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O Serogroup-Specific Touchdown-Multiplex Polymerase Chain Reaction for Detection and Identification of Vibrio cholerae O1, O139, and Non-O1/Non-O139.

Bhumiratana A, Siriphap A, Khamsuwan N, Borthong J, Chonsin K, Sutheinkul O - Biochem Res Int (2014)

Bottom Line: A novel, sensitive locus-specific touchdown-multiplex polymerase chain reaction (TMPCR), which is based on two-stage amplification pertaining to multiplex PCR and conditional touchdown strategy, was used in detecting and differentiating Vibrio cholerae serogroups.The TMPCR amplification efficiency yields either equally or unequally detectable duplex DNA bands of the O1 (588 and 364 bp) and O139 (588 and 256 bp) or a DNA fragment of non-O1/non-O139 (588 bp) while providing no false positive identifications using the genomic DNA templates of the other vibrios and Enterobacteriaceae.The reciprocal analysis of two-template combinations demonstrated that, using V. cholerae O1, O139, or equally mixed O1 and O139, the TMPCR had a detection limit of as low as 100 pg of the O1, O139, or non-O1/non-O139 in reactions containing unequally or equally mixed gDNAs.

View Article: PubMed Central - PubMed

Affiliation: Department of Parasitology and Entomology, Faculty of Public Health, Mahidol University, Bangkok 10400, Thailand.

ABSTRACT
A novel, sensitive locus-specific touchdown-multiplex polymerase chain reaction (TMPCR), which is based on two-stage amplification pertaining to multiplex PCR and conditional touchdown strategy, was used in detecting and differentiating Vibrio cholerae serogroups. A panel of molecular marker-based TMPCR method generates reproducible profiles of V. cholerae-specific (588 bp) amplicons derived from ompW gene encoding the outer membrane protein and serogroup-specific amplicons, 364 bp for the O1 and 256 bp for the O139, authentically copied from rfb genes responsible for the lipopolysaccharide biosynthesis. The TMPCR amplification efficiency yields either equally or unequally detectable duplex DNA bands of the O1 (588 and 364 bp) and O139 (588 and 256 bp) or a DNA fragment of non-O1/non-O139 (588 bp) while providing no false positive identifications using the genomic DNA templates of the other vibrios and Enterobacteriaceae. The reciprocal analysis of two-template combinations demonstrated that, using V. cholerae O1, O139, or equally mixed O1 and O139, the TMPCR had a detection limit of as low as 100 pg of the O1, O139, or non-O1/non-O139 in reactions containing unequally or equally mixed gDNAs. In addition, the O serogroup-specific TMPCR method had 100% agreement with the serotyping method when examined for the serotyped V. cholerae reference strains and those recovered from clinical samples. The potential benefit of using this TMPCR tool would augment the serotyping method used in epidemiological surveillance and monitoring of V. cholerae serogroups, O1, O139, and non-O1/non-O139 present in clinical and environmental samples.

No MeSH data available.


Related in: MedlinePlus

Agarose gel electrophoresis of the O serogroup-specific TMPCR and 16S rDNA PCR profiles using representative V. cholerae gDNAs archived from the clinical specimens. PCR products obtained by both TMPCR (5 μL) and 16S rDNA PCR (5 μL) that were separately performed are confined to the same lane: lanes 1–3, the O1 from stools; lanes 4-5, the O1 from rectal swabs; lanes 6–10, the O139 from stools; and lanes 11–15, the non-O1/non-O139 from stools. In each TMPCR employing the O1, O139, or non-O1/non-O139 gDNA, the reliable reaction represents duplex DNA fragments (588 bp and 364 bp from the O1 or 588 bp and 256 bp from the O139) and a single 588 bp DNA band from the non-O1/non-O139, as compared to 16S rDNA amplicons (663 bp) and 100 bp DNA ladder marker (lane M).
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fig4: Agarose gel electrophoresis of the O serogroup-specific TMPCR and 16S rDNA PCR profiles using representative V. cholerae gDNAs archived from the clinical specimens. PCR products obtained by both TMPCR (5 μL) and 16S rDNA PCR (5 μL) that were separately performed are confined to the same lane: lanes 1–3, the O1 from stools; lanes 4-5, the O1 from rectal swabs; lanes 6–10, the O139 from stools; and lanes 11–15, the non-O1/non-O139 from stools. In each TMPCR employing the O1, O139, or non-O1/non-O139 gDNA, the reliable reaction represents duplex DNA fragments (588 bp and 364 bp from the O1 or 588 bp and 256 bp from the O139) and a single 588 bp DNA band from the non-O1/non-O139, as compared to 16S rDNA amplicons (663 bp) and 100 bp DNA ladder marker (lane M).

Mentions: Lastly, we tested the amplification fidelity of O serogroup-specific TMPCR by using target gDNA samples archived from the stock cultures of the 148 V. cholerae clinical isolates. Overall, the TMPCR exhibited 100% concordance with serotyping method as reference whether V. cholerae cultures were obtained from stools or rectal swabs (Table 5). All the 108 cultures from the stools tested gave positive TMPCR results: 65 (60.2%) of the O1 serotype concordant with the O1 serogroup-specific TMPCR (588 and 364 bp), 15 (13.9%) of the O139 serotype concordant with the O139 serogroup-specific TMPCR (588 and 256 bp), and 28 (25.9%) of the non-O1/non-O139 serotype concordant with the O serogroup-specific TMPCR (588 bp). Meanwhile, all the 40 cultures from the rectal swabs were also consistently positive with the tests: 39 (97.5%) accordant with the O1 serotype and the O1 serogroup-specific TMPCR and only one (2.5%) accordant with the non-O1/non-O139 serotype and 588 bp specific TMPCR. Only the representative gDNAs archived from the clinically isolated V. cholerae cultures are shown in Figure 4. All samples tested by O serogroup-specific TMPCR were consistently positive with the 16S rDNA PCR.


O Serogroup-Specific Touchdown-Multiplex Polymerase Chain Reaction for Detection and Identification of Vibrio cholerae O1, O139, and Non-O1/Non-O139.

Bhumiratana A, Siriphap A, Khamsuwan N, Borthong J, Chonsin K, Sutheinkul O - Biochem Res Int (2014)

Agarose gel electrophoresis of the O serogroup-specific TMPCR and 16S rDNA PCR profiles using representative V. cholerae gDNAs archived from the clinical specimens. PCR products obtained by both TMPCR (5 μL) and 16S rDNA PCR (5 μL) that were separately performed are confined to the same lane: lanes 1–3, the O1 from stools; lanes 4-5, the O1 from rectal swabs; lanes 6–10, the O139 from stools; and lanes 11–15, the non-O1/non-O139 from stools. In each TMPCR employing the O1, O139, or non-O1/non-O139 gDNA, the reliable reaction represents duplex DNA fragments (588 bp and 364 bp from the O1 or 588 bp and 256 bp from the O139) and a single 588 bp DNA band from the non-O1/non-O139, as compared to 16S rDNA amplicons (663 bp) and 100 bp DNA ladder marker (lane M).
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4295632&req=5

fig4: Agarose gel electrophoresis of the O serogroup-specific TMPCR and 16S rDNA PCR profiles using representative V. cholerae gDNAs archived from the clinical specimens. PCR products obtained by both TMPCR (5 μL) and 16S rDNA PCR (5 μL) that were separately performed are confined to the same lane: lanes 1–3, the O1 from stools; lanes 4-5, the O1 from rectal swabs; lanes 6–10, the O139 from stools; and lanes 11–15, the non-O1/non-O139 from stools. In each TMPCR employing the O1, O139, or non-O1/non-O139 gDNA, the reliable reaction represents duplex DNA fragments (588 bp and 364 bp from the O1 or 588 bp and 256 bp from the O139) and a single 588 bp DNA band from the non-O1/non-O139, as compared to 16S rDNA amplicons (663 bp) and 100 bp DNA ladder marker (lane M).
Mentions: Lastly, we tested the amplification fidelity of O serogroup-specific TMPCR by using target gDNA samples archived from the stock cultures of the 148 V. cholerae clinical isolates. Overall, the TMPCR exhibited 100% concordance with serotyping method as reference whether V. cholerae cultures were obtained from stools or rectal swabs (Table 5). All the 108 cultures from the stools tested gave positive TMPCR results: 65 (60.2%) of the O1 serotype concordant with the O1 serogroup-specific TMPCR (588 and 364 bp), 15 (13.9%) of the O139 serotype concordant with the O139 serogroup-specific TMPCR (588 and 256 bp), and 28 (25.9%) of the non-O1/non-O139 serotype concordant with the O serogroup-specific TMPCR (588 bp). Meanwhile, all the 40 cultures from the rectal swabs were also consistently positive with the tests: 39 (97.5%) accordant with the O1 serotype and the O1 serogroup-specific TMPCR and only one (2.5%) accordant with the non-O1/non-O139 serotype and 588 bp specific TMPCR. Only the representative gDNAs archived from the clinically isolated V. cholerae cultures are shown in Figure 4. All samples tested by O serogroup-specific TMPCR were consistently positive with the 16S rDNA PCR.

Bottom Line: A novel, sensitive locus-specific touchdown-multiplex polymerase chain reaction (TMPCR), which is based on two-stage amplification pertaining to multiplex PCR and conditional touchdown strategy, was used in detecting and differentiating Vibrio cholerae serogroups.The TMPCR amplification efficiency yields either equally or unequally detectable duplex DNA bands of the O1 (588 and 364 bp) and O139 (588 and 256 bp) or a DNA fragment of non-O1/non-O139 (588 bp) while providing no false positive identifications using the genomic DNA templates of the other vibrios and Enterobacteriaceae.The reciprocal analysis of two-template combinations demonstrated that, using V. cholerae O1, O139, or equally mixed O1 and O139, the TMPCR had a detection limit of as low as 100 pg of the O1, O139, or non-O1/non-O139 in reactions containing unequally or equally mixed gDNAs.

View Article: PubMed Central - PubMed

Affiliation: Department of Parasitology and Entomology, Faculty of Public Health, Mahidol University, Bangkok 10400, Thailand.

ABSTRACT
A novel, sensitive locus-specific touchdown-multiplex polymerase chain reaction (TMPCR), which is based on two-stage amplification pertaining to multiplex PCR and conditional touchdown strategy, was used in detecting and differentiating Vibrio cholerae serogroups. A panel of molecular marker-based TMPCR method generates reproducible profiles of V. cholerae-specific (588 bp) amplicons derived from ompW gene encoding the outer membrane protein and serogroup-specific amplicons, 364 bp for the O1 and 256 bp for the O139, authentically copied from rfb genes responsible for the lipopolysaccharide biosynthesis. The TMPCR amplification efficiency yields either equally or unequally detectable duplex DNA bands of the O1 (588 and 364 bp) and O139 (588 and 256 bp) or a DNA fragment of non-O1/non-O139 (588 bp) while providing no false positive identifications using the genomic DNA templates of the other vibrios and Enterobacteriaceae. The reciprocal analysis of two-template combinations demonstrated that, using V. cholerae O1, O139, or equally mixed O1 and O139, the TMPCR had a detection limit of as low as 100 pg of the O1, O139, or non-O1/non-O139 in reactions containing unequally or equally mixed gDNAs. In addition, the O serogroup-specific TMPCR method had 100% agreement with the serotyping method when examined for the serotyped V. cholerae reference strains and those recovered from clinical samples. The potential benefit of using this TMPCR tool would augment the serotyping method used in epidemiological surveillance and monitoring of V. cholerae serogroups, O1, O139, and non-O1/non-O139 present in clinical and environmental samples.

No MeSH data available.


Related in: MedlinePlus