scholarly journals Multiplex PCR for the Detection ofLactobacillus pontis and Two Related Species in a Sourdough Fermentation

2000 ◽  
Vol 66 (5) ◽  
pp. 2113-2116 ◽  
Author(s):  
Martin R. A. Müller ◽  
Matthias A. Ehrmann ◽  
Rudi F. Vogel
Keyword(s):  
16S Rrna ◽  
Multiplex Pcr ◽  
Related Species ◽  
Lactobacillus Reuteri ◽  
Comparative Sequence Analysis ◽  
Pcr Assay ◽  
Variable Regions ◽  
Specific Pcr ◽  
Multiplex Pcr Assay ◽  
Specific Pcr Assay

ABSTRACT A specific multiplex PCR assay based on the amplification of parts of the 16S rRNA molecule was designed. Primers derived from variable regions of the 16S rRNA provided a means of easily differentiating the species Lactobacillus pontis and Lactobacillus panis. They could be clearly discriminated from the phylogenetically related species Lactobacillus vaginalis,Lactobacillus oris, and Lactobacillus reuteriand from other lactobacilli commonly known to be present in sourdough. Other strains isolated together with L. pontis from an industrial sourdough fermentation could be clearly separated from these species by comparative sequence analysis and construction of a specific PCR primer. For a fast identification a DNA isolation protocol based on the ultrasonic lysis of cells from single colonies was developed. To demonstrate the potential of such techniques for tracking these organisms in a laboratory-scale fermentation, we combined the specific PCR assay with direct DNA extraction from the organisms in the sourdough without previous cultivation.

scholarly journals Development of a multiplex PCR assay for identification of Campylobacter coli, Campylobacter fetus, Campylobacter hyointestinalis subsp. hyointestinalis, Campylobacter jejuni, Campylobacter lari and Campylobacter upsaliensis

2007 ◽  
Vol 56 (11) ◽  
pp. 1467-1473 ◽  
Author(s):  
Wataru Yamazaki-Matsune ◽  
Masumi Taguchi ◽  
Kazuko Seto ◽  
Ryuji Kawahara ◽  
Kentaro Kawatsu ◽  
...  
Keyword(s):  
Multiplex Pcr ◽  
Campylobacter Jejuni ◽  
Campylobacter Coli ◽  
Pcr Assay ◽  
Specific Pcr ◽  
Campylobacter Fetus ◽  
Multiplex Pcr Assay ◽  
Pcr Products ◽  
Campylobacter Lari ◽  
Campylobacter Upsaliensis

A multiplex PCR assay has been developed for the identification of the six common Campylobacter taxa associated with human gastroenteritis and/or septicaemia, namely Campylobacter coli, Campylobacter fetus, Campylobacter hyointestinalis subsp. hyointestinalis, Campylobacter jejuni, Campylobacter lari and Campylobacter upsaliensis. The assay was developed using a combination of newly designed and published primers. It provided a specific PCR product for each of the five Campylobacter species and the one subspecies, and each of the PCR products was sufficiently distinguished by a difference in size by agarose gel electrophoresis. On evaluation of efficacy with 142 Campylobacter strains, the assay correctly identified all strains as 1 of the 6 Campylobacter taxa. This multiplex PCR assay is a rapid, simple and practical tool for identification of the six Campylobacter taxa commonly associated with gastroenteritis and/or septicaemia in humans, and offers an effective alternative to conventional biochemical-based assays.

scholarly journals Simultaneous Detection and Identification of Common Cell Culture Contaminant and Pathogenic Mollicutes Strains by Reverse Line Blot Hybridization

2004 ◽  
Vol 70 (3) ◽  
pp. 1483-1486 ◽  
Author(s):  
Hui Wang ◽  
Fanrong Kong ◽  
Peter Jelfs ◽  
Gregory James ◽  
Gwendolyn L. Gilbert
Keyword(s):  
Cell Culture ◽  
16S Rrna ◽  
Cell Line ◽  
Intergenic Spacer ◽  
23S Rrna ◽  
Pcr Assay ◽  
Intergenic Spacer Region ◽  
Specific Pcr ◽  
Specific Pcr Assay ◽  
Species Specific

ABSTRACT We have developed a reverse line blot (RLB) hybridization assay to detect and identify the commonest mollicutes causing cell line contamination (Mycoplasma arginini, Mycoplasma fermentans, Mycoplasma hyorhinis, Mycoplasma orale, and Acholeplasma laidlawii) and human infection (Mycoplasma pneumoniae, Mycoplasma hominis, Mycoplasma genitalium, Ureaplasma parvum, and Ureaplasma urealyticum). We developed a nested PCR assay with “universal” primers targeting the mollicute 16S-23S rRNA intergenic spacer region. Amplified biotin-labeled PCR products were hybridized to membrane-bound species-specific oligonucleotide probes. The assay correctly identified reference strains of 10 mollicute species. Cell cultures submitted for detection of mollicute contamination, clinical specimens, and clinical isolates were initially tested by PCR assay targeting a presumed mollicute-specific sequence of the 16S rRNA gene. Any that were positive were assessed by the RLB assay, with species-specific PCR assay as the reference method. Initially, 100 clinical and 88 of 92 cell culture specimens gave concordant results, including 18 in which two or more mollicute species were detected by both methods. PCR and sequencing of the 16S-23S rRNA intergenic spacer region and subsequent retesting by species-specific PCR assay of the four cell culture specimens for which results were initially discrepant confirmed the original RLB results. Sequencing of amplicons from 12 cell culture specimens that were positive in the 16S rRNA PCR assay but negative by both the RLB and species-specific PCR assays failed to identify any mollicute species. The RLB hybridization assay is sensitive and specific and able to rapidly detect and identify mollicute species from clinical and cell line specimens.

A single-round multiplex PCR assay for the identification of Anopheles minimus related species infected with Plasmodium falciparum and Plasmodium vivax

2014 ◽  
Vol 63 (2) ◽  
pp. 442-449 ◽  
Author(s):  
Paiboon Eamkum ◽  
Sungsit Sungvornyothin ◽  
Onanong Kritpetcharat ◽  
Jureerat Daduang ◽  
Usa Lek-Uthai ◽  
...  
Keyword(s):  
Plasmodium Falciparum ◽  
Multiplex Pcr ◽  
Plasmodium Vivax ◽  
Related Species ◽  
Pcr Assay ◽  
Anopheles Minimus ◽  
Multiplex Pcr Assay

scholarly journals Development of a simple, rapid multiplex PCR tool kit by using 16S rRNA gene for the identification of faecal and non-faecal coli forms in the drinking waters

2021 ◽  
Author(s):  
A. Shiva Shanker ◽  
N. Rajesh ◽  
Pavan Kumar Pindi
Keyword(s):  
Drinking Water ◽  
16S Rrna ◽  
16S Rrna Gene ◽  
Multiplex Pcr ◽  
Faecal Coliform ◽  
Rrna Gene ◽  
Faecal Coliforms ◽  
Variable Regions ◽  
Total Coliforms ◽  
Multiplex Pcr Assay

Abstract A multiplex method for the detection of faecal and non-faecal coliforms in drinking water was developed using three primers from the V2, V3 and V9 variable regions of 16S rRNA gene. 194F, 474F and 1436R are the three primers designed for specific amplification of V2, V3, V9 hyper variable regions of 16S rRNA gene. Multiplex PCR allowed for differentiation of the total coliform from faecal coliform by specific amplicons: 1,285 bp of amplicon is specific for 6 non-faecal coliform genera and 1,009 bp of amplicon is specific for faecal coliform ie. E. coli. If the drinking water was contaminated with both faecal and non-faecal coliforms then two amplicons of 1,285 bp and 1,009 bp by combination of three primers are observed. The multiplex PCR assay based on 16S rRNA gene should be a beneficial tool kit for the rapid identification of the total coliforms in the large number of water samples compared with traditional methods. Results can be acquired within 3 hrs of time as compared with classic method of MPN (3–4 days). This assay will be useful in diversification and detection of seven genera of total coliforms by using variable regions of 16S rRNA.

scholarly journals Detection and differentiation of Burkholderia species with pathogenic potential in environmental soil samples

PLoS ONE ◽  
2021 ◽  
Vol 16 (1) ◽  
pp. e0245175
Author(s):  
Sujintana Janesomboon ◽  
Veerachat Muangsombut ◽  
Varintip Srinon ◽  
Chatruthai Meethai ◽  
Chayada S. Tharinjaroen ◽  
...  
Keyword(s):  
Multiplex Pcr ◽  
Capsular Polysaccharide ◽  
High Sensitivity ◽  
Pcr Primers ◽  
Soil Samples ◽  
Pcr Assay ◽  
Pathogenic Potential ◽  
Specific Pcr ◽  
Phylogenetic Cluster ◽  
Multiplex Pcr Assay

The Burkholderia pseudomallei phylogenetic cluster includes B. pseudomallei, B. mallei, B. thailandensis, B. oklahomensis, B. humptydooensis and B. singularis. Regarded as the only pathogenic members of this group, B. pseudomallei and B. mallei cause the diseases melioidosis and glanders, respectively. Additionally, variant strains of B. pseudomallei and B. thailandensis exist that include the geographically restricted B. pseudomallei that express a B. mallei-like BimA protein (BPBM), and B. thailandensis that express a B. pseudomallei-like capsular polysaccharide (BTCV). To establish a PCR-based assay for the detection of pathogenic Burkholderia species or their variants, five PCR primers were designed to amplify species-specific sequences within the bimA (Burkholderia intracellular motility A) gene. Our multiplex PCR assay could distinguish pathogenic B. pseudomallei and BPBM from the non-pathogenic B. thailandensis and the BTCV strains. A second singleplex PCR successfully discriminated the BTCV from B. thailandensis. Apart from B. humptydooensis, specificity testing against other Burkholderia spp., as well as other Gram-negative and Gram-positive bacteria produced a negative result. The detection limit of the multiplex PCR in soil samples artificially spiked with known quantities of B. pseudomallei and B. thailandensis were 5 and 6 CFU/g soil, respectively. Furthermore, comparison between standard bacterial culture and the multiplex PCR to detect B. pseudomallei from 34 soil samples, collected from an endemic area of melioidosis, showed high sensitivity and specificity. This robust, sensitive, and specific PCR assay will be a useful tool for epidemiological study of B. pseudomallei and closely related members with pathogenic potential in soil.

Application of Multiplex PCR for Monitoring Colonization of Pig Tonsils by Yersinia enterocolitica, Including Biotype 1A, and Yersinia pseudotuberculosis

2007 ◽  
Vol 70 (5) ◽  
pp. 1110-1115 ◽  
Author(s):  
BARBARA KOT ◽  
ELŻBIETA A. TRAFNY ◽  
ANTONI JAKUBCZAK
Keyword(s):  
Multiplex Pcr ◽  
Yersinia Enterocolitica ◽  
Yersinia Pseudotuberculosis ◽  
Pcr Amplification ◽  
Pcr Assay ◽  
Specific Pcr ◽  
Multiplex Pcr Assay ◽  
Control Food ◽  
Microbiological Testing ◽  
Pure Bacterial Cultures

A multiplex PCR assay was developed for the detection and differentiation of the Yersinia enterocolitica and Yersinia pseudotuberculosis isolates in both pure bacterial cultures and pig tonsils. The assay was based on the amplification of the ail, inv, yadA, and ystB genes. The PCR products, corresponding to the ail gene and the plasmid-borne yadA gene or only one product corresponding to the ail gene, were detected in Y. enterocolitica 4 biotype isolates. All of the Y. pseudotuberculosis isolates (n = 6) tested gave a positive PCR reaction for the inv gene. For all tested Y. enterocolitica 1A biotype isolates (n = 31), one product corresponding to the ystB gene was observed. The multiplex PCR assay was used to detect Y. enterocolitica and Y. pseudotuberculosis strains in pig tonsil samples obtained from 80 slaughtered pigs from three different herds. The presence of at least one of the specific PCR amplification products of ail-, ystB-, yadA-, and inv-specific sequences was observed in 11 samples (13.75%). These results of the multiplex PCR assay were compared with the results of conventional, microbiological testing. Y. enterocolitica isolates were cultured from only 3 (3.75%) of the 80 pig tonsils examined. The multiplex PCR assay was shown to be an efficient tool for differentiation between the pYV plasmid–bearing Y. enterocolitica isolates, the plasmidless Y. enterocolitica isolates, the Y. enterocolitica biotype 1A isolates, and the Y. pseudotuberculosis isolates with and without the pYV plasmid in naturally contaminated pig tonsils. This indicates that this assay is useful to control food processing and track the source of contamination.

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