scholarly journals A PCR Assay To Discriminate Human and Ruminant Feces on the Basis of Host Differences in Bacteroides-Prevotella Genes Encoding 16S rRNA

2000 ◽  
Vol 66 (10) ◽  
pp. 4571-4574 ◽  
Author(s):  
Anne E. Bernhard ◽  
Katharine G. Field
Keyword(s):  
Natural Waters ◽  
Fragment Length Polymorphism ◽  
Sequence Data ◽  
Pcr Primers ◽  
Pcr Markers ◽  
Human Feces ◽  
Inexpensive Method ◽  
Pcr Assay ◽  
Specific Pcr ◽  
Genes Encoding

ABSTRACT Our purpose was to develop a rapid, inexpensive method of diagnosing the source of fecal pollution in water. In previous research, we identified Bacteroides-Prevotella ribosomal DNA (rDNA) PCR markers based on analysis. These markers length heterogeneity PCR and terminal restriction fragment length polymorphism distinguish cow from human feces. Here, we recovered 16S rDNA clones from natural waters that were close phylogenetic relatives of the markers. From the sequence data, we designed specific PCR primers that discriminate human and ruminant sources of fecal contamination.

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

2003 ◽  
Vol 69 (12) ◽  
pp. 7430-7434 ◽  
Author(s):  
Trevor G. Phister ◽  
David A. Mills
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Pcr Primers ◽  
Rrna Gene ◽  
Dekkera Bruxellensis ◽  
Pcr Assay ◽  
Specific Pcr ◽  
Spoilage Yeast ◽  
Pcr Method ◽  
26S Rrna

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

scholarly journals Specific PCR detection of Peptostreptococcus magnus

2003 ◽  
Vol 52 (4) ◽  
pp. 309-313 ◽  
Author(s):  
M.P. Riggio ◽  
A. Lennon
Keyword(s):  
Pcr Primers ◽  
Oral Bacteria ◽  
Rrna Gene ◽  
Subgingival Plaque ◽  
Pcr Assay ◽  
Clinical Specimens ◽  
Specific Pcr ◽  
Wide Range ◽  
Pcr Products ◽  
Peptostreptococcus Magnus

Peptostreptococcus magnus is the most pathogenic and one of the most common Gram-positive anaerobic cocci found in human clinical specimens. The organism has been isolated in pure culture from a range of serious infections, including meningitis and endocarditis. However, isolation of Peptostreptococcus magnus from the oral cavity has rarely been attempted. Identification of Peptostreptococcus magnus in clinical specimens is reliant upon microbiological culture and biochemical methods, which often give ambiguous results. The aim of this study was to develop a PCR assay for the specific detection of Peptostreptococcus magnus in oral clinical specimens. PCR primers specific for Peptostreptococcus magnus DNA were derived by comparison of 16S rRNA gene sequences and selection of primers that demonstrated specificity at their 3′ ends for Peptostreptococcus magnus. PCR positivity for Peptostreptococcus magnus DNA was indicated by the amplification of a 553 bp product. The PCR assay was then used to attempt detection of Peptostreptococcus magnus DNA in subgingival plaque samples from adult periodontitis patients and pus aspirates from subjects with acute dento-alveolar abscesses. The PCR assay was demonstrated to be highly specific for Peptostreptococcus magnus DNA, since no PCR products were obtained when genomic DNA from a wide range of other oral bacteria, including closely related Peptostreptococcus species, was used in the PCR assay. Confirmation of specific amplification of Peptostreptococcus magnus DNA was obtained by digestion of PCR products with the restriction endonuclease RsaI, which gives a unique restriction profile for Peptostreptococcus magnus. Of the 33 subgingival plaque samples analysed, 2 (6 %) were positive for Peptostreptococcus magnus DNA. None of the 60 pus aspirates analysed was positive for Peptostreptococcus magnus DNA. It is concluded that Peptostreptococcus magnus is not a major pathogen in adult periodontitis or dento-alveolar abscesses. The PCR assay provides a more rapid, specific and sensitive alternative to conventional methods for identification of Peptostreptococcus magnus in clinical specimens.

scholarly journals Rare Pyrenophora teres Hybridization Events Revealed by Development of Sequence-Specific PCR Markers

2017 ◽  
Vol 107 (7) ◽  
pp. 878-884 ◽  
Author(s):  
Barsha Poudel ◽  
Simon R. Ellwood ◽  
Alison C. Testa ◽  
Mark McLean ◽  
Mark W. Sutherland ◽  
...  
Keyword(s):  
Host Plants ◽  
Fragment Length Polymorphism ◽  
Putative Hybrid ◽  
Pyrenophora Teres ◽  
Net Blotch ◽  
Pcr Markers ◽  
Chain Reaction ◽  
Specific Pcr ◽  
Polymerase Chain ◽  
Pathogenic Variability

Pyrenophora teres f. teres and P. teres f. maculata cause net form and spot form, respectively, of net blotch on barley (Hordeum vulgare). The two forms reproduce sexually, producing hybrids with genetic and pathogenic variability. Phenotypic identification of hybrids is challenging because lesions induced by hybrids on host plants resemble lesions induced by either P. teres f. teres or P. teres f. maculata. In this study, 12 sequence-specific polymerase chain reaction markers were developed based on expressed regions spread across the genome. The primers were validated using 210 P. teres isolates, 2 putative field hybrids (WAC10721 and SNB172), 50 laboratory-produced hybrids, and 7 isolates collected from barley grass (H. leporinum). The sequence-specific markers confirmed isolate WAC10721 as a hybrid. Only four P. teres f. teres markers amplified on DNA of barley grass isolates. Amplified fragment length polymorphism markers suggested that P. teres barley grass isolates are genetically different from P. teres barley isolates and that the second putative hybrid (SNB172) is a barley grass isolate. We developed a suite of markers which clearly distinguish the two forms of P. teres and enable unambiguous identification of hybrids.

scholarly journals Identification by PCR of Fusarium culmorum Strains Producing Large and Small Amounts of Deoxynivalenol

2002 ◽  
Vol 68 (11) ◽  
pp. 5472-5479 ◽  
Author(s):  
B. Bakan ◽  
C. Giraud-Delville ◽  
L. Pinson ◽  
D. Richard-Molard ◽  
E. Fournier ◽  
...  
Keyword(s):  
Intergenic Region ◽  
Sequence Data ◽  
Fusarium Culmorum ◽  
Pcr Primers ◽  
Wheat Grains ◽  
Specific Pcr ◽  
Trichodiene Synthase

ABSTRACT Thirty deoxynivalenol-producing F. culmorum strains, isolated from wheat grains, were incubated in vitro and analyzed for trichothecene production. Seventeen strains produced more than 1 ppm of deoxynivalenol and acetyldeoxynivalenol and were considered high-deoxynivalenol-producing strains, whereas 13 F. culmorum strains produced less than 0.07 ppm of trichothecenes and were considered low-deoxynivalenol-producing strains. For all strains, a 550-base portion of the trichodiene synthase gene (tri5) was amplified and sequenced. According to the tri5 data, the F. culmorum strains tested clustered into two groups that correlated with in vitro deoxynivalenol production. For three high-producing and three low-producing F. culmorum strains, the tri5-tri6 intergenic region was then sequenced, which confirmed the two separate clusters within the F. culmorum strains. According to the tri5-tri6 sequence data, specific PCR primers were designed to allow differentiation of high-producing from low-producing F. culmorum strains.

Detection ofBacillus sphaericusmosquitocidal toxin genes by multiplex colony PCR

2009 ◽  
Vol 55 (2) ◽  
pp. 207-209 ◽  
Author(s):  
Santosh C. Jagtap ◽  
Chandrakant B. Jagtap ◽  
Pradeep Kumar ◽  
R. B. Srivastava
Keyword(s):  
Bacillus Sphaericus ◽  
Pcr Assay ◽  
Single Tube ◽  
Colony Pcr ◽  
Toxin Genes ◽  
Specific Pcr ◽  
Genes Encoding ◽  
Single Tube Reaction

A multiplex colony PCR assay was developed for the detection of 5 genes encoding Bacillus sphaericus mosquito larvicidal toxins, namely binA, binB, mtx1, mtx2, and mtx3. Primers designed for these 5 genes yielded specific PCR amplicons of the expected size from type cultures of B. sphaericus. This method of detecting multiple toxin genes by colony PCR in a single tube reaction is a simple, rapid, and economical technique for identification of highly toxic environmental B. sphaericus isolates.

Molecular and morphological discrimination betweenTylospora fibrillosaandTylospora asterophoramycorrhizae

1999 ◽  
Vol 77 (1) ◽  
pp. 11-21 ◽  
Author(s):  
Ursula Eberhardt ◽  
Lutz Walter ◽  
Ingrid Kottke
Keyword(s):  
Fragment Length Polymorphism ◽  
Pcr Primers ◽  
Morphological Identification ◽  
Chain Reaction ◽  
Specific Primers ◽  
Specific Pcr ◽  
Pcr Rflp ◽  
Polymerase Chain ◽  
Species Specific ◽  
First Time

Among the mycorrhizal types of spruce, Tylospora-type mycorrhizae are the most constant and abundant. Two species of the genus Tylospora occur in Europe, Tylospora fibrillosa and Tylospora asterophora. Mycorrhizae of T. asterophora are described in detail for the first time. Sequences of the internal transcribed spacer (ITS) of the ribosomal genes were obtained from T. fibrillosa and T. asterophora mycorrhizae, sporocarps, and cultured mycelium. Discrimination and identification of the two species by ITS polymerase chain reaction - restriction fragment length polymorphism (PCR-RFLP) are discussed in the light of inter- and intra-specific variability. Species-specific PCR primers were designed to distinguish both species. Molecular screening of Tylospora-type mycorrhizae from field material led to unambiguous results, whereas morphological identification is likely to fail because of great similarity even at the microscopic level.Key words: Tylospora asterophora, Tylospora fibrillosa, ectomycorrhizae, taxon specific primers (TSOPs), ITS sequences.

scholarly journals Polymorphism of ompH gene of Pasteurella multocida serotype A strains isolated in Iran

2018 ◽  
Vol 69 (1) ◽  
pp. 847
Author(s):  
M. OULAD ◽  
Y. TAHAMTAN ◽  
N. SOHRABI
Keyword(s):  
Pasteurella Multocida ◽  
Fragment Length Polymorphism ◽  
Genetic Profile ◽  
Vaccine Formulation ◽  
Pcr Assay ◽  
Serotype A ◽  
Specific Pcr ◽  
Rflp Type ◽  
Specific Pcr Assay ◽  
Species Specific

One of the most frequent causes of respiratory infection and death in sheep and goats is Pasteurella multocida. In humans, it has been associated with diseases of the respiratory tracts, arthritis, osteomyelitis and meningitis. Outer membrane protein H (OmpH) has a role in immunogenicity and pathogenicity of P. multocida. The aim of this study was to characterize the genetic diversity of ompH gene of a panel of P. multocida serotype A strains isolated in sheep. Forty P. multocida serotype A strains isolated in previous study were selected and analyzed by restriction fragment length polymorphism (RFLP) of a species-specific PCR assay. RFLP amplified fragment produced five different cleavage patterns. On the basis of combinations resulting from ompH gene digestion, the 40 P. multocida isolates were classified in six RFLP type. It seems that isolates with variants genetic profile represent different pathogenecity. New vaccine formulation should consider multivariants of P. multocida in order to confer a wider protection.

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