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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: 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.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.

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 analysis of the PCR algorithm upon annealing temperatures and primer set concentrations that had effects on yields of three specific amplicons. In a 25 μL PCR assay using 10 ng each of purified V. cholerae O1 and O139 gDNAs, amplification was performed in triplicate with varying concentration of three primer sets (μM) (a to h) and annealing temperature increments (°C) at 54.2, 55.5, and 56.9. The 100 bp molecular marker (lane M) and serogroup-specific PCR amplicons as positive control (lane PC) were used for base pair size comparisons.
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Related In: Results  -  Collection


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fig1: Agarose gel electrophoresis analysis of the PCR algorithm upon annealing temperatures and primer set concentrations that had effects on yields of three specific amplicons. In a 25 μL PCR assay using 10 ng each of purified V. cholerae O1 and O139 gDNAs, amplification was performed in triplicate with varying concentration of three primer sets (μM) (a to h) and annealing temperature increments (°C) at 54.2, 55.5, and 56.9. The 100 bp molecular marker (lane M) and serogroup-specific PCR amplicons as positive control (lane PC) were used for base pair size comparisons.

Mentions: Primer-template specificity binding was empirically determined in reactions of which optimized PCR parameters were analyzed upon the amplification efficiency of three multiplex primer sets that specifically bind to V. cholerae gDNAs of the O1, O139, or O22/O155. We initially optimized the separate reactions containing the gDNA content of V. cholerae O1 569B or O139 MO45 alone, or equally mixed. Most reliable amplification conditions were reproducibly performed on the reactions using a 10 ng each of V. cholerae O1 569B or O139 MO45 per reaction assay, or equally mixed. The resulting algorithm upon annealing temperature above 55°C was likely to decrease amplification yields of the specific amplicons with expected sizes (data not shown). All reactions containing O22 or O155 gDNA putatively yielded a 588 bp DNA band (data not shown). Optimum annealing temperatures ranged from 53°C to 57°C. It was clear that PCR factors underlying the annealing temperatures and primer set concentrations were more likely to influence the amplification efficiency whether the O1, O139, or equally mixed gDNAs were used. As a result, optimized multiplex PCR amplification depended on annealing temperature at 55°C and primer set concentrations of 0.4 (universal), 0.4 (O1-specific), and 1.0 (O139-specific) μM (Figure 1).


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 analysis of the PCR algorithm upon annealing temperatures and primer set concentrations that had effects on yields of three specific amplicons. In a 25 μL PCR assay using 10 ng each of purified V. cholerae O1 and O139 gDNAs, amplification was performed in triplicate with varying concentration of three primer sets (μM) (a to h) and annealing temperature increments (°C) at 54.2, 55.5, and 56.9. The 100 bp molecular marker (lane M) and serogroup-specific PCR amplicons as positive control (lane PC) were used for base pair size comparisons.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig1: Agarose gel electrophoresis analysis of the PCR algorithm upon annealing temperatures and primer set concentrations that had effects on yields of three specific amplicons. In a 25 μL PCR assay using 10 ng each of purified V. cholerae O1 and O139 gDNAs, amplification was performed in triplicate with varying concentration of three primer sets (μM) (a to h) and annealing temperature increments (°C) at 54.2, 55.5, and 56.9. The 100 bp molecular marker (lane M) and serogroup-specific PCR amplicons as positive control (lane PC) were used for base pair size comparisons.
Mentions: Primer-template specificity binding was empirically determined in reactions of which optimized PCR parameters were analyzed upon the amplification efficiency of three multiplex primer sets that specifically bind to V. cholerae gDNAs of the O1, O139, or O22/O155. We initially optimized the separate reactions containing the gDNA content of V. cholerae O1 569B or O139 MO45 alone, or equally mixed. Most reliable amplification conditions were reproducibly performed on the reactions using a 10 ng each of V. cholerae O1 569B or O139 MO45 per reaction assay, or equally mixed. The resulting algorithm upon annealing temperature above 55°C was likely to decrease amplification yields of the specific amplicons with expected sizes (data not shown). All reactions containing O22 or O155 gDNA putatively yielded a 588 bp DNA band (data not shown). Optimum annealing temperatures ranged from 53°C to 57°C. It was clear that PCR factors underlying the annealing temperatures and primer set concentrations were more likely to influence the amplification efficiency whether the O1, O139, or equally mixed gDNAs were used. As a result, optimized multiplex PCR amplification depended on annealing temperature at 55°C and primer set concentrations of 0.4 (universal), 0.4 (O1-specific), and 1.0 (O139-specific) μM (Figure 1).

Bottom Line: 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.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.

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