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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 analysis of the amplification efficiency of semiquantitative TMPCR. A reciprocal amplification algorithm of detectable levels of the O1 to O139 gDNAs (experiments (a) to (e)) was described in the text, while E. coli gDNA template serving as negative control (NC) was included. (a) Lanes 1 to 6, the 100 ng O1 gDNA versus a 10-fold serially diluted O139 gDNA, 1 pg to 100 ng, and vice versa (lanes 7 to 12). (b) Lanes 1 to 3, unequal amount ratios of the O1 : O139, 100 ng to 1 pg, 10 ng to 10 pg, and 1 ng to 100 pg, and vice versa (lanes 4 to 6). Lanes 7 to 12, the 100 pg O139 gDNA versus the O1 gDNA contents of varying 100 ng, 50 ng, 25 ng, 10 ng, and 1 ng to 100 pg. (c) Lanes 1 to 6, the equal amount ratios of 10-fold serially diluted O1 to O139 gDNAs, 100 ng to 1 pg. (d) Lanes 1 to 6, 10-fold serially diluted O1 gDNA contents, 100 ng to 1 pg. (e) Lanes 1 to 6, 10-fold serially diluted O139 gDNA contents, 100 ng to 1 pg. A 100 bp DNA ladder marker (lane M) was used in comparison of the amplicons with expected size (bp).
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fig3: Agarose gel electrophoresis analysis of the amplification efficiency of semiquantitative TMPCR. A reciprocal amplification algorithm of detectable levels of the O1 to O139 gDNAs (experiments (a) to (e)) was described in the text, while E. coli gDNA template serving as negative control (NC) was included. (a) Lanes 1 to 6, the 100 ng O1 gDNA versus a 10-fold serially diluted O139 gDNA, 1 pg to 100 ng, and vice versa (lanes 7 to 12). (b) Lanes 1 to 3, unequal amount ratios of the O1 : O139, 100 ng to 1 pg, 10 ng to 10 pg, and 1 ng to 100 pg, and vice versa (lanes 4 to 6). Lanes 7 to 12, the 100 pg O139 gDNA versus the O1 gDNA contents of varying 100 ng, 50 ng, 25 ng, 10 ng, and 1 ng to 100 pg. (c) Lanes 1 to 6, the equal amount ratios of 10-fold serially diluted O1 to O139 gDNAs, 100 ng to 1 pg. (d) Lanes 1 to 6, 10-fold serially diluted O1 gDNA contents, 100 ng to 1 pg. (e) Lanes 1 to 6, 10-fold serially diluted O139 gDNA contents, 100 ng to 1 pg. A 100 bp DNA ladder marker (lane M) was used in comparison of the amplicons with expected size (bp).

Mentions: To analyze the amplification sensitivity of the TMPCR, we determined a reciprocal amplification pertaining to the algorithms that used the amount of ratios of mixed V. cholerae gDNAs (O1 : O139), in comparison to those reactions containing each gDNA template serially diluted. The resulting TMPCR experiments in which CTAB/phenol-chloroform extracted V. cholerae gDNA whether O1, O139, or O22/O155 was amplified are shown in Figure 3. As for Figure 3(a), the TMPCR could detect O serogroup-specific DNA fragments authentically derived from the O1 or the O139 in reciprocal reactions. The detection limit of as low as 1 ng of the O1 was obtained in the presence of the higher amount of O139 or 100-fold greater than the O1. Meanwhile, the detection limit of as low as 10 ng of the O139 was obtained in the presence of the higher amount of the O1 or 10-fold greater than the O139. Figure 3(b) shows that its detection limit was at 100 pg of the O1 or the O139 in inverse reactions containing unequal amount ratios of the 100 pg O1 to 1 ng O139, and vice versa. Again, the TMPCR was likely to show detection limit of as low as 100 pg each of gDNAs in the reaction containing equal amount ratio of the O1 to O139. It was clear to note that the TMPCR had the detection limit of as low as 100 pg gDNA of the O1 or O139 when examined for the equal amount ratio of the serially diluted O1 and O139 templates (Figure 3(c)). Similarly, it also had the same detection limit of as low as 100 pg gDNA of the O1 or O139 when separately copied (Figures 3(d) and 3(e)). The amount of as low as 100 pg O22 or O155 gDNA was also detected (data not shown).


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 amplification efficiency of semiquantitative TMPCR. A reciprocal amplification algorithm of detectable levels of the O1 to O139 gDNAs (experiments (a) to (e)) was described in the text, while E. coli gDNA template serving as negative control (NC) was included. (a) Lanes 1 to 6, the 100 ng O1 gDNA versus a 10-fold serially diluted O139 gDNA, 1 pg to 100 ng, and vice versa (lanes 7 to 12). (b) Lanes 1 to 3, unequal amount ratios of the O1 : O139, 100 ng to 1 pg, 10 ng to 10 pg, and 1 ng to 100 pg, and vice versa (lanes 4 to 6). Lanes 7 to 12, the 100 pg O139 gDNA versus the O1 gDNA contents of varying 100 ng, 50 ng, 25 ng, 10 ng, and 1 ng to 100 pg. (c) Lanes 1 to 6, the equal amount ratios of 10-fold serially diluted O1 to O139 gDNAs, 100 ng to 1 pg. (d) Lanes 1 to 6, 10-fold serially diluted O1 gDNA contents, 100 ng to 1 pg. (e) Lanes 1 to 6, 10-fold serially diluted O139 gDNA contents, 100 ng to 1 pg. A 100 bp DNA ladder marker (lane M) was used in comparison of the amplicons with expected size (bp).
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
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fig3: Agarose gel electrophoresis analysis of the amplification efficiency of semiquantitative TMPCR. A reciprocal amplification algorithm of detectable levels of the O1 to O139 gDNAs (experiments (a) to (e)) was described in the text, while E. coli gDNA template serving as negative control (NC) was included. (a) Lanes 1 to 6, the 100 ng O1 gDNA versus a 10-fold serially diluted O139 gDNA, 1 pg to 100 ng, and vice versa (lanes 7 to 12). (b) Lanes 1 to 3, unequal amount ratios of the O1 : O139, 100 ng to 1 pg, 10 ng to 10 pg, and 1 ng to 100 pg, and vice versa (lanes 4 to 6). Lanes 7 to 12, the 100 pg O139 gDNA versus the O1 gDNA contents of varying 100 ng, 50 ng, 25 ng, 10 ng, and 1 ng to 100 pg. (c) Lanes 1 to 6, the equal amount ratios of 10-fold serially diluted O1 to O139 gDNAs, 100 ng to 1 pg. (d) Lanes 1 to 6, 10-fold serially diluted O1 gDNA contents, 100 ng to 1 pg. (e) Lanes 1 to 6, 10-fold serially diluted O139 gDNA contents, 100 ng to 1 pg. A 100 bp DNA ladder marker (lane M) was used in comparison of the amplicons with expected size (bp).
Mentions: To analyze the amplification sensitivity of the TMPCR, we determined a reciprocal amplification pertaining to the algorithms that used the amount of ratios of mixed V. cholerae gDNAs (O1 : O139), in comparison to those reactions containing each gDNA template serially diluted. The resulting TMPCR experiments in which CTAB/phenol-chloroform extracted V. cholerae gDNA whether O1, O139, or O22/O155 was amplified are shown in Figure 3. As for Figure 3(a), the TMPCR could detect O serogroup-specific DNA fragments authentically derived from the O1 or the O139 in reciprocal reactions. The detection limit of as low as 1 ng of the O1 was obtained in the presence of the higher amount of O139 or 100-fold greater than the O1. Meanwhile, the detection limit of as low as 10 ng of the O139 was obtained in the presence of the higher amount of the O1 or 10-fold greater than the O139. Figure 3(b) shows that its detection limit was at 100 pg of the O1 or the O139 in inverse reactions containing unequal amount ratios of the 100 pg O1 to 1 ng O139, and vice versa. Again, the TMPCR was likely to show detection limit of as low as 100 pg each of gDNAs in the reaction containing equal amount ratio of the O1 to O139. It was clear to note that the TMPCR had the detection limit of as low as 100 pg gDNA of the O1 or O139 when examined for the equal amount ratio of the serially diluted O1 and O139 templates (Figure 3(c)). Similarly, it also had the same detection limit of as low as 100 pg gDNA of the O1 or O139 when separately copied (Figures 3(d) and 3(e)). The amount of as low as 100 pg O22 or O155 gDNA was also detected (data not shown).

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