Multiplex polymerase chain reaction-based assay for the specific detection of toxin-producing Vibrio cholerae in fish and fishery products.
ABSTRACT 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.
- SourceAvailable from: Eun-Young Kim[Show abstract] [Hide abstract]
ABSTRACT: Vibriosis caused by Vibrio anguillarum and edwardsiellosis caused by Edwardsiella tarda are septicemic diseases of many commercially important freshwater and marine fishes, and threaten the aquaculture industry in Korea. Early diagnosis and accurate identification of these two bacterial species could help to prevent these diseases and minimize the damage to cultured marine species. This study designed a duplex polymerase chain reaction (PCR) method for the simultaneous detection of two major fish pathogens: V. anguillarum and E. tarda. Each pair of oligonucleotide primers exclusively amplified the target groEL gene of the specific microorganism. Twenty-two Vibrio and ten non-Vibrio enteric species were used to check the specificity of the primers, which were found to be highly specific for the target species, even among closely related species. The detection limit was 400 pg for V. anguillarum and 4 ng for E. tarda when mixed purified DNA was used as the template. This assay showed high specificity and sensitivity in the simultaneous detection of V. anguillarum and E. tarda from artificially inoculated seawater and fish.Fisheries and aquatic sciences. 01/2013; 16(4).
- [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; · 2.02 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.Fisheries Science 79(2). · 0.90 Impact Factor