a reliable and sensitive method for screening the IDH mutation. Therefore, ddPCR is able to applied clinically in predicting patient prognosis and selecting effective therapeutic strategies. Our data also supported that the prognosis of Grade II and III glioma was better in patients with an IDH mutation than in those without mutation. PMID:26485760

  4. Diagnostic utility of droplet digital PCR for HIV reservoir quantification.

    PubMed

    Trypsteen, Wim; Kiselinova, Maja; Vandekerckhove, Linos; De Spiegelaere, Ward

    2016-01-01

    Quantitative real-time PCR (qPCR) is implemented in many molecular laboratories worldwide for the quantification of viral nucleic acids. However, over the last two decades, there has been renewed interest in the concept of digital PCR (dPCR) as this platform offers direct quantification without the need for standard curves, a simplified workflow and the possibility to extend the current detection limit. These benefits are of great interest in terms of the quantification of low viral levels in HIV reservoir research because changes in the dynamics of residual HIV reservoirs will be important to monitor HIV cure efforts. Here, we have implemented a systematic literature screening and text mining approach to map the use of droplet dPCR (ddPCR) in the context of HIV quantification. In addition, several technical aspects of ddPCR were compared with qPCR: accuracy, sensitivity, precision and reproducibility, to determine its diagnostic utility. We have observed that ddPCR was used in different body compartments in multiple HIV-1 and HIV-2 assays, with the majority of reported assays focusing on HIV-1 DNA-based applications (i.e. total HIV DNA). Furthermore, ddPCR showed a higher accuracy, precision and reproducibility, but similar sensitivity when compared to qPCR due to reported false positive droplets in the negative template controls with a need for standardised data analysis (i.e. threshold determination). In the context of a low level of detection and HIV reservoir diagnostics, ddPCR can offer a valid alternative to qPCR-based assays but before this platform can be clinically accredited, some remaining issues need to be resolved. PMID:27482456

  5. 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

  6. Posttreatment Follow-Up of Brucellosis by PCR Assay

    PubMed Central

    Morata, Pilar; Queipo-Ortuño, María Isabel; Reguera, José María; García-Ordoñez, Miguel Angel; Pichardo, Cristina; Colmenero, Juan de Dios

    1999-01-01

    In order to evaluate the usefulness of a peripheral blood PCR assay in the posttreatment follow-up of brucellosis, a cohort of 30 patients was studied by means of blood cultures, rose Bengal, seroagglutination, Coombs' antibrucella tests, and PCR assay at the time of diagnosis, at the end of treatment, and 2, 4, and 6 months later. Of the 29 patients whose PCR assays were initially positive, 28 (96.5%) were negative at the conclusion of the treatment. PCR was positive for the two patients who had relapses and negative for another four who had suspected but unconfirmed relapses. PCR was negative for 98.3% of the follow-up samples from those patients who had a favorable evolution. In conclusion, PCR appears to be a very useful technique, not only for the initial diagnosis of the disease, but also for posttreatment follow-up and the early detection of relapses. PMID:10565954

  7. [Development of PCR methods for detection of EAV infection].

    PubMed

    Brunner; Santschi; Gerber; Burger; Zanoni

    2014-11-01

    The goal of this work was the development of suitable (real-time) RT-PCR techniques for fast and sensitive diagnosis of EAV and for molecular-epidemiological characterisation of viral strains, as an alternative to virus isolation. To this purpose two conventional RT-PCR methods and one real-time RT-PCR were adapted to detect the broadest possible spectrum of viral strains. Several dilutions with Bucyrus strain showed a 100-fold higher sensitivity of real-time RT-PCR and heminested RT-PCR compared to simple RT-PCR. Making use of 11 cell culture supernatants of different EAV isolates and 7 semen samples of positive stallions, the suitability of the techniques could be shown. Phylogenetic analysis of sequences of the newly analysed samples compared with known sequences indicated that more EAV-lineages exist than presently described.

  8. 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. PMID:26843050

  9. 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.

  10. 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.

  11. 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.

  12. Influence of reagents formulation on real-time PCR parameters.

    PubMed

    Burgos, J S; Ramírez, C; Tenorio, R; Sastre, I; Bullido, M J

    2002-08-01

    Real-time polymerase chain reaction (PCR) techniques are increasingly used to quantify target sequences for diagnostic and research purposes. Due to its 'quantitative' character, it is very important to determine the variability of this technique correlating with several experimental conditions. The objective of this study was to analyse the effect of manufacturing lots of PCR reagents on two main PCR parameters, specificity and sensitivity. For this study, we used four different amplicons, using either mouse genomic DNA or viral DNA. Although a PCR product could be obtained in any of the conditions, we observed that there are relevant variations in sensitivity depending on the reagents formulation. We conclude that different lots of reagents may determine the analytical performance of PCR assays indicating that reagents testing are of special importance when the PCR protocol is used for quantitative purposes.

  13. 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 Central

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

    2014-01-01

    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. PMID:25437051

  14. Chimerism testing by quantitative PCR using Indel markers.

    PubMed

    Gendzekhadze, Ketevan; Gaidulis, Laima; Senitzer, David

    2013-01-01

    Engraftment monitoring is critical for patients after Hematopoietic Stem Cell Transplantation (HSCT). Complete donor chimerism is the goal; therefore, early detection of rejection and relapse is crucial for guiding the patient post HSCT treatment. Quantitative PCR for chimerism testing has been reported to be highly sensitive. In this chapter we discuss the quantitative PCR (qPCR) method using 34 Indel (Insertion and Deletion) genetic markers spread over 20 different chromosomes.

  15. [PCR-ELISA technology and its application in biomedical fields].

    PubMed

    Yan, Lin; Wang, Xiaoying; Guo, Yunchang

    2011-01-01

    Polymerase chain reaction-enzyme-linked immunosorbent assay (PCR- ELISA) technology is created by the use of the high sensitivity of PCR, nucleic acid probes of the specificity, microplate reader of objectivity; since its establishment, it has received wide attention. In this paper, a basic description of the technology was introduced, and the application of PCR-ELISA in biomedical field in recent years were reviewed.

  16. A simple, universal, efficient PCR-based gene synthesis method: sequential OE-PCR gene synthesis.

    PubMed

    Zhang, Pingping; Ding, Yingying; Liao, Wenting; Chen, Qiuli; Zhang, Huaqun; Qi, Peipei; He, Ting; Wang, Jinhong; Deng, Songhua; Pan, Tianyue; Ren, Hao; Pan, Wei

    2013-07-25

    Herein we present a simple, universal, efficient gene synthesis method based on sequential overlap extension polymerase chain reactions (OE-PCRs). This method involves four key steps: (i) the design of paired complementary 54-mer oligonucleotides with 18 bp overlaps, (ii) the utilisation of sequential OE-PCR to synthesise full-length genes, (iii) the cloning and sequencing of four positive T-clones of the synthesised genes and (iv) the resynthesis of target genes by OE-PCR with correct templates. Mispriming and secondary structure were found to be the principal obstacles preventing successful gene synthesis and were easily identified and solved in this method. Compensating for the disadvantages of being laborious and time-consuming, this method has many attractive advantages, such as the ability to guarantee successful gene synthesis in most cases and good allowance for Taq polymerase, oligonucleotides, PCR conditions and a high error rate. Thus, this method provides an alternative tool for individual gene synthesis without strict needs of the high-specialised experience. PMID:23597923

  17. 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.

  18. 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.

  19. Detection and quantification of chimerism by droplet digital PCR.

    PubMed

    George, David; Czech, Juliann; John, Bobby; Yu, Min; Jennings, Lawrence J

    2013-01-01

    Accurate quantification of chimerism and microchimerism is proving to be increasingly valuable for hematopoietic cell transplantation as well as non-transplant conditions. However, methods that are available to quantify low-level chimerism lack accuracy. Therefore, we developed and validated a method for quantifying chimerism based on digital PCR technology. We demonstrate accurate quantification that far exceeds what is possible with analog qPCR down to 0.01% with the potential to go even lower. Also, this method is inherently more informative than qPCR. We expect the advantages of digital PCR will make it the preferred method for chimerism analysis.

  20. Evaluation of Digital PCR for Absolute RNA Quantification

    PubMed Central

    Sanders, Rebecca; Mason, Deborah J.; Foy, Carole A.; Huggett, Jim F.

    2013-01-01

    Gene expression measurements detailing mRNA quantities are widely employed in molecular biology and are increasingly important in diagnostic fields. Reverse transcription (RT), necessary for generating complementary DNA, can be both inefficient and imprecise, but remains a quintessential RNA analysis tool using qPCR. This study developed a Transcriptomic Calibration Material and assessed the RT reaction using digital (d)PCR for RNA measurement. While many studies characterise dPCR capabilities for DNA quantification, less work has been performed investigating similar parameters using RT-dPCR for RNA analysis. RT-dPCR measurement using three, one-step RT-qPCR kits was evaluated using single and multiplex formats when measuring endogenous and synthetic RNAs. The best performing kit was compared to UV quantification and sensitivity and technical reproducibility investigated. Our results demonstrate assay and kit dependent RT-dPCR measurements differed significantly compared to UV quantification. Different values were reported by different kits for each target, despite evaluation of identical samples using the same instrument. RT-dPCR did not display the strong inter-assay agreement previously described when analysing DNA. This study demonstrates that, as with DNA measurement, RT-dPCR is capable of accurate quantification of low copy RNA targets, but the results are both kit and target dependent supporting the need for calibration controls. PMID:24073259

  1. Detection and identification of Lactobacillus helveticus bacteriophages by PCR.

    PubMed

    Zago, Miriam; Rossetti, Lia; Reinheimer, Jorge; Carminati, Domenico; Giraffa, Giorgio

    2008-05-01

    A PCR protocol for detection of Lactobacillus helveticus bacteriophages was optimized. PCR was designed taking into account the sequence of the lys gene of temperate bacteriophage Phi-0303 and optimized to obtain a fragment of 222 bp using different Lb. helveticus phages from our collection. PCR was applied to total phage DNA extracted from 53 natural whey starters used for the production of Grana cheese and all gave the expected fragment. The presence of actively growing phages in the cultures was verified by traditional tests. Several PCR products of the lys gene were sequenced and aligned. The resulting sequences showed variable heterogeneity between the phages. PMID:18474137

  2. 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.

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

    PubMed

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

    2016-08-01

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

  4. Prevalence of Dirofilaria immitis, Ehrlichia canis, Borrelia burgdorferi sensu lato, Anaplasma spp. and Leishmania infantum in apparently healthy and CVBD-suspect dogs in Portugal - a national serological study

    PubMed Central

    2012-01-01

    Background Canine vector-borne diseases (CVBDs) are caused by a wide range of pathogens transmitted to dogs by arthropods including ticks and insects. Many CVBD-agents are of zoonotic concern, with dogs potentially serving as reservoirs and sentinels for human infections. The present study aimed at assessing the seroprevalence of infection with or exposure to Dirofilaria immitis, Ehrlichia canis, Borrelia burgdorferi sensu lato, Anaplasma spp. and Leishmania infantum in dogs in Portugal. Methods Based on 120 veterinary medical centres from all the regions of mainland and insular Portugal, 557 apparently healthy and 628 CVBD-suspect dogs were sampled. Serum, plasma or whole blood was tested for qualitative detection of D. immitis antigen and antibodies to E. canis, B. burgdorferi s. l., Anaplasma spp. and L. infantum with two commercial in-clinic enzyme-linked immunosorbent assay kits. Odds ratios (OR) were calculated by logistic regression analysis to identify independent risk factors of exposure to the vector-borne agents. Results Total positivity levels to D. immitis, E. canis, B. burgdorferi, Anaplasma spp., L. infantum, one or more agents and mixed agents were 3.6%, 4.1%, 0.2%, 4.5%, 4.3%, 14.0% and 2.0% in the healthy group, and 8.9%, 16.4%, 0.5%, 9.2%, 25.2%, 46.3% and 11.6% in the clinically suspect group, respectively. Non-use of ectoparasiticides was a risk factor for positivity to one or more agents both in the apparently healthy (OR = 2.1) and CVBD-suspect (OR = 1.5) dogs. Seropositivity to L. infantum (OR = 7.6), E. canis (OR = 4.1) and D. immitis (OR = 2.4) were identified as risk factors for the presence of clinical signs compatible with CVBDs. Positivity to mixed agents was not found to be a risk factor for disease. Conclusions Dogs in Portugal are at risk of becoming infected with vector-borne pathogens, some of which are of zoonotic concern. CVBDs should be considered by practitioners and prophylactic measures must be put in place to protect dogs

  5. Global RT-PCR and RT-qPCR Analysis of the mRNA Expression of the Human PTPome.

    PubMed

    Nunes-Xavier, Caroline E; Pulido, Rafael

    2016-01-01

    Comprehensive comparative gene expression analysis of the tyrosine phosphatase superfamily members (PTPome) under cell- or tissue-specific growth conditions may help to define their individual and specific role in physiology and disease. Semi-quantitative and quantitative PCR are commonly used methods to analyze and measure gene expression. Here, we describe technical aspects of PTPome mRNA expression analysis by semi-quantitative RT-PCR and quantitative RT-PCR (RT-qPCR). We provide a protocol for each method consisting in reverse transcription followed by PCR using a global platform of specific PTP primers. The chapter includes aspects from primer validation to the setup of the PTPome RT-qPCR platform. Examples are given of PTP-profiling gene expression analysis using a human breast cancer cell line upon long-term or short-term treatment with cell signaling-activation agents. PMID:27514798

  6. Monitoring gene expression: quantitative real-time rt-PCR.

    PubMed

    Wagner, Elke M

    2013-01-01

    Two-step quantitative real-time RT-PCR (RT-qPCR), also known as real-time RT-PCR, kinetic RT-PCR, or quantitative fluorescent RT-PCR, has become the method of choice for gene expression analysis during the last few years. It is a fast and convenient PCR method that combines traditional RT-PCR with the phenomenon of fluorescence resonance energy transfer (FRET) using fluorogenic primers. The detection of changes in fluorescence intensity during the reaction enables the user to follow the PCR reaction in real time.RT-qPCR comprises several steps: (1) RNA is isolated from target tissue/cells; (2) mRNA is reverse-transcribed to cDNA; (3) modified gene-specific PCR primers are used to amplify a segment of the cDNA of interest, following the reaction in real time; and (4) the initial concentration of the selected transcript in a specific tissue or cell type is calculated from the exponential phase of the reaction. Relative quantification or absolute quantification compared to standards that are run in parallel can be performed.This chapter describes the entire procedure from isolation of total RNA from liver and fatty tissues/cells to the use of RT-qPCR to study gene expression in these tissues. We perform relative quantification of transcripts to calculate the fold-difference of a certain mRNA level between different samples. In addition, tips for choosing primers and performing analyses are provided to help the beginner in understanding the technique.

  7. Two unusual occurrences of trichomoniasis: rapid species identification by PCR.

    PubMed

    Bellanger, A P; Cabaret, O; Costa, J M; Foulet, F; Bretagne, S; Botterel, F

    2008-09-01

    PCR analysis in two unusual occurrences of trichomoniasis, trichomonal empyema due to Trichomonas tenax and Trichomonas vaginalis in an infant urine sample, allowed us to obtain rapid and accurate trichomonad species identification. The weak sensitivity of wet preparations and the low viability of the flagellates can be remedied by the PCR method. PMID:18632901

  8. 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...

  9. 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.

  10. 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

  11. PCR detection of polycyclic aromatic hydrocarbon-degrading mycobacteria

    SciTech Connect

    Wang, R.F.; Luneau, A.; Cao, W.W.; Cerniglia, C.E.

    1996-01-01

    Polymerase chain reaction (PCR) methods based on the 16S rRNA genes of Mycobacterium sp. PYR-1 and Mycobacterium sp. PAH135, known PAH-degrading bacteria, were developed. An efficient mycobacterial cell lysis procedure was used for the PCR assay. The PCR methods were positive with the target species, but negative for the other 45 bacterial species tested including other Mycobacterium spp. The PCR sensitivity for pure cultures was 20 cells for Mycobacterium sp. PAH135 and 200 cells for Mycobacterium sp. PYR-1. The PCR with a simple sample preparation procedure was used to monitor Mycobacterium sp. PYR-1 cell concentrations in soil slurries amended with [{sup 14}C]pyrene. The pyrene mineralization correlated with the Mycobacterium PYR-1 cell concentrations in the soil slurries. When the PCR titer (the maximum dilution for positive PCR results) reached 10{sup -4}-10{sup -5} at 5-1O days incubation, approximately 50% of the [{sup 14}C]pyrene had been mineralized to {sup 14}CO{sub 2}. However, without inoculation with Mycobacterium PYR-1 cells, both the sterile and nonsterile soils had negative PCR results and no pyrene mineralization. 25 refs., 3 figs., 2 tabs.

  12. 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...

  13. Validation of kinetics similarity in qPCR.

    PubMed

    Bar, Tzachi; Kubista, Mikael; Tichopad, Ales

    2012-02-01

    Quantitative real-time PCR (qPCR) is the method of choice for specific and sensitive quantification of nucleic acids. However, data validation is still a major issue, partially due to the complex effect of PCR inhibition on the results. If undetected PCR inhibition may severely impair the accuracy and sensitivity of results. PCR inhibition is addressed by prevention, detection and correction of PCR results. Recently, a new family of computational methods for the detection of PCR inhibition called kinetics outlier detection (KOD) emerged. KOD methods are based on comparison of one or a few kinetic parameters describing a test reaction to those describing a set of reference reactions. Modern KOD can detect PCR inhibition reflected by shift of the amplification curve by merely half a cycle with specificity and sensitivity >90%. Based solely on data analysis, these tools complement measures to improve and control pre-analytics. KOD methods do not require labor and materials, do not affect the reaction accuracy and sensitivity and they can be automated for fast and reliable quantification. This review describes the background of KOD methods, their principles, assumptions, strengths and limitations. Finally, the review provides recommendations how to use KOD and how to evaluate its performance.

  14. Diagnostic multiplex PCR for toxin genotyping of Clostridium perfringens isolates.

    PubMed

    Baums, Christoph G; Schotte, Ulrich; Amtsberg, Gunter; Goethe, Ralph

    2004-05-20

    In this study we provide a protocol for genotyping Clostridium perfringens with a new multiplex PCR. This PCR enables reliable and specific detection of the toxin genes cpa, cpb, etx, iap, cpe and cpb2 from heat lysed bacterial suspensions. The efficiency of the protocol was demonstrated by typing C. perfringens reference strains and isolates from veterinary bacteriological routine diagnostic specimens.

  15. 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

  16. Genetic relationships among strains of Xanthomonas fragariae based on random amplified polymorphic DNA PCR, repetitive extragenic palindromic PCR, and enterobacterial repetitive intergenic consensus PCR data and generation of multiplexed PCR primers useful for the identification of this phytopathogen.

    PubMed Central

    Pooler, M R; Ritchie, D F; Hartung, J S

    1996-01-01

    Genetic relationships among 25 isolates of Xanthomonas fragariae from diverse geographic regions were determined by three PCR methods that rely on different amplification priming strategies: random amplified polymorphic DNA (RAPD) PCR, repetitive extragenic palindromic (REP) PCR, and enterobacterial repetitive intergenic consensus (ERIC) PCR. The results of these assays are mutually consistent and indicate that pathogenic strains are very closely related to each other. RAPD, ERIC, and REP PCR assays identified nine, four, and two genotypes, respectively, within X. fragariae isolates. A single nonpathogenic isolate of X. fragariae was not distinguishable by these methods. The results of the PCR assays were also fully confirmed by physiological tests. There was no correlation between DNA amplification product patterns and geographic sites of isolation, suggesting that this bacterium has spread largely through exchange of infected plant germ plasm. Sequences identified through the RAPD assays were used to develop three primer pairs for standard PCR assays to identify X. fragariae. In addition, we developed a stringent multiplexed PCR assay to identify X. fragariae by simultaneously using the three independently derived sets of primers specific for pathogenic strains of the bacteria. PMID:8795198

  17. Comparison of droplet digital PCR and quantitative real-time PCR for examining population dynamics of bacteria in soil.

    PubMed

    Kim, Tae Gwan; Jeong, So-Yeon; Cho, Kyung-Suk

    2014-07-01

    The newly developed droplet digital PCR (DD-PCR) has shown promise as a DNA quantification technology in medical diagnostic fields. This study evaluated the applicability of DD-PCR as a quantitative tool for soil DNA using quantitative real-time PCR (qRT-PCR) as a reference technology. Cupriavidus sp. MBT14 and Sphingopyxis sp. MD2 were used, and a primer/TaqMan probe set was designed for each (CupMBT and SphMD2, respectively). Standard curve analyses on tenfold dilution series showed that both qRT-PCR and DD-PCR exhibited excellent linearity (R (2) = 1.00) and PCR efficiency (≥92 %) across their detectable ranges. However, DD-PCR showed a tenfold greater sensitivity than qRT-PCR. MBT14 and MD2 were added to non-sterile soil at 0 ~ 5 × 10(8) and 0 ~ 5 × 10(7) cells per gram of soil, respectively (n = 5). This bacterial load test indicated that DD-PCR was more sensitive and discriminating than qRT-PCR. For instance, DD-PCR showed a gradual DNA increase from 14 to 141,160 MBT14 rDNA copies μL DNA extract(-1) as the bacterial load increased, while qRT-PCR could quantify the DNA (6,432 copies μL DNA(-1)) at ≥5 × 10(5) MBT14 per gram of soil. When temporal DNA changes were monitored for 3 weeks in the amended soils, the two technologies exhibited nearly identical changes over time. Linearity tests (y = a · x) revealed excellent quantitative agreement between the two technologies (a = 0.98, R (2) = 0.97 in the CupMBT set and a = 0.90, R (2) = 0.94 in the SphMD2 set). These results suggest that DD-PCR is a promising tool to examine temporal dynamics of microorganisms in complex environments.

  18. 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.

  19. 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

  20. Is real-time polymerase chain reaction (PCR) more useful than a conventional PCR for the clinical management of leishmaniasis?

    PubMed

    Antinori, Spinello; Calattini, Sara; Piolini, Roberta; Longhi, Erika; Bestetti, Giovanna; Cascio, Antonio; Parravicini, Carlo; Corbellino, Mario

    2009-07-01

    It is currently unknown if the use of a real-time polymerase chain reaction (PCR) adds value to the diagnosis and follow-up prognosis of patients affected by leishmaniasis. We performed a study using a real-time PCR directed against the alpha-polymerase gene and a semiquantitative PCR that target the SSU ribosomal RNA (rRNA) gene as control for the diagnosis and quantification of parasites in patients with visceral (VL) and cutaneous (CL) leishmaniasis. Our single copy real-time PCR missed one diagnosis of VL compared with the conventional PCR, whereas both PCR methods were able to detect Leishmania parasites in CL. Under anti-leishmania treatment the kinetics of parasitemia were comparable with the two methods. The real-time PCR directed against alpha-polymerase of Leishmania despite being able to make a more accurate quantification of parasites does not add to the decision-making management compared with a semiquantitative PCR, and it is comparatively expensive.

  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. PMID:27077029

  2. 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.

  3. 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.

  4. 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

  5. Heat-activatable primers for hot-start PCR and hot-start one-step RT-PCR: endpoint and real-time experiments.

    PubMed

    Ashrafi, Elena Hidalgo; Paul, Natasha

    2009-10-01

    Hot-start PCR is a technique that improves PCR performance by reducing nonspecific amplification during the initial setup stages of the PCR. This unit describes hot-start PCR protocols which utilize primers containing temperature-sensitive modifications. The introduction of 4-oxo-tetradecyl (OXT) phosphotriester groups onto the 3' end of the primer allows for primer-based hot-start PCR that is amenable for use in a number of PCR-based applications. The protocols described in this unit utilize OXT-modified primers in applications such as standard thermal cycling PCR, fast thermal cycling PCR, multiplex PCR, and one-step reverse-transcription PCR. This method is also advantageous for instances where improved PCR specificity is desired and a hot-start polymerase suitable for your application is not available.

  6. Interaction of quantitative PCR components with polymeric surfaces.

    PubMed

    Gonzalez, Asensio; Grimes, Ronan; Walsh, Edmond J; Dalton, Tara; Davies, Mark

    2007-04-01

    This study investigated the effect of exposing a polymerase chain reaction (PCR) mixture to capillary tubing of different materials and lengths, at different contact times and flow rates and the adsorption of major reaction components into the tubing wall. Using 0.5 mm ID tubing, lengths of 40 cm and residence times up to 45 min, none of the tested polymeric materials was found to affect subsequent PCR amplification. However, after exposure of the mixture to tubing lengths of 3 m or reduction of sample volume, PCR inhibition occurred, increasing with the volume to length ratio. Different flow velocities did not affect PCR yield. When the adsorption of individual PCR components was studied, significant DNA adsorption and even more significant adsorption of the fluorescent dye Sybr Green I was found. The results indicate that PCR inhibition in polymeric tubing results from adsorption of reaction components to wall surfaces, increasing substantially with tubing length or sample volume reduction, but not with contact time or flow velocities typical in dynamic PCR amplification. The data also highlight that chemical compatibility of polymeric capillaries with DNA dyes should be carefully considered for the design of quantitative microfluidic devices. PMID:17180709

  7. Nanogold-assisted multi-round polymerase chain reaction (PCR).

    PubMed

    Pan, Jiakui; Li, Haikuo; Cao, Xueyan; Huang, Jiehuan; Zhang, Xiaodong; Fan, Chunhai; Hu, Jun

    2007-12-01

    We have previously demonstrated that nanogold effectively enhances the specificity and yield of error-prone two-round polymerase chain reaction (PCR). Here we reported that, with the assistance of nanogold, we could perform multi-round PCR. In the presence of appropriate amount of 10 nm nanogold, we could obtain the target product even after six rounds of PCR, as manifested by a single bright band in gel electrophoresis (1% agarose). In fact, we could still observe the target band even at the 7th round of PCR, which nevertheless was accompanied by smearing bands (non-specific amplification). In contrast, in the absence of nanogold, the target band was completely lost only after four rounds of amplification. This marked difference in the performance of multi-round PCR clearly showed that nanogold was a powerful enhancer for PCR. More importantly, with this nanogold-assisted multi-round PCR, it might be possible to produce a large amount of target DNA, or to amply very low copies of genomic DNA from rare sources.

  8. Rapid and simple method of qPCR primer design.

    PubMed

    Thornton, Brenda; Basu, Chhandak

    2015-01-01

    Quantitative real-time polymerase chain reaction (qPCR) is a powerful tool for analysis and quantification of gene expression. It is advantageous compared to traditional gel-based method of PCR, as gene expression can be visualized "real-time" using a computer. In qPCR, a reporter dye system is used which intercalates with DNA's region of interest and detects DNA amplification. Some of the popular reporter systems used in qPCR are the following: Molecular Beacon(®), SYBR Green(®), and Taqman(®). However, success of qPCR depends on the optimal primers used. Some of the considerations for primer design are the following: GC content, primer self-dimer, or secondary structure formation. Freely available software could be used for ideal qPCR primer design. Here we have shown how to use some freely available web-based software programs (such as Primerquest(®), Unafold(®), and Beacon designer(®)) to design qPCR primers.

  9. [Real time PCR methodology for quantification of nucleic acids].

    PubMed

    Tse, C; Capeau, J

    2003-01-01

    The polymerase chain reaction (PCR) has become an essential tool for molecular biologists and its introduction into nucleic acids detection systems has revolutionized the quantitative analysis of DNA and RNA. The technique has rapidly evolved over the last few years and the growing interest in quantitative applications of the PCR has favoured the development of real-time quantitative PCR. In this paper, we review, after presentation of the theorical aspects of PCR, the basic principles of real-time PCR with the introduction of the concept of threshold cycle. More precisely, we describe the novel assay formats that greatly simplify the protocols used for the detection of specific nucleic acids. We focus on the actual four technologies that enable sequence detection in a closed tube and that are SYBR Green I, TaqMan probes, Hybridization probes and Molecular Beacon probes. We then discuss the different quantification strategies in real time PCR and compare the competiting instruments on the market. The most important real-time PCR applications in clinical biology are also described.

  10. Evaluation of Roche Amplicor PCR assay for Mycobacterium tuberculosis.

    PubMed Central

    Wobeser, W L; Krajden, M; Conly, J; Simpson, H; Yim, B; D'costa, M; Fuksa, M; Hian-Cheong, C; Patterson, M; Phillips, A; Bannatyne, R; Haddad, A; Brunton, J L; Krajden, S

    1996-01-01

    The Roche Amplicor Mycobacterium tuberculosis PCR test (RMtb-PCR) was compared with mycobacterial culture, with the BACTEC 460 system and inoculation on Lowenstein-Jensen media. Results were interpreted with an adjusted "gold standard" incorporating clinical diagnosis. A total of 1,480 clinical specimens from 1,155 patients, including tissues and fluids, as well as 141 specimens which demonstrated a positive growth index on the BACTEC 460 system were assessed. The sensitivity, specificity, and positive and negative predictive values of RMtb-PCR compared with the adjusted gold standard for clinical specimens were 79, 99, 93, and 98%, respectively. In smear-positive specimens, the sensitivity of RMtb-PCR was 98% versus 53% for smear-negative specimens. When RMtb-PCR was performed two times per week, PCR results were available an average of 21 days before the culture results. For specimens demonstrating a positive growth index on the BACTEC 460 system, RMtb-PCR had a sensitivity and specificity of 98 and 100%, respectively. This study demonstrates the value of a commercial nucleic acid amplification kit for rapid diagnosis of M. tuberculosis, particularly in smear-positive specimens or BACTEC culture-positive specimens. PMID:8748289

  11. Development and Validation of a HPV-32 Specific PCR Assay

    PubMed Central

    Herrel, Nicholas R; Johnson, Nadia L; Cameron, Jennifer E; Leigh, Janet; Hagensee, Michael E

    2009-01-01

    Background Human Papillomavirus-32 (HPV-32) has traditionally been associated with focal-epithelial-hyperplasia (FEH). It is also present in 58% of oral warts of HIV-positive individuals whose prevalence is increasing. Current methods for the detection of HPV-32 are labor-intensive and insensitive so the goal of this work was to develop a highly sensitive and easy to use specific polymerase chain reaction (PCR) assay. Materials and methods An HPV-32 L1 specific PCR assay was developed and optimized. The sensitivity and specificity was compared to previous assays utilized for detection (PGMY and MY09/11 PCR with dot blot hybridization) using cloned HPV-32 L1, the closely related HPV-42 L1 as well as clinical samples (oral swabs and fluids from 89 HIV-positive subjects). Results The HPV-32 specific PCR assay showed improved sensitivity to 5 copies of HPV-32 as compared to the PGMY PCR, MY09/11 PCR and dot blot which had a limit of detection of approximately 3,000 copies. Using the HPV-32 dot blot hybridization assay as the gold standard, the HPV-32 specific PCR assay has a sensitivity of 95.8% and 88.9% by sample and subject, respectively, and specificity was 87.8% and 58.8% by sample and subject, respectively. The low sensitivity is due to the HPV-32 specific PCR assays ability to detect more HPV-32 positive samples and may be the new gold standard. Conclusion Due to the ease, sensitivity, and specificity the HPV-32 specific PCR assay is superior to previous assays and is ideal for detection of HPV-32 in large cohorts. This assay provides an excellent tool to study the natural history of HPV-32 infection and the development of oral warts. PMID:19558697

  12. 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

    2015-05-01

    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.

  13. Predicting Gene Structures from Multiple RT-PCR Tests

    NASA Astrophysics Data System (ADS)

    Kováč, Jakub; Vinař, Tomáš; Brejová, Broňa

    It has been demonstrated that the use of additional information such as ESTs and protein homology can significantly improve accuracy of gene prediction. However, many sources of external information are still being omitted from consideration. Here, we investigate the use of product lengths from RT-PCR experiments in gene finding. We present hardness results and practical algorithms for several variants of the problem and apply our methods to a real RT-PCR data set in the Drosophila genome. We conclude that the use of RT-PCR data can improve the sensitivity of gene prediction and locate novel splicing variants.

  14. Real-Time PCR for Gene Expression Quantification in Asthma.

    PubMed

    Segundo-Val, Ignacio San; García-Solaesa, Virginia; García-Sánchez, Asunción

    2016-01-01

    The quantitative real-time PCR (qPCR) has become the reference technique for studying gene expression in recent years. The application of qPCR to the study of asthma provides very useful information regarding the gene expression mechanisms. The quantification of RNA from cDNA can be performed by using fluorescent dyes or specific sequence probes. Here, we describe the protocol to quantify gene expression levels using SYBR Green as fluorescent dye. The protocol starts with the RNA extraction, followed by reverse transcription to obtain cDNA, quantification and finally data analysis.

  15. Quantification of transcript levels with quantitative RT-PCR.

    PubMed

    Carleton, Karen L

    2011-01-01

    Differential gene expression is a key factor driving phenotypic divergence. Determining when and where gene expression has diverged between organisms requires a quantitative method. While large-scale approaches such as microarrays or high-throughput mRNA sequencing can identify candidates, quantitative RT-PCR is the definitive method for confirming gene expression differences. Here, we describe the steps for performing qRT-PCR including extracting total RNA, reverse-transcribing it to make a pool of cDNA, and then quantifying relative expression of a few candidate genes using real-time or quantitative PCR.

  16. Real-Time PCR for Gene Expression Quantification in Asthma.

    PubMed

    Segundo-Val, Ignacio San; García-Solaesa, Virginia; García-Sánchez, Asunción

    2016-01-01

    The quantitative real-time PCR (qPCR) has become the reference technique for studying gene expression in recent years. The application of qPCR to the study of asthma provides very useful information regarding the gene expression mechanisms. The quantification of RNA from cDNA can be performed by using fluorescent dyes or specific sequence probes. Here, we describe the protocol to quantify gene expression levels using SYBR Green as fluorescent dye. The protocol starts with the RNA extraction, followed by reverse transcription to obtain cDNA, quantification and finally data analysis. PMID:27300530

  17. 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.

  18. PCR amplification on microarrays of gel immobilized oligonucleotides

    SciTech Connect

    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.

  19. 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

  20. 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.

  1. 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

  2. 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.

  3. 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.

  4. Species identification of Cannabis sativa using real-time quantitative PCR (qPCR).

    PubMed

    Johnson, Christopher E; Premasuthan, Amritha; Satkoski Trask, Jessica; Kanthaswamy, Sree

    2013-03-01

    Most narcotics-related cases in the United States involve Cannabis sativa. Material is typically identified based on the cystolithic hairs on the leaves and with chemical tests to identify of the presence of cannabinoids. Suspect seeds are germinated into a viable plant so that morphological and chemical tests can be conducted. Seed germination, however, causes undue analytical delays. DNA analyses that involve the chloroplast and nuclear genomes have been developed for identification of C. sativa materials, but they require several nanograms of template DNA. Using the trnL 3' exon-trnF intragenic spacer regions within the C. sativa chloroplast, we have developed a real-time quantitative PCR assay that is capable of identifying picogram amounts of chloroplast DNA for species determination of suspected C. sativa material. This assay provides forensic science laboratories with a quick and reliable method to identify an unknown sample as C. sativa.

  5. 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.

  6. 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-...

  7. 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...

  8. Nanodroplet real-time PCR system with laser assisted heating.

    PubMed

    Kim, Hanyoup; Dixit, Sanhita; Green, Christopher J; Faris, Gregory W

    2009-01-01

    We report the successful application of low-power (approximately 30 mW) laser radiation as an optical heating source for high-speed real-time polymerase chain reaction (PCR) amplification of DNA in nanoliter droplets dispersed in an oil phase. Light provides the heating, temperature measurement, and Taqman real-time readout in nanoliter droplets on a disposable plastic substrate. A selective heating scheme using an infrared laser appears ideal for driving PCR because it heats only the droplet, not the oil or plastic substrate, providing fast heating and completing the 40 cycles of PCR in 370 seconds. No microheaters or microfluidic circuitry were deposited on the substrate, and PCR was performed in one droplet without affecting neighboring droplets. The assay performance was quantitative and its amplification efficiency was comparable to that of a commercial instrument.

  9. Evaluation of PCR-based beef sexing methods.

    PubMed

    Zeleny, Reinhard; Bernreuther, Alexander; Schimmel, Heinz; Pauwels, Jean

    2002-07-17

    Analysis of the sex of beef meat by fast and reliable molecular methods is an important measure to ensure correct allocation of export refunds, which are considerably higher for male beef meat. Two PCR-based beef sexing methods have been optimized and evaluated. The amelogenin-type method revealed excellent accuracy and robustness, whereas the bovine satellite/Y-chromosome duplex PCR procedure showed more ambiguous results. In addition, an interlaboratory comparison was organized to evaluate currently applied PCR-based sexing methods in European customs laboratories. From a total of 375 samples sent out, only 1 false result was reported (female identified as male). However, differences in the performances of the applied methods became apparent. The collected data contribute to specify technical requirements for a common European beef sexing methodology based on PCR. PMID:12105941

  10. 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.

  11. Application of PCR-ELISA in Molecular Diagnosis

    PubMed Central

    Sue, Mei Jean; Yeap, Swee Keong; Omar, Abdul Rahman; Tan, Sheau Wei

    2014-01-01

    Polymerase chain reaction-enzyme linked immunosorbent assay (PCR-ELISA) is an immunodetection method that can quantify PCR product directly after immobilization of biotinylated DNA on a microplate. This method, which detects nucleic acid instead of protein, is a much more sensitive method compared to conventional PCR method, with shorter analytical time and lower detection limit. Its high specificity and sensitivity, together with its semiquantitative ability, give it a huge potential to serve as a powerful detection tool in various industries such as medical, veterinary, and agricultural industries. With the recent advances in PCR-ELISA, it is envisaged that the assay is more widely recognized for its fast and sensitive detection limit which could improve overall diagnostic time and quality. PMID:24971343

  12. Rapid Multiplex Nested PCR for Detection of Respiratory Viruses▿

    PubMed Central

    Lam, W. Y.; Yeung, Apple C. M.; Tang, Julian W.; Ip, Margaret; Chan, Edward W. C.; Hui, Mamie; Chan, Paul K. S.

    2007-01-01

    Respiratory tract infections can be caused by a heterogeneous group of viruses and bacteria that produce similar clinical presentations. Specific diagnosis therefore relies on laboratory investigation. This study developed and evaluated five groups of multiplex nested PCR assays that could simultaneously detect 21 different respiratory pathogens: influenza A virus (H1N1, H3N2, and H5N1); influenza B virus; parainfluenza virus types 1, 2, 3, 4a, and 4b; respiratory syncytial virus A and B; human rhinoviruses; human enteroviruses; human coronaviruses OC43 and 229E; severe acute respiratory syndrome coronavirus; human metapneumoviruses; Mycoplasma pneumoniae; Chlamydophila pneumoniae; Legionella pneumophila; and adenoviruses (A to F). These multiplex nested PCRs adopted fast PCR technology. The high speed of fast PCR (within 35 min) greatly improved the efficiency of these assays. The results show that these multiplex nested PCR assays are specific and more sensitive (100- to 1,000-fold) than conventional methods. Among the 303 clinical specimens tested, the multiplex nested PCR achieved an overall positive rate of 48.5% (95% confidence interval [CI], 42.9 to 54.1%), which was significantly higher than that of virus isolation (20.1% [95% CI, 15.6 to 24.6%]) and that of direct detection by immunofluorescence assay (13.5% [95% CI, 9.7 to 17.4%]). The improved sensitivity was partly due to the higher sensitivity of multiplex nested PCR than that of conventional methods in detecting cultivatable viruses. Moreover, the ability of the multiplex nested PCR to detect noncultivatable viruses, particularly rhinoviruses, coronavirus OC43, and metapneumoviruses, contributed a major gain (15.6%) in the overall positive rate. In conclusion, rapid multiplex nested PCR assays can improve the diagnostic yield for respiratory infections to allow prompt interventive actions to be taken. PMID:17804659

  13. 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.

  14. 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

  15. Improved PCR Amplification of Broad Spectrum GC DNA Templates

    PubMed Central

    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. PMID:27271574

  16. 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.

  17. Quantification of HEV RNA by Droplet Digital PCR.

    PubMed

    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

  18. 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.

  19. 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

  20. 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

    2016-01-01

    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

  1. 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. PMID:27451195

  2. 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.

  3. RT-PCR detection of HIV in Republic of Macedonia.

    PubMed

    Bosevska, Golubinka; Panovski, Nikola; Dokić, Eleni; Grunevska, Violeta

    2008-11-01

    The aim of the study was to detect HIV RNA in seropositive patients using RT-PCR method and thus, to establish PCR methodology in the routine laboratory works. The total of 33 examined persons were divided in two groups: 1) 13 persons seropositive for HIV; and 2) 20 healthy persons - randomly selected blood donors that made the case control group. The subjects age was between 25 and 52 years (average 38,5). ELFA test for combined detection of HIV p24 antigen and anti HIV-1+2 IgG and ELISA test for detection of antibodies against HIV-1 and HIV-2, were performed for each examined person. RNA from the whole blood was extracted using a commercial kit based on salt precipitation. Detection of HIV RNA was performed using RT-PCR kit. Following nested PCR, the product was separated by electrophoresis in 1,5 % agarose gel. The result was scored positive if the band of 210bp was visible regardless of intensity. Measures of precaution were taken during all the steps of the work and HIV infected materials were disposed of accordingly. In the group of blood donors ELFA, ELISA and RT-PCR were negative. Assuming that prevalence of HIV infection is zero, the clinical specificity of RT-PCR is 100 %. The analytical specificity of RT-PCR method was tested against Hepatitis C and B, Human Papiloma Virus, Cytomegalovirus, Herpes Simplex Virus, Rubella Virus, Mycobacterium tuberculosis, Chlamydia trachomatis. None of these templates yielded amplicon. In the group of 13 seropositive persons, 33 samples were analyzed. HIV RNA was detected in 15 samples. ELISA and ELFA test were positive in all samples. Different aliquots of the samples were tested independently and showed the same results. After different periods of storing the RNA samples at -70 masculineC, RT-PCR reaction was identical to the one performed initially. The obtained amplicons were maintained frozen at -20 masculineC for a week and the subsequently performed electrophoresis was identical to the previous one. The reaction is

  4. A strategy for detection of viruses in groundwater by PCR.

    PubMed

    Abbaszadegan, M; Stewart, P; LeChevallier, M

    1999-02-01

    We evaluated the use of the PCR for detection of enteric viruses in groundwater. To do this, we used an improved sample-processing technique and a large-volume amplification protocol. The objective of this study was to use advanced molecular techniques to develop a rapid and simple method which can be used by the water industry for detection of viral contamination in a variety of water samples. The strategy described here fulfills the water industry's need for a rapid, reliable, easily performed method for analyzing groundwater for virus contamination. Viruses were detected after concentration from at least 400 gallons (1,512 liters) of water by a filter adsorption and elution method, which resulted in a concentrate containing viruses. A total of 150 samples were analyzed by performing cell culture assays for enteroviruses and by performing reverse transcription PCR (RT-PCR) analyses for enteroviruses, hepatitis A virus, and rotavirus. Thirteen samples (8.7%) produced cellular cytopathic effects when the Buffalo green monkey cell line was used. When primers specific for enteroviruses were used in RT-PCR, 40 of 133 samples (30.1%) tested positive for the presence of enterovirus RNA. When hepatitis A virus-specific primers were used, 12 of 139 samples (8.6%) were considered positive for the presence of hepatitis A viral RNA. The RT-PCR analysis performed with rotavirus-specific primers identified 18 of 130 samples (13.8%) that were positive for rotavirus RNA sequences. Our sample-processing technique and large-volume PCR protocol (reaction volume, 300 microliter) resulted in sufficient removal or dilution of inhibitors so that more than 95% of the samples could be assayed by PCR. Because of its sensitivity for detecting viral nucleic acid sequences, PCR analysis should produce more positive results than cell culture analysis. Since either cell culture analysis or PCR can reveal only a "snapshot" of the quality of the groundwater being sampled, PCR seems to be a

  5. A survey of polymerase chain reaction (PCR) amplification studies of unicellular protists using single-cell PCR.

    PubMed

    Lynn, Denis H; Pinheiro, Marcel

    2009-01-01

    We surveyed a variety of studies that have used single-cell polymerase chain reaction (SC-PCR) to examine the gene sequences of a diversity of unicellular protists. Representatives of all the Super-Groups of eukaryotes have been subjected to SC-PCR with ciliates and dinoflagellates being most commonly examined. The SC-PCR was carried out either by directly amplifying a single lysed cell or by first extracting DNA and following this with amplification of the DNA extract. Cell lysis methods included heating, freezing, mechanical rupture, and enzyme digestion. Cells fixed or preserved with ethanol, methanol, and Lugol's have also been used successfully. Heminested or seminested PCR might follow the initial PCR, whose products were then directly sequenced or cloned and then sequenced. The methods are not complicated. This should encourage protistologists to use SC-PCR in the description of new or revised taxa, especially rare and unculturable forms, and it should also enable the probing of gene expression in relation to life history stages.

  6. 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.

  7. The methylation status of plant genomic DNA influences PCR efficiency.

    PubMed

    Kiselev, K V; Dubrovina, A S; Tyunin, A P

    2015-03-01

    During the polymerase chain reaction (PCR), which is a versatile and widely used method, certain DNA sequences are rapidly amplified through thermocycling. Although there are numerous protocols of PCR optimization for different applications, little is known about the effect of DNA modifications, such as DNA methylation, on PCR efficiency. Recent studies show that cytosine methylation alters DNA mechanical properties and suggest that DNA methylation may directly or indirectly influence the effectiveness of DNA amplification during PCR. In the present study, using plant DNA, we found that highly methylated plant DNA genomic regions were amplified with lower efficiencies compared to that for the regions methylated at a lower level. The correlation was observed when amplifying stilbene synthase (STS1, STS10) genes of Vitis amurensis, the Actin2 gene of Arabidopsis thaliana, the internal transcribed spacer (AtITS), and tRNAPro of A. thaliana. The level of DNA methylation within the analyzed DNA regions has been analyzed with bisulfite sequencing. The obtained data show that efficient PCRs of highly methylated plant DNA regions can be hampered. Proteinase K treatment of the plant DNA prior to PCR and using HotTaq DNA polymerase improved amplification of the highly methylated plant DNA regions. We suggest that increased DNA denaturation temperatures of the highly methylated DNA and contamination with DNA-binding proteins contribute to the hampered PCR amplification of highly methylated DNA. The data show that it is necessary to use current DNA purification protocols and commercial kits with caution to ensure appropriate PCR product yield and prevent bias toward unmethylated DNA amplification in PCRs.

  8. [Evaluation of minimal residual disease using allele (mutation) -specific PCR].

    PubMed

    Matsuda, Kazuyuki

    2014-06-01

    For patients with hematological malignancies, monitoring minimal residual disease (MRD) provides useful information to evaluate the therapeutic response and risk of relapse. The currently available quantitative MRD assays are fluorescence in situ hybridization of chromosomal aberrations, multiparameter flow cytometry of leukemia-associated immunophenotypes, and quantitative polymerase chain reaction (qPCR) analysis of fusion genes, immunoglobulin/T-cell receptor gene rearrangements, genetic alterations, or over-expressed genes. Single nucleotide mutations associated with leukemogenesis can be considered as applicable MRD markers. Allele-specific qPCR (AS-qPCR) using primers including mismatched bases and locked nucleic acids (LNA) can quantify not only the insertion and duplication of several nucleotides, but also single nucleotide mutation in the presence of an excess amount of wild-type nucleotides. The AS-qPCR for analyzing single nucleotide mutations contributes to the monitoring of MRD in patients without recurrent fusion genes throughout the clinical course and, thus, broadens the spectrum of patients in whom MRD can be monitored. In addition to the evaluation of MRD, AS-qPCR can provide insight into the development of leukemia and the sequential acquisition of gene mutations.

  9. 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

  10. PCR Assay for Species-Specific Identification of Bacteroides thetaiotaomicron

    PubMed Central

    Teng, Lee-Jene; Hsueh, Po-Ren; Tsai, Jui-Chang; Chiang, Feng-Lin; Chen, Ching-Yi; Ho, Shen-Wu; Luh, Kwen-Tay

    2000-01-01

    Bacteroides thetaiotaomicron is the second most frequently encountered species of the anaerobes isolated from clinical specimens. We developed a PCR-based assay for the rapid identification of B. thetaiotaomicron. Specific primers were based on shared amplicons of about 1.2 kb generated from B. thetaiotaomicron by randomly amplified polymorphic DNA. This 1.2-kb fragment was sequenced and then used to design a set of PCR amplification primers. This PCR generated an amplification product of 721 bp, which was unique to all 65 isolates of B. thetaiotaomicron tested. There was no amplification with isolates of other bacterial species. Restriction enzyme digestion of the amplification product and dot blot hybridization further verified the specificity of the assay. These results suggest that this PCR assay targets a nucleotide sequence that is strongly conserved in B. thetaiotaomicron. This simple and rapid PCR assay provides a rapid and accurate method for identification of B. thetaiotaomicron and shows promise for the detection of B. thetaiotaomicron in clinical samples. PMID:10747167

  11. 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

  12. Multiplex PCR: Optimization and Application in Diagnostic Virology

    PubMed Central

    Elnifro, Elfath M.; Ashshi, Ahmed M.; Cooper, Robert J.; Klapper, Paul E.

    2000-01-01

    PCR has revolutionized the field of infectious disease diagnosis. To overcome the inherent disadvantage of cost and to improve the diagnostic capacity of the test, multiplex PCR, a variant of the test in which more than one target sequence is amplified using more than one pair of primers, has been developed. Multiplex PCRs to detect viral, bacterial, and/or other infectious agents in one reaction tube have been described. Early studies highlighted the obstacles that can jeopardize the production of sensitive and specific multiplex assays, but more recent studies have provided systematic protocols and technical improvements for simple test design. The most useful of these are the empirical choice of oligonucleotide primers and the use of hot start-based PCR methodology. These advances along with others to enhance sensitivity and specificity and to facilitate automation have resulted in the appearance of numerous publications regarding the application of multiplex PCR in the diagnosis of infectious agents, especially those which target viral nucleic acids. This article reviews the principles, optimization, and application of multiplex PCR for the detection of viruses of clinical and epidemiological importance. PMID:11023957

  13. Detection of adenovirus using PCR and molecular beacon.

    PubMed

    Poddar, S K

    1999-09-01

    The polymerase chain reaction (PCR) and a molecular beacon probe were used for the detection of Adenovirus. A 307 bp DNA fragment from a conserved region of the hexon gene was amplified. The specific molecular beacon was characterized with respect to its efficiency of quenching, and signal to noise ratio by spectrofluorometric analysis of its hybridization with virus specific complementary single stranded oligonucleotide target. Amplification was carried out in the presence of the molecular beacon probe, and the amplified target was detected by measurement of fluorescence signal in the post PCR sample. Separately, a 32P-labeled linear probe (having the same sequence as that of molecular beacon probe) was liquid-phase hybridized with the product of PCR performed in the absence of the molecular beacon. The virus specific target was then detected by electrophoresis of the hybridized product in a nondenaturing polyacrylamide gel and subsequent autoradiographic analysis. The detection limit of adenovirus by PCR in the presence of the molecular beacon probe was found to be similar to that obtained by labeled linear probe hybridization following PCR.

  14. Real-Time Quantitative PCR for Human Herpesvirus 6 DNA

    PubMed Central

    Locatelli, Giuseppe; Santoro, Fabio; Veglia, Fabrizio; Gobbi, Alberto; Lusso, Paolo; Malnati, Mauro S.

    2000-01-01

    The diagnosis of human herpesvirus 6 (HHV-6) infection represents a complex issue because the most widely used diagnostic tools, such as immunoglobulin G antibody titer determination and qualitative DNA PCR with blood cells, are unable to distinguish between latent (clinically silent) and active (often clinically relevant) infection. We have developed a new, highly sensitive, quantitative PCR assay for the accurate measurement of HHV-6 DNA in tissue-derived cell suspensions and body fluids. The test uses a 5′ nuclease, fluorogenic assay combined with real-time detection of PCR amplification products with the ABI PRISM 7700 sequence detector system. The sensitivity of this method is equal to the sensitivity of a nested PCR protocol (lower detection limit, 1 viral genome equivalent/test) for both the A and the B HHV-6 subgroups and shows a wider dynamic range of detection (from 1 to 106 viral genome equivalents/test) and a higher degree of accuracy, repeatability, and reproducibility compared to those of a standard quantitative-competitive PCR assay developed with the same reference DNA molecule. The novel technique is versatile, showing the same sensitivity and dynamic range with viral DNA extracted from different fluids (i.e., culture medium or plasma) or from tissue-derived cell suspensions. Furthermore, by virtue of its high-throughput format, this method is well suited for large epidemiological surveys. PMID:11060066

  15. Minimizing DNA recombination during long RT-PCR.

    PubMed

    Fang, G; Zhu, G; Burger, H; Keithly, J S; Weiser, B

    1998-12-01

    Recent developments have made it possible to reverse transcribe RNA and amplify cDNA molecules of > 10 kb in length, including the HIV-1 genome. To use long reverse transcription combined with polymerase chain reaction (RT-PCR) to best advantage, it is necessary to determine the frequency of recombination during the combined procedure and then take steps to reduce it. We investigated the requirements for minimizing DNA recombination during long RT-PCR of HIV-1 by experimenting with three different aspects of the procedure: conditions for RT, conditions for PCR, and the molar ratios of different templates. We used two distinct HIV-1 strains as templates and strain-specific probes to detect recombination. The data showed that strategies aimed at completing DNA strand synthesis and the addition of proofreading function to the PCR were most effective in reducing recombination during the combined procedure. This study demonstrated that by adjusting reaction conditions, the recombination frequency during RT-PCR can be controlled and greatly reduced.

  16. A comparative analysis of different molecular targets using PCR for diagnosis of old world leishmaniasis.

    PubMed

    Koltas, Ismail S; Eroglu, Fadime; Uzun, Soner; Alabaz, Derya

    2016-05-01

    The different sensitivity values were obtained in each study conducted for the diagnosis of leishmaniasis with the polymerase chain reaction (PCR). However, a standardized PCR target for the diagnosis of leishmaniasis does not exist. The aim of the current study, the most ideal PCR target was determined for diagnosis of leishmaniasis. A total of 72 smear and 48 bone marrow samples were analyzed with six different molecular targets to determine their potential as a tool for the specific molecular diagnosis of leishmaniasis using PCR. The positivity-negativity value and the sensitivity-specificity of each PCR targets were calculated. The positivity value of PCR targets were sequenced in different levels in the diagnosis of leishmaniasis from highest to lowest in the order of kDNA-PCR > SSU rRNA-PCR > ITS2-PCR > ITS1-PCR > ME-PCR > HSP70-PCR. The sensitivities of PCR targets except ITS1-PCR, ME-PCR and HSP70-PCR were found to be 100% in cutaneous leishmaniasis (CL) and visceral leishmaniasis (VL) cases as compared to microscopic examination accepted as a gold standard. The sensitivities of ITS1-PCR, ME-PCR and HSP70-PCR were found 96.6%, 90.0% and 86.6%, respectively, in CL-cases. In addition, the sensitivities of ITS1-PCR, ME-PCR and HSP70-PCR were found 90.0%, 70.0% and 60.0%, respectively, in VL-cases. The kDNA genomic region was the most sensitive for routine diagnosis of leishmaniasis. ITS1-PCR restriction fragment length polymorphism, the alternative method for the identification of Old World Leishmania species, did not require culturing of the parasites.

  17. 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.

  18. 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

  19. [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.

  20. [PCR studies in laboratory diagnosis: problems of quality assurance].

    PubMed

    Tvorogova, M G; Gushchin, A E

    2006-12-01

    The determination of DNA and RNA of causative agents by the nucleic acid amplification techniques (NAT) presented in the Russian laboratory diagnosis of infections solely by polymerase chain reaction (PCR) have currently found the widest application in world practice and, in some cases, is a basic procedure for detecting an infectious agent, particularly in the diagnosis of infections caused by human immunodeficiency virus, hepatitis C virus, hepatitis B virus, cytomegalovirus, Chlamydia trachomatis, Neisseria gonorrhoeae, and others. The paper presents the data available in the literature on the main reasons for false positive and false negative results in PCR studies, international and national programs for the outside quality control (OQC) of detection of causative agents for various diseases, by applying NAT. It is emphasized that the regular participation in OQC programs is likely to be useful in improving the quality of PCR studies.

  1. [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. PMID:25612419

  2. 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.

  3. 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.

  4. An improved, PCR-based strategy for the detection of Trypanosoma cruzi in human blood samples.

    PubMed

    Ribeiro-dos-Santos, G; Nishiya, A S; Sabino, E C; Chamone, D F; Saez-Alquézar, A

    1999-10-01

    Attempts were made to improve the PCR-based detection of Trypanosoma cruzi in blood samples, primarily for screening blood donors. Samples were obtained from candidate donors who were reactive in one or two of three serological tests for Chagas disease (and therefore considered 'indeterminate') or in all three tests (3+). Each sample was then examined using three different, PCR-based techniques: 'PCR-I' (in which the target DNA is a nuclear repetitive sequence); 'PCR-II' [amplifying a conserved region of the T. cruzi kinetoplast DNA (kDNA)]; and 'PCR-III' (a new strategy in which the target kDNA is amplified by 'nested' PCR). Among the samples from 3+ individuals, PCR-I, PCR-II and PCR-III amplified two (3.8%) out of 52, four (4.5%) out of 88, and 27 (25.7%) out of 105 samples tested, respectively. Seven, 69 and 70 samples from 'indeterminate' subjects were tested by PCR-I, PCR-II and PCR-III, respectively; there was not a single positive result by PCR-I or PCR-II, but three (4.3%) of the samples tested by PCR-III were positive. In a reconstruction experiment, in conditions in which PCR-I and PCR-II could not detect 10,000 parasites/ml, PCR-III was able to detect one parasite/ml. Although all three PCR-based strategies examined had rather poor sensitivities, PCR-III was far more sensitive than PCR-I or PCR-II. PMID:10715696

  5. Detection of Grapevine leafroll-associated virus 7 using real time qRT-PCR and conventional RT-PCR.

    PubMed

    Al Rwahnih, Maher; Osman, Fatima; Sudarshana, Mysore; Uyemoto, Jerry; Minafra, Angelantonio; Saldarelli, Pasquale; Martelli, Giovanni; Rowhani, Adib

    2012-02-01

    Nine isolates of Grapevine leafroll-associated virus 7 (GLRaV-7) from diverse geographical regions were sequenced to design more sensitive molecular diagnostic tools. The coat protein (CP) and heat shock protein 70 homologue (HSP70h) genes of these nine isolates were sequenced. Sequences were then used to design more sensitive molecular diagnostic tools. Sequence identity among these isolates ranged between 90 to 100% at the nucleotide and amino acid levels. One RT-PCR and two qRT-PCR assays were used to survey 86 different grapevines from the University of California, Davis Grapevine Virus Collection, the Foundation Plant Services collection and the USDA National Clonal Germplasm Repository, Davis, CA with primers designed in conserved regions of the CP and HSP70h genes. Results revealed that qRT-PCR assays designed in the HSP70h gene was more sensitive (29.07% positives) than that designed in the CP gene (22.09% positives) and both qRT-PCR assays proved to be more sensitive than RT-PCR.

  6. Quantification of mRNA using real-time reverse transcription PCR (RT-PCR): trends and problems.

    PubMed

    Bustin, S A

    2002-08-01

    The fluorescence-based real-time reverse transcription PCR (RT-PCR) is widely used for the quantification of steady-state mRNA levels and is a critical tool for basic research, molecular medicine and biotechnology. Assays are easy to perform, capable of high throughput, and can combine high sensitivity with reliable specificity. The technology is evolving rapidly with the introduction of new enzymes, chemistries and instrumentation. However, while real-time RT-PCR addresses many of the difficulties inherent in conventional RT-PCR, it has become increasingly clear that it engenders new problems that require urgent attention. Therefore, in addition to providing a snapshot of the state-of-the-art in real-time RT-PCR, this review has an additional aim: it will describe and discuss critically some of the problems associated with interpreting results that are numerical and lend themselves to statistical analysis, yet whose accuracy is significantly affected by reagent and operator variability.

  7. Role of real-time PCR (RT-PCR) in rapid diagnosis of tuberculous mycobacteria in different clinical samples.

    PubMed

    2014-02-01

    The study was aimed for molecular detection of mycobacterial DNA in different clinical samples using real-time polymerase chain reaction (RT-PCR) system and rapid diagnosis of tuberculosis. A total of 508 clinical specimens (blood 343, menstrual fluid 53, endometrial tissue 43, body fluid 36, pus from lymph nodes 18, sputum 8, urine 5 and semen 2) were included in this study. We extracted DNA using QIAamp DNA Mini Kit (QIAGEN, Germany) and performed real-time assay using Rotor-Gene Q machine from Corbett Research, Australia for specific amplification of IS6110 sequence of mycobacterial genome. The RT-PCR result was also compared with bacterial culture and acid-fast bacillus staining. RT-PCR assay showed positivity in 52 cases and negative in 456 cases. Corresponding positive results in culture and acid-fast bacillus staining methods were 49 cases and 24 cases respectively. The sensitivity and specificity of detecting Mycobacterium tuberculosis by RT-PCR were 93.87% and 98.69% respectively taking positive culture results as reference standards. The overall positive and negative predictive values were 88.46% and 99.34% respectively. RT-PCR is a useful diagnostic tool for rapid and sensitive detection of mycobacteria in different clinical samples. The easy processing, fast reporting and relative lack of contamination issues make it worthy as a possible replacement to time consuming culture techniques. Moreover, it has added advantage of quantification of mycobacterial DNA, hence bacterial load.

  8. 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

  9. PCR Amplicon Prediction from Multiplex Degenerate Primer and Probe Sets

    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 theremore » are also TaqMan/Luminex probe matches within predicted amplicons.« less

  10. 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.

  11. 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.

  12. 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.

  13. 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.

  14. 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.

  15. 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. PMID:25455748

  16. Comparison of PCR and quantitative real-time PCR methods for the characterization of ruminant and cattle fecal pollution sources.

    PubMed

    Raith, Meredith R; Kelty, Catherine A; Griffith, John F; Schriewer, Alexander; Wuertz, Stefan; Mieszkin, Sophie; Gourmelon, Michele; Reischer, Georg H; Farnleitner, Andreas H; Ervin, Jared S; Holden, Patricia A; Ebentier, Darcy L; Jay, Jennifer A; Wang, Dan; Boehm, Alexandria B; Aw, Tiong Gim; Rose, Joan B; Balleste, E; Meijer, W G; Sivaganesan, Mano; Shanks, Orin C

    2013-11-15

    The State of California has mandated the preparation of a guidance document on the application of fecal source identification methods for recreational water quality management. California contains the fifth highest population of cattle in the United States, making the inclusion of cow-associated methods a logical choice. Because the performance of these methods has been shown to change based on geography and/or local animal feeding practices, laboratory comparisons are needed to determine which assays are best suited for implementation. We describe the performance characterization of two end-point PCR assays (CF128 and CF193) and five real-time quantitative PCR (qPCR) assays (Rum2Bac, BacR, BacCow, CowM2, and CowM3) reported to be associated with either ruminant or cattle feces. Each assay was tested against a blinded set of 38 reference challenge filters (19 duplicate samples) containing fecal pollution from 12 different sources suspected to impact water quality. The abundance of each host-associated genetic marker was measured for qPCR-based assays in both target and non-target animals and compared to quantities of total DNA mass, wet mass of fecal material, as well as Bacteroidales, and enterococci determined by 16S rRNA qPCR and culture-based approaches (enterococci only). Ruminant- and cow-associated genetic markers were detected in all filters containing a cattle fecal source. However, some assays cross-reacted with non-target pollution sources. A large amount of variability was evident across laboratories when protocols were not fixed suggesting that protocol standardization will be necessary for widespread implementation. Finally, performance metrics indicate that the cattle-associated CowM2 qPCR method combined with either the BacR or Rum2Bac ruminant-associated methods are most suitable for implementation.

  17. Use of Treponema pallidum PCR in Testing of Ulcers for Diagnosis of Primary Syphilis1

    PubMed Central

    Sednaoui, Patrice; Lautenschlager, Stephan; Ferry, Tristan; Toutous-Trellu, Laurence; Cavassini, Matthias; Yassir, Fatima; Martinez de Tejada, Begoña; Emonet, Stéphane; Combescure, Christophe; Schrenzel, Jacques; Perneger, Thomas

    2015-01-01

    Treponema pallidum PCR (Tp-PCR) has been noted as a valid method for diagnosing syphilis. We compared Tp-PCR to a combination of darkfield microscopy (DFM), the reference method, and serologic testing in a cohort of 273 patients from France and Switzerland and found the diagnostic accuracy of Tp-PCR was higher than that for DFM. PMID:25531672

  18. Use of Treponema pallidum PCR in testing of ulcers for diagnosis of primary syphilis.

    PubMed

    Gayet-Ageron, Angèle; Sednaoui, Patrice; Lautenschlager, Stephan; Ferry, Tristan; Toutous-Trellu, Laurence; Cavassini, Matthias; Yassir, Fatima; Martinez de Tejada, Begoña; Emonet, Stéphane; Combescure, Christophe; Schrenzel, Jacques; Perneger, Thomas

    2015-01-01

    Treponema pallidum PCR (Tp-PCR) has been noted as a valid method for diagnosing syphilis. We compared Tp-PCR to a combination of darkfield microscopy (DFM), the reference method, and serologic testing in a cohort of 273 patients from France and Switzerland and found the diagnostic accuracy of Tp-PCR was higher than that for DFM. PMID:25531672

  19. 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.

  20. Comparative analysis of two broad-range PCR assays for pathogen detection in positive-blood-culture bottles: PCR-high-resolution melting analysis versus PCR-mass spectrometry.

    PubMed

    Jeng, Kevin; Gaydos, Charlotte A; Blyn, Lawrence B; Yang, Samuel; Won, Helen; Matthews, Heather; Toleno, Donna; Hsieh, Yu-Hsiang; Carroll, Karen C; Hardick, Justin; Masek, Billy; Kecojevic, Alexander; Sampath, Rangarajan; Peterson, Stephen; Rothman, Richard E

    2012-10-01

    Detection of pathogens in bloodstream infections is important for directing antimicrobial treatment, but current culture-based approaches can be problematic. Broad-range PCR assays which target conserved genomic motifs for postamplification amplicon analysis permit detection of sepsis-causing pathogens. Comparison of different broad-range assays is important for informing future implementation strategies. In this study, we compared positive-blood-culture bottles processed by PCR coupled to high-resolution melting curve analysis (PCR/HRMA) and PCR coupled to electrospray ionization-mass spectrometry (PCR/ESI-MS) to microbiology culture results. Genus-level concordance was 90% (confidence interval [CI], 80 to 96%) for PCR/HRMA and 94% (CI, 85 to 98%) for PCR/ESI-MS. Species-level concordance was 90% (CI, 80 to 96%) for PCR/HRMA and 86% (CI, 75 to 93%) for PCR/ESI-MS. Unlike PCR/HRMA, PCR/ESI-MS was able to resolve polymicrobial samples. Our results demonstrated that the two assays have similar overall concordance rates but may have different roles as potential adjunctive tests with standard blood culture, since each method has different capabilities, advantages, and disadvantages.

  1. 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,...

  2. 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

  3. 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.

  4. 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…

  5. 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.

  6. 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...

  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. 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. 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.

  10. Multiplex PCR Method for Identifying Recombinant Vaccine-Related Polioviruses

    PubMed Central

    Kilpatrick, David R.; Ching, Karen; Iber, Jane; Campagnoli, Ray; Freeman, Christopher J.; Mishrik, Nada; Liu, Hong-Mei; Pallansch, Mark A.; Kew, Olen M.

    2004-01-01

    The recent discovery of recombinant circulating vaccine-derived poliovirus (recombinant cVDPV) has highlighted the need for enhanced global poliovirus surveillance to assure timely detection of any future cVDPV outbreaks. Six pairs of Sabin strain-specific recombinant primers were designed to permit rapid screening for VDPV recombinants by PCR. PMID:15365031

  11. 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 ...

  12. 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,…

  13. 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

  14. Nested PCR Assay for Detection of Granulocytic Ehrlichiae

    PubMed Central

    Massung, Robert F.; Slater, Kim; Owens, Jessica H.; Nicholson, William L.; Mather, Thomas N.; Solberg, Victoria B.; Olson, James G.

    1998-01-01

    A sensitive and specific nested PCR assay was developed for the detection of granulocytic ehrlichiae. The assay amplifies the 16S rRNA gene and was used to examine acute-phase EDTA-blood and serum samples obtained from seven humans with clinical presentations compatible with human granulocytic ehrlichiosis. Five of the seven suspected cases were positive by the PCR assay using DNA extracted from whole blood as the template, compared with a serologic assay that identified only one positive sample. The PCR assay using DNA extracted from the corresponding serum samples as the template identified three positive samples. The sensitivity of the assay on human samples was examined, and the limit of detection was shown to be fewer than 2 copies of the 16S rRNA gene. The application of the assay to nonhuman samples demonstrated products amplified from template DNA extracted from Ixodes scapularis ticks collected in Rhode Island and from EDTA-blood specimens obtained from white-tailed deer in Maryland. All PCR products were sequenced and identified as specific to granulocytic ehrlichiae. A putative variant granulocytic ehrlichia 16S rRNA gene sequence was detected among products amplified from both the ticks and the deer blood specimens. PMID:9542943

  15. PCR-mediated detection of acidophilic, bioleaching-associated bacteria.

    PubMed Central

    De Wulf-Durand, P; Bryant, L J; Sly, L I

    1997-01-01

    The detection of acidophilic microorganisms from mining environments by culture methods is time consuming and unreliable. Several PCR approaches were developed to amplify small-subunit rRNA sequences from the DNA of six bacterial phylotypes associated with acidic mining environments, permitting the detection of the target DNA at concentrations as low as 10 fg. PMID:9212441

  16. Internally controlled PCR system for detection of airborne microorganisms.

    PubMed

    Usachev, Evgeny V; Agranovski, Igor E

    2012-05-01

    Recently, we reported the outcomes of feasibility studies of a technological approach allowing rapid detection of a wide range of bioaerosols by combining a personal bioaerosol sampler with a real-time PCR technology. The protocol was found suitable for detection of targeted microorganisms within relatively short time periods. Considering the crucial importance of the PCR procedure quality control, the current paper reports the results of the development of an internally controlled PCR system for utilization by the above technology. The suggested strategy is based on utilization of only two fluorescent dyes, which are used respectively for target and internal amplification control (IAC) DNA amplification. A bacteriophage T4 and recombinant phage fd (M13) were used in this research as target and IAC, respectively. The constructed IAC was added directly to the collection liquid of the personal bioaerosol sampler enabling quality control to be present throughout the entire sampling-analysis procedures. For performance evaluation, serial ten-fold dilutions of T4 phage were aerosolized and sampled over a 10 minutes time period. The results showed that T4 phage could be reliably detected at the concentration of around 200 PFU per litre of air over the 10 minutes sampling period. The developed PCR assay demonstrated high specificity and no cross reaction. It is concluded that the recombinant phage fd is suitable for utilization as an internal control enabling to significantly minimize false negative results for bioaerosol detection procedures. PMID:22565862

  17. PCR detection of Helicobacter pylori in clinical samples.

    PubMed

    Rimbara, Emiko; Sasatsu, Masanori; Graham, David Y

    2013-01-01

    Helicobacter pylori is an important pathogen whose primary niche is the human stomach. H. pylori is etiologically 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.

  18. Facilitated Molecular Typing of Shigella Isolates Using ERIC-PCR

    PubMed Central

    Kosek, Margaret; Yori, Pablo Peñataro; Gilman, Robert H.; Vela, Henry; Olortegui, Maribel Paredes; Chavez, Cesar Banda; Calderon, Maritza; Bao, Juan Perez; Hall, Eric; Maves, Ryan; Burga, Rosa; Sanchez, Graciela Meza

    2012-01-01

    To evaluate the performance of enterobacterial repetitive intergenic sequence-based polymerase chain reaction (ERIC-PCR) typing versus the current standard for the typing of Shigella pulsed gel electrophoresis (PFGE), we typed 116 Shigella isolates from a village in an endemic setting over a 20-month period using both methods. PFGE identified 37 pulse types and had a discrimination index of 0.925 (95% confidence interval = 0.830–1.00), whereas ERIC-PCR identified 42 types and had a discrimination index of 0.961 (95% confidence interval = 0.886–1.00). PFGE and ERIC-PCR showed a 90.4% correlation in the designation of isolates as clonal or non-clonal in pairwise comparisons. Both systems were highly reproducible and provided highly similar and supplementary data compared with serotyping regarding the transmission dynamics of shigellosis in this community. ERIC-PCR is considerably more rapid and inexpensive than PFGE and may have a complementary role to PFGE for initial investigations of hypothesized outbreaks in resource-limited settings. PMID:22665611

  19. 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. PMID:21391499

  20. 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) ...

  1. 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.

  2. 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.

  3. Determination of DQB1 alleles using PCR amplification and allele-specific primers.

    PubMed

    Lepage, V; Ivanova, R; Loste, M N; Mallet, C; Douay, C; Naoumova, E; Charron, D

    1995-10-01

    Molecular genotyping of HLA class II genes is commonly carried out using polymerase chain reaction (PCR) in combination with sequence-specific oligotyping (PCR-SSO) or a combination of the PCR and restriction fragment length polymorphism methods (PCR-RFLP). However, the identification of the DQB1 type by PCR-SSO and PCR-RFLP is very time-consuming which is disadvantageous for the typing of cadaveric organ donors. We have developed a DQB1 typing method using PCR in combination with allele-specific amplification (PCR-ASA), which allows the identification of the 17 most frequent alleles in one step using seven amplification mixtures. PCR allele-specific amplification HLA-DQB1 typing is easy to perform, and the results are easy to interpret in routine clinical practice. The PCR-ASA method is therefore better suited to DQB1 typing for organ transplantation than other methods.

  4. 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.

  5. PCR for bioaerosol monitoring: sensitivity and environmental interference.

    PubMed

    Alvarez, A J; Buttner, M P; Stetzenbach, L D

    1995-10-01

    The PCR technique has potential for use in detection of low concentrations of airborne microorganisms. In this study, the sensitivity of PCR and its susceptibility to environmental interference were assessed with Escherichia coli DH1 as the target organism. Air samples, containing environmental bioaerosols, were collected with AGI-30 samplers and seeded with E. coli DH1 cells. Parallel studies were performed with cells seeded into the sampler prior to collection of air samples to determine the effects of environmental inhibition and sampling stress on the PCR assay. Baseline studies were also performed without environmental challenge or sampling stress to compare two protocols for cell lysis, solid phase and freeze-thaw. Amplification of a plasmid target sequence resulted in a detection limit of a single bacterial cell by the freeze-thaw and solid-phase methods within 5 and 9 h, respectively. With a genomic target, the sensitivity of the solid-phase method was 10-fold lower than that of freeze-thaw. Samples which contained 10(3) to 10(4) CFU of environmental organisms per m3 inhibited amplification; however, a 1/10 dilution of these samples resulted in successful amplifications. No difference in sensitivity of the PCR assay was obtained as a result of sampling stress, although a 10-fold decrease in culturability was observed. A field validation of the protocol with genomic primers demonstrated the presence of airborne E. coli and/or Shigella spp. in outdoor samples. This study indicates that the PCR method for detection of airborne microorganisms is rapid and sensitive and can be used as an alternative method for air quality monitoring. PMID:7487000

  6. 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.

  7. Band smearing of PCR amplified bacterial 16S rRNA genes: dependence on initial PCR target diversity.

    PubMed

    Zrimec, Jan; Kopinč, Rok; Rijavec, Tomaž; Zrimec, Tatjana; Lapanje, Aleš

    2013-11-01

    Band smearing in agarose gels of PCR amplified bacterial 16S rRNA genes is understood to comprise amplicons of varying sizes arising from PCR errors, and requires elimination. We consider that with amplified heterogeneous DNA, delayed electro-migration is caused not by PCR errors but by dsDNA structures that arise from imperfect strand pairing. The extent of band smearing was found to be proportional to the sequence heterogeneity in 16S rRNA variable regions. Denaturing alkaline gels showed that all amplified DNA was of the correct size. A novel bioinformatic approach was used to reveal that band smearing occurred due to imperfectly paired strands of the amplified DNA. Since the smear is a structural fraction of the correct size PCR product, it carries important information on richness and diversity of the target DNA. For accurate analysis, the origin of the smear must first be identified before it is eliminated by examining the amplified DNA in denaturing alkaline gels.

  8. 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. PMID:27120637

  9. 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

  10. Quantification of type II procollagen splice forms using Alternative Transcript-qPCR (AT-qPCR)

    PubMed Central

    McAlinden, Audrey; Shim, Kyu-Hwan; Wirthlin, Louisa; Ravindran, Soumya; Hering, Thomas M.

    2012-01-01

    During skeletal development, the onset of chondrogenic differentiation is marked by expression of the α1(II) procollagen Col2a1) gene. Exon 2 of Col2a1 codes for a cysteine-rich von Willebrand factor C-like domain. Chondroprogenitors express the exon 2-containing IIA and IID splice forms by utilizing adjacent 5′ splice sites separated by 3 base pairs. There is a shift to expression of the shorter, exon 2-lacking IIB splice form with further differentiation. Alternative splicing analysis of Col2a1 splice forms has often relied upon semi-quantitative PCR, using a single set of PCR primers to amplify multiple splice forms. We show that this widely used method is inaccurate due to mismatched amplification efficiency of different-sized PCR products. We have developed the TaqMan®-based AT-qPCR (Alternative Transcript-qPCR) assay to more accurately quantify alternatively spliced mRNA, and demonstrate the measurement of Col2a1 splice form expression in differentiating ATDC5 cells in vitro and in wild type mouse embryonic and postnatal cartilage in vivo. The AT-qPCR assay is based on the use of a multiple amplicon standard (MAS) plasmid, containing a chemically synthesized cluster of splice site-spanning PCR amplicons, to quantify alternative splice forms by standard curve-based qPCR. The MAS plasmid designed for Col2a1 also contained an 18S rRNA amplicon for sample normalization, and an amplicon corresponding to a region spanning exon 52-53 to measure total Col2a1 mRNA. In mouse E12.5 to P70 cartilage, we observed the expected switch between the IIA and IIB splice forms; no IID or IIC splice products were observed. However, in the ATDC5 cultures, predominant expression of the IIA and IID splice forms was found at all times in culture. Additionally, we observed that the sum of the IIA, IIB and IID splice forms comprises only a small fraction of Col2a1 transcripts containing the constitutive exon 52-53 junction. We conclude from our results that the majority of ATDC5

  11. Identification of weakly beta-hemolytic porcine spirochetes by biochemical reactions, PCR-based restriction fragment length polymorphism analysis and species-specific PCR.

    PubMed

    Ohya, Tatsuo; Araki, Hiroshi; Sueyoshi, Masuo

    2008-08-01

    We examined the usefulness of PCR-based restriction fragment length polymorphism (PCR-RFLP) and species-specific PCR combined with a newly devised rapid biochemical test using microplates for identifying weakly beta-hemolytic intestinal spirochetes (WBHIS) isolated from pigs. WBHIS strains showing atypical biochemical characteristics were decisively identified at the species level by PCR-RFLP and species-specific PCR. Identification of WBHIS at the species level in routine diagnostic work will certainly contribute to clarifying the pathogenicity of WBHIS.

  12. 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

  13. 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

  14. 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.

  15. 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.

  16. 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

  17. Species identification of cattle and buffalo fat through PCR assay.

    PubMed

    Vaithiyanathan, S; Kulkarni, V V

    2016-04-01

    A method was standardized to isolate quality DNA from cattle and buffalo fat for species identification using QIAamp DNA stool mini kit. The quality of the DNA was sufficient enough to amplify universal primers viz., mt 12S rRNA and mt 16S rRNA, and species specific D loop primers for cattle and buffalo. The sensitivity of the PCR assay in the species specific D loop primer amplification was with a detection level of 0. 47 ng cattle DNA and 0.23 ng buffalo DNA in simplex and, 0. 47 ng cattle DNA and 0.12 ng buffalo DNA in duplex PCR. It is a potentially reliable method for DNA detection to authenticate animal fat. PMID:27413237

  18. Measuring Digital PCR Quality: Performance Parameters and Their Optimization.

    PubMed

    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

  19. PCR and blot hybridization for rapid identification of Haloferax species.

    PubMed

    Asker, Dalal; Ohta, Yoshiyuki

    2002-05-01

    Based on the amplification of a 16S rDNA, a PCR assay for the identification of species of Haloferax to genus level was performed. Two variable regions of the 16S rDNA in Haloferax spp. were selected as genus-specific primers for the PCR assay and hybridization probe. Five genera of halophilic Archaea and Escherichia coli were examined as outside groups. Using this approach, all strains of Haloferax spp. were positive. In contrast, all species belonging to the most closely related genera, including Natrinema, Halorubrum, Halobacterium, and Haloarcula, were negative. In addition, the mass bloom of halophilic Archaea that develops in the El-Mallahet saltern of Alexandria City was positive using the same approach. This assay, which does not require pure cultures of microorganisms, is a specific and rapid method for identifying Haloferax spp. in hypersaline environments.

  20. Measuring Digital PCR Quality: Performance Parameters and Their Optimization.

    PubMed

    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.

  1. pPCV, a versatile vector for cloning PCR products.

    PubMed

    Janner, Christiane R; Brito, Ana Lívia P; Moraes, Lidia Maria P; Reis, Viviane Cb; Torres, Fernando Ag

    2013-01-01

    The efficiency of PCR product cloning depends on the nature of the DNA polymerase employed because amplicons may have blunt-ends or 3' adenosines overhangs. Therefore, for amplicon cloning, available commercial vectors are either blunt-ended or have a single 3' overhanging thymidine. The aim of this work was to offer in a single vector the ability to clone both types of PCR products. For that purpose, a minimal polylinker was designed to include restriction sites for EcoRV and XcmI which enable direct cloning of amplicons bearing blunt-ends or A-overhangs, respectively, still offering blue/white selection. When tested, the resulting vector, pPCV, presented high efficiency cloning of both types of amplicons. PMID:24058893

  2. Adaptive DNA Computing Algorithm by Using PCR and Restriction Enzyme

    NASA Astrophysics Data System (ADS)

    Kon, Yuji; Yabe, Kaoru; Rajaee, Nordiana; Ono, Osamu

    In this paper, we introduce an adaptive DNA computing algorithm by using polymerase chain reaction (PCR) and restriction enzyme. The adaptive algorithm is designed based on Adleman-Lipton paradigm[3] of DNA computing. In this work, however, unlike the Adleman- Lipton architecture a cutting operation has been introduced to the algorithm and the mechanism in which the molecules used by computation were feedback to the next cycle devised. Moreover, the amplification by PCR is performed in the molecule used by feedback and the difference concentration arisen in the base sequence can be used again. By this operation the molecules which serve as a solution candidate can be reduced down and the optimal solution is carried out in the shortest path problem. The validity of the proposed adaptive algorithm is considered with the logical simulation and finally we go on to propose applying adaptive algorithm to the chemical experiment which used the actual DNA molecules for solving an optimal network problem.

  3. 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. PMID:15567226

  4. 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. PMID:18385011

  5. Molecular beacon real-time PCR detection of swine viruses.

    PubMed

    McKillen, John; Hjertner, Bernt; Millar, Andrena; McNeilly, Francis; Belák, Sándor; Adair, Brian; Allan, Gordon

    2007-03-01

    Rapid and reliable detection of viral pathogens is critical for the management of the diseases threatening the economic competitiveness of the swine farming industry worldwide. Molecular beacon assays are one type of real-time polymerase chain reaction (PCR) technology capable of fast, specific, sensitive, and reliable viral detection. In this paper, the development of molecular beacon assays as novel tools for the rapid detection of Aujeszky's disease virus, African swine fever virus, porcine circovirus type 2 and porcine parvovirus is described. The assays are capable of rapidly detecting 2 x 10(1) copies of target and are linear between 2 x 10(9) and 2 x 10(2) copies. They can detect virus specifically in clinical samples such as whole blood, serum and tissue. In comparison to conventional PCR they are either as sensitive or more sensitive. As such these molecular beacon assays represent a powerful tool for the detection of these viruses in swine.

  6. 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

  7. 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.

  8. Actuation method and apparatus, micropump, and PCR enhancement method

    SciTech Connect

    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.

  9. 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.

  10. 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.

  11. Monitoring Piscirickettsia salmonis by denaturant gel electrophoresis and competitive PCR.

    PubMed

    Heath, S; Pak, S; Marshall, S; Prager, E M; Orrego, C

    2000-05-25

    Reported strains of Piscirickettsia salmonis, a pathogen of salmonid fishes, were analyzed by amplifying part of the internal transcribed spacer (ITS) of the ribosomal RNA (rRNA) operon followed by denaturing gradient gel electrophoresis (DGGE) of the amplicons. All amplified fragments differing in sequence were distinguished by migration during DGGE. A simpler format, constant denaturant gel electrophoresis (CDGE), allowed the same diagnostic distinctions among strains. Sampling during 1997 and 1998 of salmonids from 5 different sites on and near Chiloé Island in southern Chile displaying piscirickettsiosis revealed only P. salmonis resembling LF-89, the type strain first isolated in 1989. These observations are encouraging for control strategies, which might otherwise be compromised by unpredictable shifts of P. salmonis types in salmon farms. A competitive PCR assay offered insight about the power of PCR for quantification and about specific tissue invasiveness by this intracellular pathogen. This approach revealed that the PCR could amplify approximately 1 to 10 P. salmonis genome equivalents against a background of > 99.9% salmonid DNA. It also raised the possibility that the salmonid brain is an important site for P. salmonis survival, with its bacterial load in 1 individual having been about 100 times the loads observed in liver and kidney. Pathogen detection by competitive PCR in a surface seawater sample from a netpen in use indicated a density of about 3000 to 4000 P. salmonis cells (or their DNA remnants) 1(-1). Such quantitative estimates should aid decisions about disease prevention and management as, for example, choice of netpen sites following fallow periods and certification of ova, which are known conduits of infection.

  12. Single-tube nested PCR in the diagnosis of tuberculosis.

    PubMed Central

    Chan, C M; Yuen, K Y; Chan, K S; Yam, W C; Yim, K H; Ng, W F; Ng, M H

    1996-01-01

    AIMS: To evaluate the usefulness of a single-tube nested polymerase chain reaction (PCR) assay in the diagnosis of tuberculosis in 1497 pulmonary and 536 extrapulmonary specimens. METHODS: A single-tube nested PCR, utilising two sets of primers with different melting temperatures (88 degrees C for external primers; 70 degrees C for internal primers) to augment sensitivity and specificity without increasing the risk of amplicon contamination, was evaluated. Specimens were initially tested for the repetitive IS6110 sequences and if negative, retested for the universal 38 kilodalton sequence and for inhibitors. dUTP/Uracil-N-glycosylase and Instagene treatment were used to minimise contamination and the effect of inhibitors, respectively. RESULTS: Using culture as the gold standard, the overall sensitivity of the assay was 89% for pulmonary and 42% for extrapulmonary specimens. Sensitivity varied greatly with respect to sample type (92% for follow up specimens from a chest hospital and 70% for non-follow up specimens from a general hospital). The smear positivity rates were 15% for extrapulmonary specimens, and 69% and 45%, respectively, for follow up and non-follow up specimens from pulmonary sites. Specificity was 99.7%. Inhibitors were present more frequently in extrapulmonary than in pulmonary specimens (13.4% v 2.7%). CONCLUSION: Despite the high sensitivity of the PCR assay for the diagnosis of tuberculosis in pulmonary specimens, it was less effective in the extrapulmonary samples. This is probably because of the lower bacterial load in extrapulmonary specimens, the presence of more inhibitors adversely affecting the PCR assay and the higher volume of specimens used for culture. Images PMID:8655703

  13. Performance of two real-time PCR assays for hepatitis B virus DNA detection and quantitation.

    PubMed

    Kania, Dramane; Ottomani, Laure; Meda, Nicolas; Peries, Marianne; Dujols, Pierre; Bolloré, Karine; Rénier, Wendy; Viljoen, Johannes; Ducos, Jacques; Van de Perre, Philippe; Tuaillon, Edouard

    2014-06-01

    In-house developed real-time PCR (qPCR) techniques could be useful conjunctives to the management of hepatitis B virus (HBV) infection in resource-limited settings with high prevalence. Two qPCR assays (qPCR1 and qPCR2), based on primers/probes targeting conserved regions of the X and S genes of HBV respectively, were evaluated using clinical samples of varying HBV genotypes, and compared to the commercial Roche Cobas AmpliPrep/Cobas TaqMan HBV Test v2.0. The lower detection limit (LDL) was established at 104 IU/ml for qPCR1, and 91 IU/ml for qPCR2. Good agreement and correlation were obtained between the Roche assay and both qPCR assays (r = 0.834 for qPCR1; and r = 0.870 for qPCR2). Differences in HBV DNA load of > 0.5 Log10 IU/ml between the Roche and the qPCR assays were found in 49/122 samples of qPCR1, and 35/122 samples of qPCR2. qPCR1 tended to underestimate HBV DNA quantity in samples with a low viral load and overestimate HBV DNA concentration in samples with a high viral load when compared to the Roche test. Both molecular tools that were developed, used on an open real-time PCR system, were reliable for HBV DNA detection and quantitation. The qPCR2 performed better than the qPCR1 and had the additional advantage of various HBV genotype detection and quantitation. This low cost quantitative HBV DNA PCR assay may be an alternative solution when implementing national programmes to diagnose, monitor and treat HBV infection in low- to middle-income countries where testing for HBV DNA is not available in governmental health programmes.

  14. Analytical Sensitivity Comparison between Singleplex Real-Time PCR and a Multiplex PCR Platform for Detecting Respiratory Viruses

    PubMed Central

    Parker, Jayme; Fowler, Nisha; Walmsley, Mary Louise; Schmidt, Terri; Scharrer, Jason; Kowaleski, James; Grimes, Teresa; Hoyos, Shanann; Chen, Jack

    2015-01-01

    Multiplex PCR methods are attractive to clinical laboratories wanting to broaden their detection of respiratory viral pathogens in clinical specimens. However, multiplexed assays must be well optimized to retain or improve upon the analytic sensitivity of their singleplex counterparts. In this experiment, the lower limit of detection (LOD) of singleplex real-time PCR assays targeting respiratory viruses is compared to an equivalent panel on a multiplex PCR platform, the GenMark eSensor RVP. LODs were measured for each singleplex real-time PCR assay and expressed as the lowest copy number detected 95–100% of the time, depending on the assay. The GenMark eSensor RVP LODs were obtained by converting the TCID50/mL concentrations reported in the package insert to copies/μL using qPCR. Analytical sensitivity between the two methods varied from 1.2–1280.8 copies/μL (0.08–3.11 log differences) for all 12 assays compared. Assays targeting influenza A/H3N2, influenza A/H1N1pdm09, influenza B, and human parainfluenza 1 and 2 were most comparable (1.2–8.4 copies/μL, <1 log difference). Largest differences in LOD were demonstrated for assays targeting adenovirus group E, respiratory syncytial virus subtype A, and a generic assay for all influenza A viruses regardless of subtype (319.4–1280.8 copies/μL, 2.50–3.11 log difference). The multiplex PCR platform, the GenMark eSensor RVP, demonstrated improved analytical sensitivity for detecting influenza A/H3 viruses, influenza B virus, human parainfluenza virus 2, and human rhinovirus (1.6–94.8 copies/μL, 0.20–1.98 logs). Broader detection of influenza A/H3 viruses was demonstrated by the GenMark eSensor RVP. The relationship between TCID50/mL concentrations and the corresponding copy number related to various ATCC cultures is also reported. PMID:26569120

  15. Improved sensitivity of PCR for Chlamydophila using pmp genes.

    PubMed

    Laroucau, K; Souriau, A; Rodolakis, A

    2001-09-20

    Primers targeting the conserved pmp gene family of Chlamydophila abortus were evaluated for their ability to improve the polymerase chain reaction (PCR) sensitivity. In purified DNA, specific pmp primers (named CpsiA and CpsiB) allowed at least a 10-fold increase of the PCR sensitivity compared to the specific ompA primers for C. abortus, but also for C. psittaci and C. caviae strains. No amplification was observed on C. felis, C. pecorum, C. pneumoniae and Chlamydia trachomatis strains. Tested on contaminated specimens such as genital swabs, the PCR sensitivity observed with CpsiA/CpsiB was also better than with the ompA primers. This study demonstrated that these specific pmp primers could serve as valuable, sensitive and common tools for a specific Chlamydophila diagnosis in ruminant, avian and human diseases. Digestion by AluI of the CpsiA/CpsiB fragments allowed a specific discrimination of the strains in function of their hosts and/or their serotypes.

  16. 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.

  17. Validation of chimerism in pediatric recipients of allogeneic hematopoietic stem cell transplantation (HSCT) a comparison between two methods: real-time PCR (qPCR) vs. variable number tandem repeats PCR (VNTR PCR).

    PubMed

    Kletzel, Morris; Huang, Wei; Olszewski, Marie; Khan, Sana

    2013-01-01

    Post-hematopoietic stem cell transplantation (HSCT) chimerism monitoring is important to assess relapse and therapeutic intervention. The purpose of our study is to compare two methods variable number tandem repeats (VNTR) vs. quantitative real- time polymerase chain reaction (qPCR) in terms of determining chimerism. 127 (peripheral blood n=112, bone marrow n=15) samples were simultaneously tested by VNTR using APO-B, D1S80, D1S111, D17S30, gene loci SRY and ZP3 and qPCR using 34 assays (CA001-CA034) that are designed to a bi-allelic insertion/deletion (indel) polymorphism in the human genome. Samples were separated in three subsets: total WBC, T-cell and Myeloid cells. Extraction of DNA was performed then quantified. We analyzed column statistics, paired t-test and regression analysis for both methods. There was complete correlation between the two methods. The simplicity and rapidity of the test results from the qPCR method is more efficient and accurate to assess chimerism.

  18. 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.

  19. Rat Gene Mapping Using Pcr-Analyzed Microsatellites

    PubMed Central

    Serikawa, T.; Kuramoto, T.; Hilbert, P.; Mori, M.; Yamada, J.; Dubay, C. J.; Lindpainter, K.; Ganten, D.; Guenet, J. L.; Lathrop, G. M.; Beckmann, J. S.

    1992-01-01

    One hundred and seventy-four rat loci which contain short tandem repeat sequences were extracted from the GenBank or EMBL data bases and used to define primers for amplification by the polymerase chain reaction (PCR) of the microsatellite regions, creating PCR-formatted sequence-tagged microsatellite sites (STMSs). One hundred and thirty-four STMSs for 118 loci, including 6 randomly cloned STMSs, were characterized: (i) PCR-analyzed loci were assigned to specific chromosomes using a panel of rat X mouse somatic cell hybrid clones. (ii) Length variation of the STMSs among 8 inbred rat strains could be visualized at 85 of 107 loci examined (79.4%). (iii) A genetic map, integrating biochemical, coat color, mutant and restriction fragment length polymorphism loci, was constructed based on the segregation of 125 polymorphic markers in seven rat backcrosses and in two F(2) crosses. Twenty four linkage groups were identified, all of which were assigned to a defined chromosome. As a reflection of the bias for coding sequences in the public data bases, the STMSs described herein are often associated with genes. Hence, the genetic map we report coincides with a gene map. The corresponding map locations of the homologous mouse and human genes are also listed for comparative mapping purposes. PMID:1628813

  20. 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

  1. Authentication of Atlantic cod (Gadus morhua) using real time PCR.

    PubMed

    Herrero, Beatriz; Madriñán, María; Vieites, Juan M; Espiñeira, Montserrat

    2010-04-28

    This work describes the development of a real-time polymerase chain reaction (RT-PCR) system for the detection and identification of Atlantic cod (Gadus morhua). Among the advantages of this technique, it is worth highlighting that this is reliable in terms of specificity and sensitivity. The TaqMan real-time PCR is the simplest, fastest testing process and has the highest potential for automation, therefore representing the currently most suitable method for screening, allowing the detection of fraudulent or unintentional mislabeling of this species. The method can be applied to all kinds of products, fresh, frozen, and processed products, including those undergoing intensive processes of transformation. The developed methodology using specific primer-probe set was validated and further applied to 40 commercial samples labeled as cod in order to determinate if the species used for their manufacturing corresponded to G. morhua, detecting 20% that were incorrectly labeled. A C(t) value of about 19 was obtained when G. morhua was present. In samples with a species mixture, all samples that had a fluorescence signal were positive (C(t) < 30) for the presence of G. morhua by conventional end-point RT-PCR, and the estimated limit of detection for these type of samples was of 20 pg of DNA. The methodology herein developed is useful to check the fulfilment of labeling regulations for seafood products and verify the correct traceability in commercial trade and for fisheries control.

  2. Microdroplet-based PCR amplification for large scale targeted sequencing

    PubMed Central

    Tewhey, Ryan; Warner, Jason; Nakano, Masakazu; Libby, Brian; Medkova, Martina; David, Patricia; Kotsopoulos, Steve; Samuels, Michael; Hutchison, J. Brian; Larson, Jonathan W.; Topol, Eric J.; Weiner, Michael P.; Harismendy, Olivier; Olson, Jeff; Link, Darren R.; Frazer, Kelly A.

    2009-01-01

    Targeted sequencing of specific loci of the human genome is a promising approach for maximizing the efficiency of second-generation sequencing technologies for population-based studies of genetic variation. Here we describe microdroplet PCR, which performs 1.5 million separate amplifications in parallel, as an approach for enriching targeted sequences in the human genome. We initially designed primers to 435 exons of 47 genes that were selected for having a broad spectrum of sequence characteristics. Using this primer set we amplified the same six samples by both microdroplet and traditional singleplex PCR and sequenced the products using the Illumina GAII demonstrating that both methods generate similarly high quality data; 84% of the uniquely mapping reads fell within the targeted sequences, uniform coverage of ~90% of the targeted bases, greater than 99% accuracy in sequence variant calls, and high reproducibility between different samples (r2=0.9). We next scaled the microdroplet PCR to 3976 amplicons totaling 1.49 Mb of sequence, sequenced the resulting sample on both the Illumina GAII and Roche 454 platforms, and obtained data with equally high specificity and sensitivity quality. Our results demonstrate that microdroplet technology is well suited for processing DNA for massively parallel amplification of specific subsets of the human genome for targeted sequencing. PMID:19881494

  3. Determination of Arabidopsis thaliana telomere length by PCR

    PubMed Central

    Vaquero-Sedas, María I.; Vega-Palas, Miguel A.

    2014-01-01

    In humans, telomere length studies have acquired great relevance because the length of telomeres has been related to natural processes like disease, aging and cancer. However, very little is known about the influence of telomere length on the biology of wild type plants. The length of plant telomeres has been usually studied by Terminal Restriction Fragment (TRF) analyses. This technique requires high amounts of tissue, including multiple cell types, which might be the reason why very little is known about the influence of telomere length on plant natural processes. In contrast, many of the human telomere length studies have focused on homogenous cell populations. Most of these studies have been performed by PCR, using telomeric degenerated primers, which allow the determination of telomere length from small amounts of human cells. Here, we have adapted the human PCR procedure to analyze the length of Arabidopsis thaliana telomeres. This PCR approach will facilitate the analysis of telomere length from low amounts of tissue. We have used it to determine that CG and non CG DNA methylation positively regulates Arabidopsis telomere length. PMID:24986269

  4. 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

  5. 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.

  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. Pure chromosome-specific PCR libraries from single sorted chromosomes.

    PubMed Central

    VanDevanter, D R; Choongkittaworn, N M; Dyer, K A; Aten, J; Otto, P; Behler, C; Bryant, E M; Rabinovitch, P S

    1994-01-01

    Chromosome-specific DNA libraries can be very useful in molecular and cytogenetic genome mapping studies. We have developed a rapid and simple method for the generation of chromosome-specific DNA sequences that relies on polymerase chain reaction (PCR) amplification of a single flow-sorted chromosome or chromosome fragment. Previously reported methods for the development of chromosome libraries require larger numbers of chromosomes, with preparation of pure chromosomes sorted by flow cytometry, generation of somatic cell hybrids containing targeted chromosomes, or a combination of both procedures. These procedures are labor intensive, especially when hybrid cell lines are not already available, and this has limited the generation of chromosome-specific DNA libraries from nonhuman species. In contrast, a single sorted chromosome is a pure source of DNA for library production even when flow cytometric resolution of chromosome populations is poor. Furthermore, any sorting cytometer may be used with this technique. Using this approach, we demonstrate the generation of PCR libraries suitable for both molecular and fluorescence in situ hybridization studies from individual baboon and canine chromosomes, separate human homologues, and a rearranged marker chromosome from a transformed cell line. PCR libraries specific to subchromosomal regions have also been produced by sorting a small chromosome fragment. This simple and rapid technique will allow generation of nonhuman linkage maps and probes for fluorescence in situ hybridization and the characterization of marker chromosomes from solid tumors. In addition, allele-specific libraries generated by this strategy may also be useful for mapping genetic diseases. Images PMID:8016078

  8. Determination of Arabidopsis thaliana telomere length by PCR.

    PubMed

    Vaquero-Sedas, María I; Vega-Palas, Miguel A

    2014-07-02

    In humans, telomere length studies have acquired great relevance because the length of telomeres has been related to natural processes like disease, aging and cancer. However, very little is known about the influence of telomere length on the biology of wild type plants. The length of plant telomeres has been usually studied by Terminal Restriction Fragment (TRF) analyses. This technique requires high amounts of tissue, including multiple cell types, which might be the reason why very little is known about the influence of telomere length on plant natural processes. In contrast, many of the human telomere length studies have focused on homogenous cell populations. Most of these studies have been performed by PCR, using telomeric degenerated primers, which allow the determination of telomere length from small amounts of human cells. Here, we have adapted the human PCR procedure to analyze the length of Arabidopsis thaliana telomeres. This PCR approach will facilitate the analysis of telomere length from low amounts of tissue. We have used it to determine that CG and non CG DNA methylation positively regulates Arabidopsis telomere length.

  9. Widespread use of real-time PCR for rickettsial diagnosis.

    PubMed

    Renvoisé, Aurélie; Rolain, Jean-Marc; Socolovschi, Cristina; Raoult, Didier

    2012-02-01

    We report 2 years of experience with rickettsial molecular diagnosis using real-time PCR at the French National Reference Center. All Rickettsia genomes available were compared to discover specific sequences to design new sets of primers and probes. The specificity was verified in silico and against a panel of 30 rickettsial species. Sensitivity was determined using 10-fold serial dilutions. Finally, primers and probes that were both specific and sensitive were routinely used for the diagnosis of rickettsial infections from clinical specimens. We retained sets of primers and probes to detect spotted fever group Rickettsia, typhus group Rickettsia,Rickettsia conorii,Rickettsia slovaca,Rickettsia africae and Rickettsia australis; 643 clinical samples were screened for the presence of Rickettsia DNA. Overall, 45 positive samples were detected, including 15 Rickettsia africae, nine R. conorii, five Rickettsia sibirica mongolitimonae, four R. slovaca, two R. australis, four Rickettsia massiliae, one Rickettsia honei, one Rickettsia typhi and eight Rickettsia sp. Positive samples were detected mainly from cutaneous biopsies and swabs (31/45). Widespread use of real-time PCR is inexpensive and reduces delay in the diagnosis of rickettsial infections. These real-time PCR assays could be implemented easily in laboratories that have molecular facilities and may be added to existing molecular tools as a point-of-care strategy.

  10. Large scale multiplex PCR improves pathogen detection by DNA microarrays

    PubMed Central

    2009-01-01

    Background Medium density DNA microchips that carry a collection of probes for a broad spectrum of pathogens, have the potential to be powerful tools for simultaneous species identification, detection of virulence factors and antimicrobial resistance determinants. However, their widespread use in microbiological diagnostics is limited by the problem of low pathogen numbers in clinical specimens revealing relatively low amounts of pathogen DNA. Results To increase the detection power of a fluorescence-based prototype-microarray designed to identify pathogenic microorganisms involved in sepsis, we propose a large scale multiplex PCR (LSplex PCR) for amplification of several dozens of gene-segments of 9 pathogenic species. This protocol employs a large set of primer pairs, potentially able to amplify 800 different gene segments that correspond to the capture probes spotted on the microarray. The LSplex protocol is shown to selectively amplify only the gene segments corresponding to the specific pathogen present in the analyte. Application of LSplex increases the microarray detection of target templates by a factor of 100 to 1000. Conclusion Our data provide a proof of principle for the improvement of detection of pathogen DNA by microarray hybridization by using LSplex PCR. PMID:19121223

  11. PCR-based typing of DNA extracted from cigarette butts.

    PubMed

    Hochmeister, M N; Budowle, B; Jung, J; Borer, U V; Comey, C T; Dirnhofer, R

    1991-01-01

    Limited genetic marker information can be obtained from saliva by typing by conventional serological means. Thus, the application of PCR-based DNA typing methods was investigated as a potential approach for typing genetic markers in saliva. DNA was isolated from 200 cigarettes smoked by 10 different individuals (20 cigarettes per individual) and from 3 cigarette butts recovered from 2 crime scenes (adjudicated cases) using a Chelex 100 extraction procedure. The amount of recovered human DNA was quantified by slot-blot analysis and ranged from approximately less than 2-160 ng DNA per cigarette butt for the 200 samples, and 8 ng, 50 ng, and 100 ng for the cigarette butts from the adjudicated cases. The DNA was successfully amplified by the polymerase chain reaction (PCR) for the HLA-DQ alpha locus (99 out of 100 samples) as well as for the variable number of tandem repeat (VNTR) locus D1S80 (99 out of 100 samples). Amplification and typing of DNA was successful on all samples recovered from the crime scenes. The results suggest that PCR-based typing of DNA offers a potential method for genetically characterizing traces of saliva on cigarette butts.

  12. Diagnostic approaches for oculoglandular tularemia: advantages of PCR

    PubMed Central

    Kantardjiev, Todor; Padeshki, Plamen; Ivanov, Ivan N

    2007-01-01

    Aim The authors describe a diagnostic approach that proved to be particularly valuable in rare cases of ocular tularemia registered during the tularemia outbreak in 1997–2005 in Bulgaria. The authors describe the laboratory findings and diagnosis of four cases with an oculoglandular form of infection. Methods Several different specimens from each patient were analysed. Oculoglandular tularemia was diagnosed in four patients either by culture, immunofluorescent antibody analysis (IFA), serology or by a polymerase chain reaction (PCR) assay. Results and Discussion Three F tularensis strains were isolated and characterised. One of these was isolated from a conjuctival swab specimen obtained from a seronegative patient. The authors report for the first time a successful application of diagnostic PCR performed directly on conjuctival swab specimen. From all analysed specimens IFA was diagnostically effective only in the case of lymph node aspirates and was not sensitive enough for conjuctival swabs or blood samples. The authors also describe the histological picture of a conjunctival granuloma in course of infection. All patients were successfully treated with ciprofloxacin. Conclusions Some of the proposed laboratory diagnostic strategies (swab PCR) are not invasive and could represent a new approach for resolving rare and hard‐to‐diagnose cases of oculoglandular tularemia. PMID:17475710

  13. Determination of Arabidopsis thaliana telomere length by PCR.

    PubMed

    Vaquero-Sedas, María I; Vega-Palas, Miguel A

    2014-01-01

    In humans, telomere length studies have acquired great relevance because the length of telomeres has been related to natural processes like disease, aging and cancer. However, very little is known about the influence of telomere length on the biology of wild type plants. The length of plant telomeres has been usually studied by Terminal Restriction Fragment (TRF) analyses. This technique requires high amounts of tissue, including multiple cell types, which might be the reason why very little is known about the influence of telomere length on plant natural processes. In contrast, many of the human telomere length studies have focused on homogenous cell populations. Most of these studies have been performed by PCR, using telomeric degenerated primers, which allow the determination of telomere length from small amounts of human cells. Here, we have adapted the human PCR procedure to analyze the length of Arabidopsis thaliana telomeres. This PCR approach will facilitate the analysis of telomere length from low amounts of tissue. We have used it to determine that CG and non CG DNA methylation positively regulates Arabidopsis telomere length. PMID:24986269

  14. An unusual presentation of herpes simplex encephalitis with negative PCR.

    PubMed

    Buerger, Kelly J; Zerr, Kayleigh; Salazar, Richard

    2015-01-01

    A 74-year-old man presented with acute right-sided hemiparesis and epilepsia partialis continua in association with fever and confusion. Initial workup revealed possible cerebritis in the left medial frontal lobe without involvement of the temporal lobes. Cerebrospinal fluid (CSF) analysis revealed minimal lymphocytic pleocytosis but negative real-time herpes simplex virus (HSV) PCR. Acyclovir was discontinued on day 5 due to a negative infectious workup and clinical improvement. On day 9 his condition deteriorated and he was transferred to a higher level of acuity for advanced supportive care. Worsening encephalopathy and refractory status epilepticus ensued despite medical care. Repeat CSF analysis showed mild lymphocytic pleocytosis with negative real-time HSV PCR. Brain MRI revealed progression of cortical enhancement. Immunosuppressive therapy and plasma exchange were attempted without clinical response. On day 24, another lumbar puncture showed only mild lymphocytic pleocytosis. Brain MRI showed involvement of the right medial temporal lobe. Subsequently, acyclovir was resumed. The HSV-1 PCR result was positive on day 30. Unfortunately, the patient expired.

  15. [Multiplex PCR for detecting genotypes of deletional alpha-thalassemia].

    PubMed

    Wu, Jie-Ying; Liao, Can; Li, Jian; Huang, Yi-Ning

    2004-08-01

    To investigate the clinical application of multiplex PCR in detecting genotypes of deletional alpha-thalassemia in South China and observe the distribution frequency of alpha-globin gene deletion, 145 patients with silent carrier, alpha thalassemia trait or HbH were identified by M-PCR and 1.2% agarose gel electrophoresis. There are 1.3, 1.6, 1.8 and 2.0 kb bands which indicate --(SEA), -alpha(4.2), alphaalpha and -alpha(3.7), respectively. The results showed that among 145 patients, 100 patients with --(SEA)/alphaalpha (68.9%), 15 with -alpha(3.7)/alphaalpha (10.3%), 8 with -alpha(4.2)/alphaalpha (5.52%), 2 with -alpha(3.7)/-alpha(4.2) (1.38%), 1 with -alpha(3.7)/-alpha(3.7) (0.69%), 1 with -alpha(4.2)/-alpha(4.2) (0.69%), 14 with --(SEA)/-alpha(3.7) (9.65%), 2 with --(SEA)/-alpha(4.2) (1.38%) were found. Two patients prenatal diagnosed were confirmed with Bart's hydrops fetuses. In conclusion, M-PCR analysis is a simple, rapid and accurate method for detection of alpha-thalassemia gene deletion. This technique is helpful in screening, carrier identification and prenatal diagnosis of deletional alpha-thalassemia.

  16. 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. PMID:24937806

  17. De novo DNA synthesis using single molecule PCR

    PubMed Central

    Yehezkel, Tuval Ben; Linshiz, Gregory; Buaron, Hen; Kaplan, Shai; Shabi, Uri; Shapiro, Ehud

    2008-01-01

    The throughput of DNA reading (sequencing) has dramatically increased recently due to the incorporation of in vitro clonal amplification. The throughput of DNA writing (synthesis) is trailing behind, with cloning and sequencing constituting the main bottleneck. To overcome this bottleneck, an in vitro alternative for in vivo DNA cloning must be integrated into DNA synthesis methods. Here we show how a new single molecule PCR (smPCR)-based procedure can be employed as a general substitute to in vivo cloning thereby allowing for the first time in vitro DNA synthesis. We integrated this rapid and high fidelity in vitro procedure into our earlier recursive DNA synthesis and error correction procedure and used it to efficiently construct and error-correct a 1.8-kb DNA molecule from synthetic unpurified oligos completely in vitro. Although we demonstrate incorporating smPCR in a particular method, the approach is general and can be used in principle in conjunction with other DNA synthesis methods as well. PMID:18667587

  18. [Identification for genetically modified maize T14/T25 with real time fluorescent PCR method].

    PubMed

    Cao, Ji-Juan; Qin, Wen; Zhu, Shui-Fang; Cao, Yuan-Yin

    2004-09-01

    To identify genetically modified (GM) maize T14/T25 lines, a real-time fluorescent PCR (RTF PCR) assay was performed in this study. Primers and Taqman probes specific for inserted genes in the T14/T25 were used to conduct the real-time fluorescent (RTF) PCR and PCR assays. The RTF PCR method was established to detect and identify GM maize lines. The results show that the TaqMan probe could identify T14/T25 maize used, while other GM and NO-GM maize didn't be detected. The RTF PCR could be a new method for detecting other genetically modified organism.

  19. 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

  20. 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.

  1. 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.

  2. Selection of Reference Genes for Quantitative Real Time PCR (qPCR) Assays in Tissue from Human Ascending Aorta

    PubMed Central

    Rueda-Martínez, Carmen; Lamas, Oscar; Mataró, María José; Robledo-Carmona, Juan; Sánchez-Espín, Gemma; Jiménez-Navarro, Manuel; Such-Martínez, Miguel; Fernández, Borja

    2014-01-01

    Dilatation of the ascending aorta (AAD) is a prevalent aortopathy that occurs frequently associated with bicuspid aortic valve (BAV), the most common human congenital cardiac malformation. The molecular mechanisms leading to AAD associated with BAV are still poorly understood. The search for differentially expressed genes in diseased tissue by quantitative real-time PCR (qPCR) is an invaluable tool to fill this gap. However, studies dedicated to identify reference genes necessary for normalization of mRNA expression in aortic tissue are scarce. In this report, we evaluate the qPCR expression of six candidate reference genes in tissue from the ascending aorta of 52 patients with a variety of clinical and demographic characteristics, normal and dilated aortas, and different morphologies of the aortic valve (normal aorta and normal valve n = 30; dilated aorta and normal valve n = 10; normal aorta and BAV n = 4; dilated aorta and BAV n = 8). The expression stability of the candidate reference genes was determined with three statistical algorithms, GeNorm, NormFinder and Bestkeeper. The expression analyses showed that the most stable genes for the three algorithms employed were CDKN1β, POLR2A and CASC3, independently of the structure of the aorta and the valve morphology. In conclusion, we propose the use of these three genes as reference genes for mRNA expression analysis in human ascending aorta. However, we suggest searching for specific reference genes when conducting qPCR experiments with new cohort of samples. PMID:24841551

  3. Single-step PCR in molecular diagnosis of hepatitis C virus infection.

    PubMed

    Farma, E; Boeri, E; Bettini, P; Repetto, C M; McDermott, J; Lillo, F B; Varnier, O E

    1996-12-01

    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.

  4. Comparison of standard, quantitative and digital PCR in the detection of enterotoxigenic Bacteroides fragilis

    PubMed Central

    Purcell, Rachel V.; Pearson, John; Frizelle, Frank A.; Keenan, Jacqueline I.

    2016-01-01

    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. PMID:27686415

  5. Rapid molecular typing of Prototheca zopfii by high resolution melting real-time PCR (PCR-HRM).

    PubMed

    Sobukawa, Hideto; Ibaraki, Masato; Kano, Rui; Ito, Takaaki; Suzuki, Kazuyuki; Kamata, Hiroshi; Hasegawa, Atsuhiko

    2013-01-01

    Prototheca zopfii is an achlorophyllic alga that is ubiquitous around cow sheds. The alga is associated with bovine mastitis, which causes a reduction in milk production and secretion of thin watery milk containing white flakes. Isolates of P. zopfii from bovine mastitis were almost all identified as P. zopfii genotype 2, suggesting that it is the main causative agent of bovine protothecal mastitis. The ability to differentiate between genotype 1 and genotype 2 is therefore very important for preventing bovine mastitis. In this study, high resolution melting real-time PCR (PCR-HRM) analysis of the protothecal 18S rDNA domain successfully differentiated between genotypes of P. zopfii in less than 3 hours, while conventional sequence analysis requires more than 48 hours to differentiate between genotypes. PCR-HRM analysis clustered P. zopfii genotype 1 isolates separately from P. zopfii genotype 2 isolates, indicating that this molecular typing method is an effective tool for rapidly diagnosing bovine protothecal mastitis. PMID:24292136

  6. 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.

  7. Microbial Pollution Tracking of Dairy Farm with a Combined PCR-DGGE and qPCR Approach.

    PubMed

    Xi, Xiaoxia; Zhang, Jiachao; Kwok, Laiyu; Huo, Dongxue; Feng, Shuzhen; Zhang, Heping; Sun, Tiansong

    2015-12-01

    Animal husbandry is a traditional industry with regional characteristic in the Inner Mongolia of China. Recent years, animal breeding has been one of the main pollution sources in this area, followed by domestic sewage and industrial wastewater. The pollution of livestock farm feces may accelerate the development of pathogens and antibiotic resistance genes which pose health risks to humans and animals. In present research, culture-independent molecular ecological methods based on DGGE combined with qPCR were used to investigate the pollution to surrounding environment with different degrees of livestock farm. The cluster analysis of DGGE patterns showed that the livestock farm feces from point pollution source flowed with wastewater discharge has resulted in an impacted range of at least 3000 m, but it did not cause pollution to residential water delivered from upstream of sewage drain outlet. qPCR results revealed that 5 common pathogens (Escherichia coli, Enterococcus, Staphylococcus aureus, Shigella, and Salmonella) presented decreased trend as the sampled distance from point pollution source increased. Also, qPCR assays of 10 common antibiotic resistance genes (tetO, tetL, rpp, rpoB, sul2, sulA, floR, yidY, mphA, and ermC) which cause resistance to tetracycline, rifampicin, fluoroquinolone, quinolone, and erythromycin have been found in the environmental samples. This study clearly indicates the livestock farm discharge pollutants contaminated to the surrounding environment. Our data have provided important information to pollution control in the future. PMID:26341923

  8. Microbial Pollution Tracking of Dairy Farm with a Combined PCR-DGGE and qPCR Approach.

    PubMed

    Xi, Xiaoxia; Zhang, Jiachao; Kwok, Laiyu; Huo, Dongxue; Feng, Shuzhen; Zhang, Heping; Sun, Tiansong

    2015-12-01

    Animal husbandry is a traditional industry with regional characteristic in the Inner Mongolia of China. Recent years, animal breeding has been one of the main pollution sources in this area, followed by domestic sewage and industrial wastewater. The pollution of livestock farm feces may accelerate the development of pathogens and antibiotic resistance genes which pose health risks to humans and animals. In present research, culture-independent molecular ecological methods based on DGGE combined with qPCR were used to investigate the pollution to surrounding environment with different degrees of livestock farm. The cluster analysis of DGGE patterns showed that the livestock farm feces from point pollution source flowed with wastewater discharge has resulted in an impacted range of at least 3000 m, but it did not cause pollution to residential water delivered from upstream of sewage drain outlet. qPCR results revealed that 5 common pathogens (Escherichia coli, Enterococcus, Staphylococcus aureus, Shigella, and Salmonella) presented decreased trend as the sampled distance from point pollution source increased. Also, qPCR assays of 10 common antibiotic resistance genes (tetO, tetL, rpp, rpoB, sul2, sulA, floR, yidY, mphA, and ermC) which cause resistance to tetracycline, rifampicin, fluoroquinolone, quinolone, and erythromycin have been found in the environmental samples. This study clearly indicates the livestock farm discharge pollutants contaminated to the surrounding environment. Our data have provided important information to pollution control in the future.

  9. 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. PMID:24743571

  10. Detection of Mycobacterium tuberculosis in extrapulmonary biopsy samples using PCR targeting IS6110, rpoB, and nested-rpoB PCR Cloning

    PubMed Central

    Meghdadi, Hossein; Khosravi, Azar D.; Ghadiri, Ata A.; Sina, Amir H.; Alami, Ameneh

    2015-01-01

    Present study was aimed to examine the diagnostic utility of polymerase chain reaction (PCR) and nested PCR techniques for the detection of Mycobacterium tuberculosis (MTB) DNA in samples from patients with extra pulmonary tuberculosis (EPTB). In total 80 formalin-fixed, paraffin-embedded (FFPE) samples comprising 70 samples with definite diagnosis of EPTB and 10 samples from known non- EPTB on the basis of histopathology examination, were included in the study. PCR amplification targeting IS6110, rpoB gene and nested PCR targeting the rpoB gene were performed on the extracted DNAs from 80 FFPE samples. The strong positive samples were directly sequenced. For negative samples and those with weak band in nested-rpoB PCR, TA cloning was performed by cloning the products into the plasmid vector with subsequent sequencing. The 95% confidence intervals (CI) for the estimates of sensitivity and specificity were calculated for each method. Fourteen (20%), 34 (48.6%), and 60 (85.7%) of the 70 positive samples confirmed by histopathology, were positive by rpoB-PCR, IS6110-PCR, and nested-rpoB PCR, respectively. By performing TA cloning on samples that yielded weak (n = 8) or negative results (n = 10) in the PCR methods, we were able to improve their quality for later sequencing. All samples with weak band and 7 out of 10 negative samples, showed strong positive results after cloning. So nested-rpoB PCR cloning revealed positivity in 67 out of 70 confirmed samples (95.7%). The sensitivity of these combination methods was calculated as 95.7% in comparison with histopathology examination. The CI for sensitivity of the PCR methods were calculated as 11.39–31.27% for rpoB-PCR, 36.44–60.83% for IS6110- PCR, 75.29–92.93% for nested-rpoB PCR, and 87.98–99.11% for nested-rpoB PCR cloning. The 10 true EPTB negative samples by histopathology, were negative by all tested methods including cloning and were used to calculate the specificity of the applied methods. The CI for 100

  11. A-T linker adapter polymerase chain reaction for determining flanking sequences by rescuing inverse PCR or thermal asymmetric interlaced PCR products.

    PubMed

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

    2014-12-01

    The polymerase chain reaction (PCR)-based genome walking method has been extensively used to isolate unknown flanking sequences, whereas nonspecific products are always inevitable. To resolve these problems, we developed a new strategy to isolate the unknown flanking sequences by combining A-T linker adapter PCR with inverse PCR (I-PCR) or thermal asymmetric interlaced PCR (TAIL-PCR). The result showed that this method can be efficiently achieved with the flanking sequence from the Arabidopsis mutant and papain gene. Our study provides researchers with an additional method for determining genomic DNA flanking sequences to identify the target band from bulk of bands and to eliminate the cloning step for sequencing.

  12. Multiplex SYBR® green-real time PCR (qPCR) assay for the detection and differentiation of Bartonella henselae and Bartonella clarridgeiae in cats.

    PubMed

    Staggemeier, Rodrigo; Pilger, Diogo André; Spilki, Fernando Rosado; Cantarelli, Vlademir Vicente

    2014-01-01

    A novel SYBR® green-real time polymerase chain reaction (qPCR) was developed to detect two Bartonella species, B. henselae and B. clarridgeiae, directly from blood samples. The test was used in blood samples obtained from cats living in animal shelters in Southern Brazil. Results were compared with those obtained by conventional PCR targeting Bartonella spp. Among the 47 samples analyzed, eight were positive using the conventional PCR and 12 were positive using qPCR. Importantly, the new qPCR detected the presence of both B. henselae and B. clarridgeiae in two samples. The results show that the qPCR described here may be a reliable tool for the screening and differentiation of two important Bartonella species.

  13. 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

  14. 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...

  15. 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.

  16. 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. PMID:22649939

  17. 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.

  18. 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. PMID:26410309

  19. 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

  20. 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

  1. 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

  2. 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.

  3. [Detection of bacterial DNA using the polymerase chain reaction (PCR)].

    PubMed

    Höfler, G

    1994-01-01

    Enzymatic amplification of DNA using the polymerase chain reaction (PCR) is a very sensitive and rapid way of detecting specific DNA sequences. Bacterial DNA can be detected in a wide variety of samples provided at least partial sequence information is available. For a great number of bacteria PCR detection methods have been published. Most important for the pathologist are mycobacteriae (M. tuberculosis, avium, etc.). Borellia burgdorferi, Listeria monozytogenes and chlamydiae (Ce. trachomatis, C. psittaci). Fresh or fixed paraffin embedded tissues, exfoliated cells, whole blood, serum, sputum, urine, ascites or pleural fluid etc. can be analyzed. The time needed to perform the analysis varies between 5 hours and 2 days mostly depending on the DNA extraction method. Several potential pitfalls have to be avoided. The most common problem is contamination of reagents with target DNA. Amplification of DNA from biological samples may be prevented by enzyme inhibitors (salts, proteins). This problem can at least partially be avoided by changing the DNA purification method. Several additional problems may arise if bacterial DNA has to be amplified. Bacterial walls may have to be disrupted using heat or detergent for accessibility of target DNA. Positive results have to be judged carefully. Unlike the situation in retroviral infections with the virus sometimes present in the absence of disease, in the majority of bacterial infections the presence of bacteria signals manifest disease. A possible exception may be the finding of mycobacterial DNA in sarcoidosis patients who can be treated with steroids without provoking tuberculosis. PCR is especially useful in situations where rapid results are necessary or only fixed tissue is available.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:7533972

  4. Detection of Saccharopolyspora rectivirgula by quantitative real-time PCR.

    PubMed

    Schäfer, Jenny; Kämpfer, Peter; Jäckel, Udo

    2011-07-01

    The thermophilic actinomycete species Saccharopolyspora rectivirgula has been associated with the exogen allergic alveolitis (EAA). EAA is caused by the inhalation of high amounts of airborne spores that can be found for example in environments of agricultural production, compost facilities, mushroom cultivation rooms, or rooms with technical air moistening. Because of the medical relevance of S. rectivirgula, a reliable detection system is needed. Therefore, a quantitative real-time polymerase chain reaction (qPCR) primer system was designed, targeting the 16S rRNA gene of the type strain S. rectivirgula DSM 43747(T) and six other S. rectivirgula reference strains. Our investigation showed that S. rectivirgula presumably own four operons of the 16S rRNA gene, which has to be considered for estimation of cell equivalents. Furthermore, the DNA recovery efficiency from these strains was tested in combination with bioaerosol or material sample as well as the influence of non-target DNA to the recovery rate. Results showed a recovery DNA efficiency of 7-55%. The recovery rate of DNA in a mixture with non-target DNA resulted in ∼87%. In summary, a high amplification efficiency using real-time PCR was found, for which estimated concentrations revealed cell numbers of 2.7 × 10(5) cells m(-3) in bioaerosol and 2.8 × 10(6) cells g(-1) fw(-1) in material samples from a duck house. The specificity of the new developed quantification system was shown by generation of two clone libraries from bioarosol samples, from a duck house, and from a composting plant. Totally, the results clearly show the specificity and practicability of the established qPCR assay for detection of S. rectivirgula.

  5. 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). PMID:27423733

  6. 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).

  7. Performance of Droplet Digital PCR in Non-Invasive Fetal RHD Genotyping - Comparison with a Routine Real-Time PCR Based Approach

    PubMed Central

    Hořínek, Aleš; Novotná, Michaela; Calda, Pavel; Korabečná, Marie

    2015-01-01

    Detection and characterization of circulating cell-free fetal DNA (cffDNA) from maternal circulation requires an extremely sensitive and precise method due to very low cffDNA concentration. In our study, droplet digital PCR (ddPCR) was implemented for fetal RHD genotyping from maternal plasma to compare this new quantification alternative with real-time PCR (qPCR) as a golden standard for quantitative analysis of cffDNA. In the first stage of study, a DNA quantification standard was used. Clinical samples, including 10 non-pregnant and 35 pregnant women, were analyzed as a next step. Both methods’ performance parameters—standard curve linearity, detection limit and measurement precision—were evaluated. ddPCR in comparison with qPCR has demonstrated sufficient sensitivity for analysing of cffDNA and determination of fetal RhD status from maternal circulation, results of both methods strongly correlated. Despite the more demanding workflow, ddPCR was found to be slightly more precise technology, as evaluated using quantitative standard. Regarding the clinical samples, the precision of both methods equalized with decreasing concentrations of tested DNA samples. In case of cffDNA with very low concentrations, variance parameters of both techniques were comparable. Detected levels of fetal cfDNA in maternal plasma were slightly higher than expected and correlated significantly with gestational age as measured by both methods (ddPCR r = 0.459; qPCR r = 0.438). PMID:26562517

  8. The new LM-PCR/shifter method for the genotyping of microorganisms based on the use of a class IIS restriction enzyme and ligation mediated PCR.

    PubMed

    Krawczyk, Beata; Leibner-Ciszak, Justyna; Stojowska, Karolina; Kur, Józef

    2011-12-01

    This study details and examines a novel ligation-mediated polymerase chain reaction (LM-PCR) method. Named the LM-PCR/Shifter, it relies on the use of a Class IIS restriction enzyme giving restriction fragments with different 4-base, 5' overhangs, this being the Shifter, and the ligation of appropriate oligonucleotide adapters. A sequence of 4-base, 5' overhangs of the adapter and a 4- base sequence of the 3' end of the primer(s) determine a subset of the genomic restriction fragments, which are amplified by PCR. The method permits the differentiation of bacterial species strains on the basis of the different DNA band patterns obtained after electrophoresis in polyacrylamide gels stained with ethidium bromide and visualized in UV light. The usefulness of the LM-PCR/ Shifter method for genotyping is analyzed by a comparison with the restriction endonuclease analysis of chromosomal DNA by the pulsed-field gel electrophoresis (REA-PFGE) and PCR melting profile (PCR MP) methods for isolates of clinical origin. The clustering of the LM-PCR/Shifter fingerprinting data matched those of the REA-PFGE and PCR MP methods. We found that the LM-PCR/Shifter is rapid, and offers good discriminatory power and excellent reproducibility, making it a method that may be effectively applied in epidemiological studies.

  9. [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.

  10. A PCR amplification strategy for unrestricted generation of chimeric genes.

    PubMed

    Vos, Michel J; Kampinga, Harm H

    2008-09-15

    For analyzing protein function, protein dynamics, or protein-protein interactions, the use of chimeric proteins has become an indispensable tool. The generation of DNA constructs coding for such fused proteins can, however, be a tedious process. Currently used strategies often make use of available endonuclease sites, leading to limitations in the choice of the site of fusion between two genes and problems in maintaining protein secondary structure. We have developed a cloning strategy to get around these disadvantages that is based on a single round of PCR amplification followed by antibiotic-resistant gene complementation. PMID:18555003

  11. A PCR amplification strategy for unrestricted generation of chimeric genes.

    PubMed

    Vos, Michel J; Kampinga, Harm H

    2008-09-15

    For analyzing protein function, protein dynamics, or protein-protein interactions, the use of chimeric proteins has become an indispensable tool. The generation of DNA constructs coding for such fused proteins can, however, be a tedious process. Currently used strategies often make use of available endonuclease sites, leading to limitations in the choice of the site of fusion between two genes and problems in maintaining protein secondary structure. We have developed a cloning strategy to get around these disadvantages that is based on a single round of PCR amplification followed by antibiotic-resistant gene complementation.

  12. Whole genome amplification using single-primer PCR.

    PubMed

    Lao, Kaiqin; Xu, Nan Lan; Straus, Neil A

    2008-03-01

    Comprehensive genomic molecular analyses require relatively large DNA amounts that are often not available from forensic, clinical and other crucial biological samples. Numerous methods to amplify the whole genome have been proposed for cancer, forensic and taxonomic research. Unfortunately, when using truly random primers for the initial priming step, all of these procedures suffer from high background problems for sub-nanogram quantities of input DNA. Here we report an approach to eliminate this problem for PCR-based methods even at levels of DNA approaching that of a single cell.

  13. First Australian isolation of epidemic Clostridium difficile PCR ribotype 027.

    PubMed

    Riley, Thomas V; Thean, Sarah; Hool, Graham; Golledge, Clayton L

    2009-06-15

    We report the first isolation in Australia of a hypervirulent epidemic strain of Clostridium difficile, PCR ribotype 027. It was isolated from a 43-year-old woman with a permanent ileostomy, who appears to have been infected while travelling in the United States. The isolate was positive for toxin A, toxin B and binary toxin, and resistant to fluoroquinolone antimicrobials, and had characteristic deletions in the tcdC gene. All diagnostic laboratories and health care facilities in Australia should now be on high alert for this organism. PMID:19527210

  14. 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...

  15. Inactivation conditions for human Norovirus measured by an in situ capture-qRT-PCR Method

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Human noroviruses (HuNoVs) are the major cause of epidemic non-bacterial gastroenteritis. Due to the inability to cultivate HuNoVs, it has been a challenge to determine their infectivity. Quantitative real-time RT-PCR (qRT-PCR) is widely used in detecting HuNoVs. However, qRT-PCR only detects the...

  16. Principles and applications of Ligation Mediated PCR methods for DNA-based typing of microbial organisms.

    PubMed

    Krawczyk, Beata; Kur, Józef; Stojowska-Swędrzyńska, Karolina; Śpibida, Marta

    2016-01-01

    A significant number of DNA-based techniques has been introduced into the field of microorganisms' characterization and taxonomy. These genomic fingerprinting methods were developed to detect DNA sequence polymorphisms by using general principles, such as restriction endonuclease analysis, molecular hybridization, and PCR amplification. In recent years, some alternative techniques based on ligation of oligonucleotide adapters before DNA amplification by PCR, known as Ligation-Mediated PCR methods (LM PCR), have been successfully applied for the typing of microorganisms below the species level. These molecular methods include: Amplified Fragment Length Polymorphism (AFLP), Amplification of DNA fragments Surrounding Rare Restriction Sites (ADSRRS), PCR Melting Profiles (PCR MP), Ligation Mediated PCR/Shifter (LM PCR/Shifter), Infrequent-Restriction-Site Amplification (IRS PCR), double digestion Ligation Mediated Suppression PCR (ddLMS PCR). These techniques are now applied more and more often because they involve less time, are comparably inexpensive, and require only standard lab equipment. Here, we present a general review of this group of methods showing their possibilities and limitations. We also identify questions and propose solutions which may be helpful in choosing a particular LM PCR method for the achievement of the required goal.

  17. Clinical evaluation of a type III secretion system real-time PCR assay for diagnosing melioidosis.

    PubMed

    Meumann, Ella M; Novak, Ryan T; Gal, Daniel; Kaestli, Mirjam E; Mayo, Mark; Hanson, Joshua P; Spencer, Emma; Glass, Mindy B; Gee, Jay E; Wilkins, Patricia P; Currie, Bart J

    2006-08-01

    A Burkholderia pseudomallei type III secretion system real-time PCR assay was evaluated on clinical specimens in a region where melioidosis is endemic. The PCR was positive in 30/33 (91%) patients with culture-confirmed melioidosis. All six patients with melioidosis septic shock were blood PCR positive, suggesting potential for rapid diagnosis and commencement of appropriate therapy.

  18. DEVELOPMENT OF AN IMPROVED PCR-BASED TECHNIQUE FOR DETECTION OF PHYTOPHTHORA CACTORUM IN STRAWBERRY PLANTS

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Specific and rapid plant pathogen detection methods can aid in strawberry disease management decisions. PCR-based diagnostics for Phytophthora cactorum and other strawberry pathogens are hindered by PCR inhibitors and lack of species-specific PCR primers. We developed a DNA extraction and purificati...

  19. Effects of prolonged chlorine exposures upon PCR detection of Helicobacter pylori DNA.

    EPA Science Inventory

    The effect of low doses of free chlorine on the detection by qPCR of Helicobacter pylori (H. pylori) cells by qPCR in tap water was monitored. H. pylori target sequences (within suspended, intact cells at densities of 102 to 103 cells /ml) were rendered undetectable by qPCR an...

  20. Principles and applications of Ligation Mediated PCR methods for DNA-based typing of microbial organisms.

    PubMed

    Krawczyk, Beata; Kur, Józef; Stojowska-Swędrzyńska, Karolina; Śpibida, Marta

    2016-01-01

    A significant number of DNA-based techniques has been introduced into the field of microorganisms' characterization and taxonomy. These genomic fingerprinting methods were developed to detect DNA sequence polymorphisms by using general principles, such as restriction endonuclease analysis, molecular hybridization, and PCR amplification. In recent years, some alternative techniques based on ligation of oligonucleotide adapters before DNA amplification by PCR, known as Ligation-Mediated PCR methods (LM PCR), have been successfully applied for the typing of microorganisms below the species level. These molecular methods include: Amplified Fragment Length Polymorphism (AFLP), Amplification of DNA fragments Surrounding Rare Restriction Sites (ADSRRS), PCR Melting Profiles (PCR MP), Ligation Mediated PCR/Shifter (LM PCR/Shifter), Infrequent-Restriction-Site Amplification (IRS PCR), double digestion Ligation Mediated Suppression PCR (ddLMS PCR). These techniques are now applied more and more often because they involve less time, are comparably inexpensive, and require only standard lab equipment. Here, we present a general review of this group of methods showing their possibilities and limitations. We also identify questions and propose solutions which may be helpful in choosing a particular LM PCR method for the achievement of the required goal. PMID:26885774

  1. Quantitative analysis of food and feed samples with droplet digital PCR.

    PubMed

    Morisset, Dany; Štebih, Dejan; Milavec, Mojca; Gruden, Kristina; Žel, Jana

    2013-01-01

    In this study, the applicability of droplet digital PCR (ddPCR) for routine analysis in food and feed samples was demonstrated with the quantification of genetically modified organisms (GMOs). Real-time quantitative polymerase chain reaction (qPCR) is currently used for quantitative molecular analysis of the presence of GMOs in products. However, its use is limited for detecting and quantifying very small numbers of DNA targets, as in some complex food and feed matrices. Using ddPCR duplex assay, we have measured the absolute numbers of MON810 transgene and hmg maize reference gene copies in DNA samples. Key performance parameters of the assay were determined. The ddPCR system is shown to offer precise absolute and relative quantification of targets, without the need for calibration curves. The sensitivity (five target DNA copies) of the ddPCR assay compares well with those of individual qPCR assays and of the chamber digital PCR (cdPCR) approach. It offers a dynamic range over four orders of magnitude, greater than that of cdPCR. Moreover, when compared to qPCR, the ddPCR assay showed better repeatability at low target concentrations and a greater tolerance to inhibitors. Finally, ddPCR throughput and cost are advantageous relative to those of qPCR for routine GMO quantification. It is thus concluded that ddPCR technology can be applied for routine quantification of GMOs, or any other domain where quantitative analysis of food and feed samples is needed. PMID:23658750

  2. Quantitative analysis of food and feed samples with droplet digital PCR.

    PubMed

    Morisset, Dany; Štebih, Dejan; Milavec, Mojca; Gruden, Kristina; Žel, Jana

    2013-01-01

    In this study, the applicability of droplet digital PCR (ddPCR) for routine analysis in food and feed samples was demonstrated with the quantification of genetically modified organisms (GMOs). Real-time quantitative polymerase chain reaction (qPCR) is currently used for quantitative molecular analysis of the presence of GMOs in products. However, its use is limited for detecting and quantifying very small numbers of DNA targets, as in some complex food and feed matrices. Using ddPCR duplex assay, we have measured the absolute numbers of MON810 transgene and hmg maize reference gene copies in DNA samples. Key performance parameters of the assay were determined. The ddPCR system is shown to offer precise absolute and relative quantification of targets, without the need for calibration curves. The sensitivity (five target DNA copies) of the ddPCR assay compares well with those of individual qPCR assays and of the chamber digital PCR (cdPCR) approach. It offers a dynamic range over four orders of magnitude, greater than that of cdPCR. Moreover, when compared to qPCR, the ddPCR assay showed better repeatability at low target concentrations and a greater tolerance to inhibitors. Finally, ddPCR throughput and cost are advantageous relative to those of qPCR for routine GMO quantification. It is thus concluded that ddPCR technology can be applied for routine quantification of GMOs, or any other domain where quantitative analysis of food and feed samples is needed.

  3. Insulated Isothermal Reverse Transcriptase PCR (iiRT-PCR) for Rapid and Sensitive Detection of Classical Swine Fever Virus.

    PubMed

    Lung, O; Pasick, J; Fisher, M; Buchanan, C; Erickson, A; Ambagala, A

    2016-10-01

    Classical swine fever (CSF) is an OIE-listed disease that can have a severe impact on the swine industry. User-friendly, sensitive, rapid diagnostic tests that utilize low-cost field-deployable instruments for CSF diagnosis can be useful for disease surveillance and outbreak monitoring. In this study, we describe validation of a new probe-based insulated isothermal reverse transcriptase PCR (iiRT-PCR) assay for rapid detection of classical swine fever virus (CSFV) on a compact, user-friendly device (POCKIT(™) Nucleic Acid Analyzer) that does not need data interpretation by the user. The assay accurately detected CSFV RNA from a diverse panel of 33 CSFV strains representing all three genotypes plus an additional in vitro-transcribed RNA from cloned sequences representing a vaccine strain. No cross-reactivity was observed with a panel of 18 viruses associated with livestock including eight other pestivirus strains (bovine viral diarrhoea virus type 1 and type 2, border disease virus, HoBi atypical pestivirus), African swine fever virus, swine vesicular disease virus, swine influenza virus, porcine respiratory and reproductive syndrome virus, porcine circovirus 1, porcine circovirus 2, porcine respiratory coronavirus, vesicular exanthema of swine virus, bovine herpes virus type 1 and vesicular stomatitis virus. The iiRT-PCR assay accurately detected CSFV as early as 2 days post-inoculation in RNA extracted from serum samples of experimentally infected pigs, before appearance of clinical signs. The limit of detection (LOD95% ) calculated by probit regression analysis was 23 copies per reaction. The assay has a sample to answer turnaround time of less than an hour using extracted RNA or diluted or low volume of neat serum. The user-friendly, compact device that automatically analyses and displays results could potentially be a useful tool for surveillance and monitoring of CSF in a disease outbreak. PMID:25644051

  4. Analysis of PCR Thermocycling by Rayleigh-Bénard Convection

    NASA Astrophysics Data System (ADS)

    Sharma, Ruchi; Ugaz, Victor

    2004-03-01

    In previous studies, we demonstrated a novel device employing the circulatory flow field established by Rayleigh-Bénard convection to perform amplification of a 295 base target region from a human genomic DNA template inside a 35 uL cylindrical cavity using the polymerase chain reaction (PCR) [Krishnan, Ugaz & Burns, Science, Vol. 298, 2002, p. 793]. This design eliminates the need for dynamic external temperature control required in conventional thermocyclers that repeatedly heat and cool static sample volumes to denaturation, annealing, and extension temperatures. In this paper, we extend these studies by demonstrating the design and operation of a multiwell convective flow device capable of achieving amplification of a 191 base pair fragment associated with membrane channel proteins M1 and M2 of the influenza-A virus in as little as 15 minutes with performance comparable to a conventional thermocycler. We also study the effect of initial template concentration and observe no degradation in performance over four orders of magnitude of initial template loading dilution, consistent with conventional thermocycler results. These results illustrate the ability of convective flow PCR systems to achieve performance equal to or exceeding conventional thermocycling hardware, and demonstrate their suitability for use in rapid biodetection assays.

  5. Fast detection of deletion breakpoints using quantitative PCR

    PubMed Central

    Abildinova, Gulshara; Abdrakhmanova, Zhanara; Tuchinsky, Helena; Nesher, Elimelech; Pinhasov, Albert; Raskin, Leon

    2016-01-01

    Abstract The routine detection of large and medium copy number variants (CNVs) is well established. Hemizygotic deletions or duplications in the large Duchenne muscular dystrophy DMD gene responsible for Duchenne and Becker muscular dystrophies are routinely identified using multiple ligation probe amplification and array-based comparative genomic hybridization. These methods only map deleted or duplicated exons, without providing the exact location of breakpoints. Commonly used methods for the detection of CNV breakpoints include long-range PCR and primer walking, their success being limited by the deletion size, GC content and presence of DNA repeats. Here, we present a strategy for detecting the breakpoints of medium and large CNVs regardless of their size. The hemizygous deletion of exons 45-50 in the DMD gene and the large autosomal heterozygous PARK2 deletion were used to demonstrate the workflow that relies on real-time quantitative PCR to narrow down the deletion region and Sanger sequencing for breakpoint confirmation. The strategy is fast, reliable and cost-efficient, making it amenable to widespread use in genetic laboratories. PMID:27560363

  6. Fast detection of deletion breakpoints using quantitative PCR.

    PubMed

    Abildinova, Gulshara; Abdrakhmanova, Zhanara; Tuchinsky, Helena; Nesher, Elimelech; Pinhasov, Albert; Raskin, Leon

    2016-01-01

    The routine detection of large and medium copy number variants (CNVs) is well established. Hemizygotic deletions or duplications in the large Duchenne muscular dystrophy DMD gene responsible for Duchenne and Becker muscular dystrophies are routinely identified using multiple ligation probe amplification and array-based comparative genomic hybridization. These methods only map deleted or duplicated exons, without providing the exact location of breakpoints. Commonly used methods for the detection of CNV breakpoints include long-range PCR and primer walking, their success being limited by the deletion size, GC content and presence of DNA repeats. Here, we present a strategy for detecting the breakpoints of medium and large CNVs regardless of their size. The hemizygous deletion of exons 45-50 in the DMD gene and the large autosomal heterozygous PARK2 deletion were used to demonstrate the workflow that relies on real-time quantitative PCR to narrow down the deletion region and Sanger sequencing for breakpoint confirmation. The strategy is fast, reliable and cost-efficient, making it amenable to widespread use in genetic laboratories. PMID:27560363

  7. Identifying of meat species using polymerase chain reaction (PCR)

    NASA Astrophysics Data System (ADS)

    Foong, Chow Ming; Sani, Norrakiah Abdullah

    2013-11-01

    Meat has been widely consumed as an important protein source in daily life of human. Furthermore, with busy and intense urban lifestyle, processed food is now one of the main protein sources of one's diet. Consumers rely on the food labeling to decide if the meat product purchased is safe and reliable. Therefore, it is important to ensure the food labeling is done in a correct manner to avoid consumer fraud. More consumers are now concern about the food quality and safety as compared to before. This study described the meat species identification and detection method using Polymerase Chain Reaction (PCR) in 8 types of meats (cattle, buffalo, goat, sheep, chicken, duck, pork and horse). The objective of this study is to decide on the specificity of oligonucleotide sequences obtained from previous study. There were 5 proposed oligonucleotide primer in this study. The main important finding in this work is the specificity of oligonucleotide primers to raw meats. It if found that the oligonucleotide primers proposed were not specific to the local raw meat species. Therefore, further study is needed to obtain a species-specific oligonucletide primers for PCR, in order to be applied in food product testing.

  8. Sequetyping: serotyping Streptococcus pneumoniae by a single PCR sequencing strategy.

    PubMed

    Leung, Marcus H; Bryson, Kevin; Freystatter, Kathrin; Pichon, Bruno; Edwards, Giles; Charalambous, Bambos M; Gillespie, Stephen H

    2012-07-01

    The introduction of pneumococcal conjugate vaccines necessitates continued monitoring of circulating strains to assess vaccine efficacy and replacement serotypes. Conventional serological methods are costly, labor-intensive, and prone to misidentification, while current DNA-based methods have limited serotype coverage requiring multiple PCR primers. In this study, a computer algorithm was developed to interrogate the capsulation locus (cps) of vaccine serotypes to locate primer pairs in conserved regions that border variable regions and could differentiate between serotypes. In silico analysis of cps from 92 serotypes indicated that a primer pair spanning the regulatory gene cpsB could putatively amplify 84 serotypes and differentiate 46. This primer set was specific to Streptococcus pneumoniae, with no amplification observed for other species, including S. mitis, S. oralis, and S. pseudopneumoniae. One hundred thirty-eight pneumococcal strains covering 48 serotypes were tested. Of 23 vaccine serotypes included in the study, most (19/22, 86%) were identified correctly at least to the serogroup level, including all of the 13-valent conjugate vaccine and other replacement serotypes. Reproducibility was demonstrated by the correct sequetyping of different strains of a serotype. This novel sequence-based method employing a single PCR primer pair is cost-effective and simple. Furthermore, it has the potential to identify new serotypes that may evolve in the future.

  9. PCR Primers for Metazoan Mitochondrial 12S Ribosomal DNA Sequences

    PubMed Central

    Machida, Ryuji J.; Kweskin, Matthew; Knowlton, Nancy

    2012-01-01

    Background Assessment of the biodiversity of communities of small organisms is most readily done using PCR-based analysis of environmental samples consisting of mixtures of individuals. Known as metagenetics, this approach has transformed understanding of microbial communities and is beginning to be applied to metazoans as well. Unlike microbial studies, where analysis of the 16S ribosomal DNA sequence is standard, the best gene for metazoan metagenetics is less clear. In this study we designed a set of PCR primers for the mitochondrial 12S ribosomal DNA sequence based on 64 complete mitochondrial genomes and then tested their efficacy. Methodology/Principal Findings A total of the 64 complete mitochondrial genome sequences representing all metazoan classes available in GenBank were downloaded using the NCBI Taxonomy Browser. Alignment of sequences was performed for the excised mitochondrial 12S ribosomal DNA sequences, and conserved regions were identified for all 64 mitochondrial genomes. These regions were used to design a primer pair that flanks a more variable region in the gene. Then all of the complete metazoan mitochondrial genomes available in NCBI's Organelle Genome Resources database were used to determine the percentage of taxa that would likely be amplified using these primers. Results suggest that these primers will amplify target sequences for many metazoans. Conclusions/Significance Newly designed 12S ribosomal DNA primers have considerable potential for metazoan metagenetic analysis because of their ability to amplify sequences from many metazoans. PMID:22536450

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

    PubMed

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

    2001-06-01

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

  11. Digital PCR on an integrated self-priming compartmentalization chip.

    PubMed

    Zhu, Qiangyuan; Qiu, Lin; Yu, Bingwen; Xu, Yanan; Gao, Yibo; Pan, Tingting; Tian, Qingchang; Song, Qi; Jin, Wei; Jin, Qinhan; Mu, Ying

    2014-03-21

    An integrated on-chip valve-free and power-free microfluidic digital PCR device is for the first time developed by making use of a novel self-priming compartmentalization and simple dehydration control to realize 'divide and conquer' for single DNA molecule detection. The high gas solubility of PDMS is exploited to provide the built-in power of self-priming so that the sample and oil are sequentially sucked into the device to realize sample self-compartmentalization based on surface tension. The lifespan of its self-priming capability was about two weeks tested using an air-tight packaging bottle sealed with a small amount of petroleum jelly, which is significant for a practical platform. The SPC chip contains 5120 independent 5 nL microchambers, allowing the samples to be compartmentalized completely. Using this platform, three different abundances of lung cancer related genes are detected to demonstrate the feasibility and flexibility of the microchip for amplifying a single nucleic acid molecule. For maximal accuracy, within less than 5% of the measurement deviation, the optimal number of positive chambers is between 400 and 1250 evaluated by the Poisson distribution, which means one panel can detect an average of 480 to 4804 template molecules. This device without world-to-chip connections eliminates the constraint of the complex pipeline control, and is an integrated on-chip platform, which would be a significant improvement to digital PCR automation and more user-friendly.

  12. Multiplex real-time PCR assay for Legionella species.

    PubMed

    Kim, Seung Min; Jeong, Yoojung; Sohn, Jang Wook; Kim, Min Ja

    2015-12-01

    Legionella pneumophila serogroup 1 (sg1) accounts for the majority of infections in humans, but other Legionella species are also associated with human disease. In this study, a new SYBR Green I-based multiplex real-time PCR assay in a single reaction was developed to allow the rapid detection and differentiation of Legionella species by targeting specific gene sequences. Candidate target genes were selected, and primer sets were designed by referring to comparative genomic hybridization data of Legionella species. The Legionella species-specific groES primer set successfully detected all 30 Legionella strains tested. The xcpX and rfbA primers specifically detected L. pneumophila sg1-15 and L. pneumophila sg1, respectively. In addition, this assay was validated by testing clinical samples and isolates. In conclusion, this novel multiplex real-time PCR assay might be a useful diagnostic tool for the rapid detection and differentiation of Legionella species in both clinical and epidemiological studies.

  13. PCR-based identification of drowning: four case reports.

    PubMed

    Rácz, Evelin; Könczöl, Franciska; Tóth, Dénes; Patonai, Zoltán; Porpáczy, Zoltán; Kozma, Zsolt; Poór, Viktor S; Sipos, Katalin

    2016-09-01

    Proper diagnosis in drowning victims is often difficult due to the lack of signs specific to drowning. The diatom test is a widely used procedure for the diagnosis. Some types of water contain only minimal amounts of diatom cells which may provide false-negative results, while a negative diatom test result does not exclude drowning. In proving drowning, we used a polymerase chain reaction (PCR)-based biological method in addition to the conventional methods. DNA was extracted from postmortem spleen tissues and water of the drowning site. Samples were tested with algae (diatoms and small green algae)- and cyanobacteria (blue-green algae)-specific primers. We present here multiple drowning cases in which diatom tests of the postmortem tissue samples and the water were negative. In each case, the presence of phytoplanktonic DNA strengthened the autopsy diagnosis of drowning even in the absence of visible diatoms. In the future, the PCR method may be of consideration as a possible supplement of the diatom test in the examination of presumed drowning cases.

  14. PCR diagnostic methods for Ascosphaera infections in bees.

    PubMed

    James, R R; Skinner, J S

    2005-10-01

    Fungi in the genus Ascosphaera are the causative agents of chalkbrood, a major disease affecting bee larval viability. Identification of individual Ascosphaera species based on morphological features has been difficult due to a lack of distinguishing characteristics. Most identifications are based on the size and shape of the ascomata, spore balls and conidia. Unfortunately, much overlap occurs in the size of these structures, and some Ascosphaera species will not produce sexual structures in vitro. We report a quick and reliable diagnostic method for identifying Ascosphaera infections in Megachile bees (leafcutting bees) using PCR markers that employ genus-specific primers for Ascosphaera, and species-specific primers for species known to be associated with Megachile spp. Using these methods, species identifications can be performed directly on bees, including asymptomatic individuals. Furthermore, the PCR markers can detect co-infections of multiple Ascosphaera species in a single host. We also identified a marker for Ascosphaera apis, the predominant cause of chalkbrood in Apis mellifera, the honey bee. Our diagnostic methods eliminate the need for culturing samples, and could be used to process a large number of field collected bee larvae. PMID:16214164

  15. Monitoring the immune response using real-time PCR.

    PubMed

    Stordeur, Patrick

    2009-01-01

    Induction of an immune response to a particular antigen is the basis of vaccination. This has been done for years to prevent infectious diseases, and has the potential for the treatment of cancer. The immune response is nowadays more precisely modulated rather than simply induced, like in case of immunotherapy of allergic diseases. Likewise, autoimmune diseases are associated with an inappropriate immune response, and many efforts are made for specifically inhibiting this unwanted response. A possible line of attack is the induction of an antigen-specific immune tolerance, which also has a use in the field of transplantation, where allogeneic responses are deleterious for the graft. In all of these fields of fundamental and clinical medicine, the modulation of immune response requires the assistance of laboratory tests, among which real-time PCR appears more and more helpful. This chapter describes a protocol to quantify immune-related mRNAs using reverse transcription-real-time PCR. The transcripts can be quantified in cultured cells or in cultured whole blood, after an incubation period in the presence of the antigen to which the immune response is analyzed. This is the typical approach to evaluate the efficacy of a vaccine. The transcripts can also be quantified directly in the biological sample, giving information about the in vivo immune status of the individual. The techniques to achieve these different methods are described, and are illustrated by the analysis of the response against the toxoid tetanus antigen.

  16. Identifying of meat species using polymerase chain reaction (PCR)

    SciTech Connect

    Foong, Chow Ming; Sani, Norrakiah Abdullah

    2013-11-27

    Meat has been widely consumed as an important protein source in daily life of human. Furthermore, with busy and intense urban lifestyle, processed food is now one of the main protein sources of one’s diet. Consumers rely on the food labeling to decide if the meat product purchased is safe and reliable. Therefore, it is important to ensure the food labeling is done in a correct manner to avoid consumer fraud. More consumers are now concern about the food quality and safety as compared to before. This study described the meat species identification and detection method using Polymerase Chain Reaction (PCR) in 8 types of meats (cattle, buffalo, goat, sheep, chicken, duck, pork and horse). The objective of this study is to decide on the specificity of oligonucleotide sequences obtained from previous study. There were 5 proposed oligonucleotide primer in this study. The main important finding in this work is the specificity of oligonucleotide primers to raw meats. It if found that the oligonucleotide primers proposed were not specific to the local raw meat species. Therefore, further study is needed to obtain a species-specific oligonucletide primers for PCR, in order to be applied in food product testing.

  17. Development of PCR and TaqMan PCR Assays to Detect Pseudomonas coronafaciens, a Causal Agent of Halo Blight of Oats

    PubMed Central

    An, Ji-Hye; Noh, Young-Hee; Kim, Yong-Eon; Lee, Hyok-In; Cha, Jae-Soon

    2015-01-01

    Pseudomonas coronafaciens causes halo blight on oats and is a plant quarantine bacterium in many countries, including the Republic of Korea. Using of the certificated seed is important for control of the disease. Since effective detection method of P. coronafaciens is not available yet, PCR and TaqMan PCR assays for specific detection of P. coronafaciens were developed in this study. PCR primers were designed from the draft genome sequence of P. coronafaciens LMG 5060 which was obtained by the next-generation sequencing in this study. The PCR primer set Pc-12-F/Pc-12-R specifically amplified 498 bp from the 13 strains of P. coronafaciens isolated in the seven different countries (Canada, Japan, United Kingdom, Zimbabwe, Kenya, Germany, and New Zealand) and the nested primer set Pc-12-ne-F/Pc-12-ne-R specifically amplified 298 bp from those strains. The target-size PCR product was not amplified from the non-target bacteria with the PCR and nested primer sets. TaqMan PCR with Pc-12-ne-F/Pc-12-ne-R and a TaqMan probe, Pc-taqman, which were designed inside of the nested PCR amplicon, generated Ct values which in a dose-dependent manner to the amount of the target DNA and the Ct values of all the P. coronafaciens strains were above the threshold Ct value for positive detection. The TaqMan PCR generated positive Ct values from the seed extracts of the artificially inoculated oat seeds above 10 cfu/ml inoculation level. PCR and TaqMan PCR assays developed in this study will be useful tools to detect and identify the plant quarantine pathogen, P. coronafaciens. PMID:25774107

  18. Comparison of the conventional multiplex RT-PCR, real time RT-PCR and Luminex xTAG® RVP fast assay for the detection of respiratory viruses.

    PubMed

    Choudhary, Manohar L; Anand, Siddharth P; Tikhe, Shamal A; Walimbe, Atul M; Potdar, Varsha A; Chadha, Mandeep S; Mishra, Akhilesh C

    2016-01-01

    Detection of respiratory viruses using polymerase chain reaction (PCR) is sensitive, specific and cost effective, having huge potential for patient management. In this study, the performance of an in-house developed conventional multiplex RT-PCR (mRT-PCR), real time RT-PCR (rtRT-PCR) and Luminex xTAG(®) RVP fast assay (Luminex Diagnostics, Toronto, Canada) for the detection of respiratory viruses was compared. A total 310 respiratory clinical specimens predominantly from pediatric patients, referred for diagnosis of influenza A/H1N1pdm09 from August 2009 to March 2011 were tested to determine performance characteristic of the three methods. A total 193 (62.2%) samples were detected positive for one or more viruses by mRT-PCR, 175 (56.4%) samples by real time monoplex RT-PCR, and 138 (44.5%) samples by xTAG(®) RVP fast assay. The overall sensitivity of mRT-PCR was 96.9% (95% CI: 93.5, 98.8), rtRT-PCR 87.9% (95% CI: 82.5, 92.1) and xTAG(®) RVP fast was 68.3% (95% CI: 61.4, 74.6). Rhinovirus was detected most commonly followed by respiratory syncytial virus group B and influenza A/H1N1pdm09. The monoplex real time RT-PCR and in-house developed mRT-PCR are more sensitive, specific and cost effective than the xTAG(®) RVP fast assay.

  19. Quantitative real-time PCR (qPCR)--based tool for detection and quantification of Cordyceps militaris in soil.

    PubMed

    Saragih, Syaiful Amri; Takemoto, S; Hisamoto, Y; Fujii, M; Sato, H; Kamata, N

    2015-01-01

    A quantitative real-time PCR using a primer pair CM2946F/CM3160R was developed for specific detection and quantification of Cordyceps militaris from soil. Standard curves were obtained for genomic DNA and DNA extracts from autoclaved soil with a certain dose of C. militaris suspension. C. militaris was detected from two forest soil samples out of ten that were collected when fruit bodies of C. militaris were found. This method seemed effective in detection of C. militaris in the soil and useful for rapid and reliable quantification of C. militaris in different ecosystems. PMID:25446034

  20. Quantitative real-time PCR (qPCR)--based tool for detection and quantification of Cordyceps militaris in soil.

    PubMed

    Saragih, Syaiful Amri; Takemoto, S; Hisamoto, Y; Fujii, M; Sato, H; Kamata, N

    2015-01-01

    A quantitative real-time PCR using a primer pair CM2946F/CM3160R was developed for specific detection and quantification of Cordyceps militaris from soil. Standard curves were obtained for genomic DNA and DNA extracts from autoclaved soil with a certain dose of C. militaris suspension. C. militaris was detected from two forest soil samples out of ten that were collected when fruit bodies of C. militaris were found. This method seemed effective in detection of C. militaris in the soil and useful for rapid and reliable quantification of C. militaris in different ecosystems.