A multiplex polymerase chain reaction (MPCR)-based assay was developed for the simultaneous detection of Vibrios using the genus-specific RNA polymerase subunit A (rpoA) gene and specific detection of toxin-producing Vibrio cholerae strains using two sets of primer based on cholera toxin subunit A (ctxA) and repeat in toxin subunit A (RtxA)-producing genes. The MPCR method developed is applicable to both the simultaneous and the two-step detection of genus Vibrio total and toxigenic V. cholerae species. This assay was specific as no amplification occurred with the other bacterial pathogens tested. The sensitivity of the assay was tested by artificially spiking the shrimp homogenate with the toxigenic strain of V. cholerae (NICED 16582) in different dilutions. The developed MPCR assay could detect three cells of V. cholerae in 12 h pre-enrichment in APW. The proposed method is rapid, sensitive, and specific for the detection of Vibrio genus as well as toxin-producing V. cholerae strains in environmental samples.
"Most Aeromonas hemolysins described to date are related to one of these two toxins and are reasonable predictors of human diarrhoeal disease in both the A. hydrophila and A. sobria (Heuzenroeder et al. 1999). There are several conventional as well as rapid systems including PCR that have been tried for the detection of seafood borne pathogens (Kumar et al. 2006; Jeyasekaran et al. 2011; Raj et al. 2011). Although Aeromonas spp. "
[Show abstract][Hide abstract] ABSTRACT: Hemolytic strains of Aeromonas spp. from fish and fishery products were detected by multiplex PCR. The selected primers for the amplification of segments of ahh1, asa1 and 16S rRNA gene yielded products with the size of 130 bp, 249 bp and 356 bp, respectively. This assay was found to be highly sensitive, as it could detect 7 and 9 cells of Aeromonas hydrophila and A. sobria with a detection limit of 1 pg of pure genomic DNA. The assay, when screened for 73 commercial fish and fishery product samples consisting of freshwater, marine fish and shellfish, showed 56 % positive for Aeromonas spp., 16 % for Aeromonas hydrophila and 13 % for A. sobria. This assay provides specific and reliable results and can be a powerful tool for the simultaneous detection of hemolytic strains of A. hydrophila A. sobria and other Aeromonas spp. from fish and fishery products.
Journal of Food Science and Technology -Mysore- 02/2014; 51(2). DOI:10.1007/s13197-013-1190-9 · 2.20 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Vibrio
cholerae and V. vulnificus are of major concern due to their effect on public health throughout the world. It is therefore imperative to identify a gene and method that are suitable for the accurate species-specific detection of these two species. A duplex polymerase chain reaction (PCR) assay was developed using two sets of primers targeting the groEL gene for the accurate simultaneous detection of V. cholerae and V. vulnificus. The nucleotide sequence of the groEL gene was compared with the sequences of other Vibrio and non-Vibrio species. The specificity of two primer sets for duplex PCR was checked using 24 Vibrio and 8 non-Vibrio species. The primer sets were found to be specific for these two species and could detect both of the target bacterial species without any ambiguity, even when comparing closely related species. For both species, the detection limit was 100 pg of purified genomic DNA. The duplex PCR showed high specificity and sensitivity for each species and was sufficient for the detection of V. cholerae and V. vulnificus from artificially infected shellfish tissue, flounder, and even inoculated seawater. This method is simple and cost-effective, and can be utilized for the simultaneous detection of both species, thus representing an effective tool for both epidemiologist and ecologist.
[Show abstract][Hide abstract] ABSTRACT: Aims:
To develop an effective multiplex polymerase chain reaction (PCR) for the simultaneous detection of three important Vibrio species, Vibrio cholerae (Vc), V. parahaemolyticus (Vp) and V. vulnificus (Vv) using the groEL gene, a potential phylogenetic marker.
Methods and results:
Three species-specific primer sets were designed to target Vc, Vp and Vv. A total of 131 Vibrio and non-Vibrio strains were used to determine the specificity and sensitivity of primers. The primers produced specific PCR fragments from all target species strains and did not cross-react with other Vibrio and non-Vibrio species. This PCR method showed good efficiency in detecting coexisting target species in the same sample with a detection limit of 100 pg of Vc, Vp and Vv from mixed purified DNA. Detection of three target species was also possible from artificially inoculated shellfish, flounder and sea water.
The groEL gene is a potential marker for accurate simultaneous detection of Vc, Vp and Vv and could be used to detect these species in environmental and clinical samples.
Significance and impact of the study:
This newly developed multiplex PCR is a useful and cost-effective method that is applicable in a disease-outbreak prediction system and may provide an effective tool for both the epidemiologist and ecologist.
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