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.

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 Development of a multiplex PCR test-system for detection of BHV-1, BVDV, Chlamydia spp. and Mycoplasma spp.

2019 ◽  
Vol 5 (3) ◽  
pp. 19-21
Author(s):  
M. M. Isakov
Keyword(s):  
Multiplex Pcr ◽  
Pcr Primers ◽  
Pcr Assay ◽  
Bovine Herpesvirus 1 ◽  
Diarrhea Virus ◽  
Dna And Rna ◽  
Multiplex Pcr Assay ◽  
Herpesvirus 1 ◽  
Viral Diarrhea Virus ◽  
Mycoplasma Spp

This study describes development of a multiplex PCR assay for detection of BHV-1, BVDV, Chlamydia spp. and Mycoplasma spp. infections in bovines. The assay was developed using genomic DNA and RNA and four sets of PCR primers targeting 16S rRNA genes of Chlamydia spp., Mycoplasma spp., 5’-UTR of Bovine viral diarrhea virus, gE of Bovine herpesvirus-1, respectively. A total of 100 tissue samples were collected from cattle suspected to be infected with the viral and bacterial pathogens (BVDV, BHV-1, Chlamydia spp. and Mycoplasma spp.) from different regions of Ukraine. A part of sample was stored at –50°C for isolation of genomic DNA and RNA. The multiplex PCR assay was optimized in the study. The specific primers designed and used in the study were found sensitive and specific in amplifying target genes viz. 16S rRNA, gE, 5’-UTR of Chlamydia spp., Mycoplasma spp., BHV-1 and BVDV, respectively. The PCR primers used in the optimization of multiplex PCR assay for detection of Bovine viral diarrhea virus, Bovine herpesvirus-1, Chlamydia spp., Mycolasma spp. could amplify 221 bp, 111 bp, 386 bp, 279 bp products, respectively. Non specific amplification was not observed

scholarly journals Molecular Characterization of BacillusStrains Involved in Outbreaks of Anthrax in France in 1997

1998 ◽  
Vol 36 (11) ◽  
pp. 3412-3414 ◽  
Author(s):  
Guy Patra ◽  
Josée Vaissaire ◽  
Martine Weber-Levy ◽  
Claudine Le Doujet ◽  
Michèle Mock
Keyword(s):  
Multiplex Pcr ◽  
Tandem Repeat ◽  
Resistant Strain ◽  
Variable Number Tandem Repeat ◽  
Variable Number ◽  
Soil Samples ◽  
Pcr Assay ◽  
Multiplex Pcr Assay ◽  
Biological Methods

Outbreaks of anthrax zoonose occurred in two regions of France in 1997. Ninety-four animals died, and there were three nonfatal cases in humans. The diagnosis of anthrax was rapidly confirmed by bacteriological and molecular biological methods. The strains ofBacillus anthracis in animal and soil samples were identified by a multiplex PCR assay. They all belonged to the variable-number tandem repeat (VNTR) group (VNTR)3. A penicillin-resistant strain was detected. Nonvirulent bacilli related to B. anthracis, of all VNTR types, were also found in the soil.

scholarly journals Multiplex PCR for Identification of Two Capsular Types in Epidemic KPC-Producing Klebsiella pneumoniae Sequence Type 258 Strains

2014 ◽  
Vol 58 (7) ◽  
pp. 4196-4199 ◽  
Author(s):  
Liang Chen ◽  
Kalyan D. Chavda ◽  
Jacqueline Findlay ◽  
Gisele Peirano ◽  
Katie Hopkins ◽  
...  
Keyword(s):  
Klebsiella Pneumoniae ◽  
Multiplex Pcr ◽  
Capsular Polysaccharide ◽  
Sequence Type ◽  
Pcr Assay ◽  
Content Type ◽  
Multiplex Pcr Assay ◽  
Polysaccharide Synthesis ◽  
Sequence Types

ABSTRACTWe developed a multiplex PCR assay capable of identifying two capsular polysaccharide synthesis sequence types (sequence type 258 [ST258]cps-1andcps-2) in epidemicKlebsiella pneumoniaeST258 strains. The assay performed with excellent sensitivity (100%) and specificity (100%) for identifyingcpstypes in 60 ST258K. pneumoniaesequenced isolates. The screening of 419 ST258 clonal isolates revealed a significant association betweencpstype andK. pneumoniaecarbapenemase (KPC) variant:cps-1is largely associated with KPC-2, whilecps-2is primarily associated with KPC-3.

scholarly journals Detection and Identification ofActinobacillus pleuropneumoniae Serotype 5 by Multiplex PCR

1998 ◽  
Vol 36 (6) ◽  
pp. 1704-1710 ◽  
Author(s):  
Terry M. Lo ◽  
Christine K. Ward ◽  
Thomas J. Inzana
Keyword(s):  
Early Detection ◽  
Multiplex Pcr ◽  
Sensitivity And Specificity ◽  
Dna Sequences ◽  
Capsular Polysaccharide ◽  
Actinobacillus Pleuropneumoniae ◽  
Pcr Assay ◽  
Multiplex Pcr Assay ◽  
Detection And Identification ◽  
Serotype 5

Serotyping of Actinobacillus pleuropneumoniae is based on detection of the serotype-specific capsular antigen. However, not all isolates can be serotyped, and some may cross-react with multiple serotyping reagents. To improve sensitivity and specificity of serotyping and for early detection, a multiplex PCR assay was developed for detection of A. pleuropneumoniae and identification of serotype 5 isolates. DNA sequences specific to the conserved export and serotype-specific biosynthesis regions of the capsular polysaccharide of A. pleuropneumoniaeserotype 5 were used as primers to amplify 0.7- and 1.1-kb DNA fragments, respectively. The 0.7-kb fragment was amplified from all strains of A. pleuropneumoniae tested with the exception of serotype 4. The 0.7-kb fragment was not amplified from any heterologous species that are also common pathogens or commensals of swine. In contrast, the 1.1-kb fragment was amplified from all serotype 5 strains only. The assay was capable of amplifying DNA from less than 102 CFU. The A. pleuropneumoniaeserotype 5 capsular DNA products were readily amplified from lung tissues obtained from infected swine, although the 1.1-kb product was not amplified from some tissues stored frozen for 6 years. The multiplex PCR assay enabled us to detect A. pleuropneumoniae rapidly and to distinguish serotype 5 strains from other serotypes. The use of primers specific to the biosynthesis regions of other A. pleuropneumoniae serotypes would expand the diagnostic and epidemiologic capabilities of this assay.

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.

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.

Design of a multiplex PCR assay for the simultaneous detection and confirmation of Neisseria gonorrhoeae

2010 ◽  
Vol 63 (5) ◽  
pp. 431-433 ◽  
Author(s):  
Isabelle O'Callaghan ◽  
Daniel Corcoran ◽  
Brigid Lucey
Keyword(s):  
Neisseria Gonorrhoeae ◽  
Multiplex Pcr ◽  
Internal Control ◽  
Limit Of Detection ◽  
Simultaneous Detection ◽  
Pcr Primers ◽  
Real Potential ◽  
Pcr Assay ◽  
Neisseria Species ◽  
Multiplex Pcr Assay

ObjectivesTo improve the detection of Neisseria gonorrhoeae by designing a multiplex PCR assay using two N gonorrhoeae-specific genes as targets, thereby providing detection and confirmation of a positive result simultaneously.MethodsPCR primers were designed to detect two N gonorrhoeae genes, namely porA and pgi1. Primers for an internal control targeting the ompW gene of Vibrio cholerae were also designed and incorporated in the assay. The DNA of 45 clinical isolates including 33 N gonorrhoeae isolates, seven non-gonococcal Neisseria species, and five non-Neisseria species was tested using the multiplex PCR assay.ResultsAll 33 N gonorrhoeae isolates were successfully detected by the assay and none of the non-gonococcal isolates was detected. The assay showed a sensitivity and specificity of 100%, and a limit of detection of 1.25 ng of DNA.ConclusionThis multiplex PCR assay offers a sensitive and specific assay suitable for the detection of N gonorrhoeae, and offers real potential for diagnostic use.

Usefulness of a Multiplex PCR for Detection of Diarrheagenic Escherichia coli in a Diagnostic Microbiology Laboratory Setting

2016 ◽  
Vol 1 (2) ◽  
pp. 38-42 ◽  
Author(s):  
Khairun Nessa ◽  
Dilruba Ahmed ◽  
Johirul Islam ◽  
FM Lutful Kabir ◽  
M Anowar Hossain
Keyword(s):  
Escherichia Coli ◽  
Multiplex Pcr ◽  
Clinical Laboratory ◽  
Proteinase K ◽  
Microbiology Laboratory ◽  
Pcr Assay ◽  
E Coli ◽  
Diarrheagenic Escherichia Coli ◽  
Multiplex Pcr Assay ◽  
Diagnostic Microbiology

A multiplex PCR assay was evaluated for diagnosis of diarrheagenic Escherichia coli in stool samples of patients with diarrhoea submitted to a diagnostic microbiology laboratory. Two procedures of DNA template preparationproteinase K buffer method and the boiling method were evaluated to examine isolates of E. coli from 150 selected diarrhoeal cases. By proteinase K buffer method, 119 strains (79.3%) of E. coli were characterized to various categories by their genes that included 55.5% enteroaggregative E. coli (EAEC), 18.5% enterotoxigenic E. coli (ETEC), 1.7% enteropathogenic E. coli (EPEC), and 0.8% Shiga toxin-producing E. coli (STEC). Although boiling method was less time consuming (<24 hrs) and less costly (<8.0 US $/ per test) but was less efficient in typing E. coli compared to proteinase K method (41.3% vs. 79.3% ; p<0.001). The sensitivity and specificity of boiling method compared to proteinase K method was 48.7% and 87.1% while the positive and negative predictive value was 93.5% and 30.7%, respectively. The majority of pathogenic E. coli were detected in children (78.0%) under five years age with 53.3% under one year, and 68.7% of the children were male. Children under 5 years age were frequently infected with EAEC (71.6%) compared to ETEC (24.3%), EPEC (2.7%) and STEC (1.4%). The multiplex PCR assay could be effectively used as a rapid diagnostic tool for characterization of diarrheagenic E. coli using a single reaction tube in the clinical laboratory setting.Bangladesh J Med Microbiol 2007; 01 (02): 38-42

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