Multiplex polymerase chain reaction-based assay for the specific detection of toxin-producing Vibrio cholerae in fish and fishery products

Jeyasekaran, G.; Raj, K. Thirumalai; Shakila, R. Jeya; Thangarani, A. Jemila; Sukumar, D.

doi:10.1007/s00253-011-3175-9

Cited by 14 publications

(11 citation statements)

References 39 publications

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“…Enrichment procedures can minimize the interference of the PCR inhibitors and increase the concentration of the target micro-organisms. Enrichment for 5-12 h was applied during the detection of Vibrio species from fish, fishery products, shellfish and water (Hossain et al 2011;Jeyasekaran et al 2011;Malayil et al 2011;Wong et al 2012). In this study, we extracted DNA from inoculated shellfish homogenates and sea water before and after enrichment for 5 h. Detection was possible in sea water samples before and after enrichment, but weak amplicons were observed in shellfish samples before enrichment.…”

Section: Discussionmentioning

confidence: 99%

Development of agroELgene-based species-specific multiplex polymerase chain reaction assay for simultaneous detection ofVibrio cholerae,Vibrio parahaemolyticusandVibrio vulnificus

Hossain

Kim

et al. 2012

J Appl Microbiol

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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. Conclusions: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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Section: Discussionmentioning

confidence: 99%

Development of agroELgene-based species-specific multiplex polymerase chain reaction assay for simultaneous detection ofVibrio cholerae,Vibrio parahaemolyticusandVibrio vulnificus

Hossain

Kim

et al. 2012

J Appl Microbiol

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“…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.…”

Section: Introductionmentioning

confidence: 99%

Detection of hemolytic strains of Aeromonas hydrophila and A. sobria along with other Aeromonas spp. from fish and fishery products by multiplex PCR

et al. 2013

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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.

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“…All 209 isolates tested positive for rpoA gene yielding a 242-bp fragment (Figure 2). The rpoA gene detection is considered a specific, sensitive and fast method for Vibrio identification (Thompson et al, 2005;Dalmasso et al, 2009;Jeyasekaran et al, 2011).…”

Section: Resultsmentioning

confidence: 99%

Detection of virulence and antibiotic resistance genes in environmental strains of Vibrio spp. from mussels along the coast of Rio de Janeiro State, Brazil

Oliva¹,

Bronzato²,

Soares³

et al. 2016

Afr. J. Microbiol. Res.

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Mussels have a filter system enabling them to take up nutrients from the water, so a microbiological analysis of these bivalve mollusks can show the contamination levels of their surrounding aquatic environment. The present work aimed to isolate Vibrio species from two hundred samples of mussels (Perna perna) incrusted on rocks of the Santana Archipelago and from longline mariculture in Ilha Grande Bay in Angra dos Reis and from Arraial do Cabo, all of which are in Rio de Janeiro state, Brazil. A total of 209 Vibrio were isolated. The most prevalent species was Vibrio parahaemolyticus (44.66%) followed by Vibrio alginolyticus (19.62%) and Vibrio vulnificus (12.44%). All 209 Vibrio isolates tested positive for the RNA polymerase alpha gene (rpoA). The tlh gene (thermolabile hemolysin), a genetic marker for V. parahaemolyticus, and vvhA (cytolysin hemolysin) of V. vulnificus were detected in 85 and 26 isolates, respectively. The MALDI-TOF MS proteomic technique was used to confirm the identification of the 41 V. alginolyticus isolates. Our most important finding was the detection of the tdh virulence gene in 68.20% (58/85) of V. parahaemolyticus environmental strains. Besides the circulation of the virulence gene, the spread of antimicrobial resistance was evaluated and 91.3% (191/209) of the isolates showed resistance to ampicillin, 23.9% (50/209) to ciprofloxacin, 18.6% (39/209) to nitrofurantoin, 5.7% (12/209) to tetracycline, 4.3% (9/209) to pefloxacin and 3.3% (7/209) to chloramphenicol. These findings indicate that environmental isolates can act as reservoirs of virulence and antibiotic resistance genes.

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Multiplex polymerase chain reaction-based assay for the specific detection of toxin-producing Vibrio cholerae in fish and fishery products

Cited by 14 publications

References 39 publications

Development of agroELgene-based species-specific multiplex polymerase chain reaction assay for simultaneous detection ofVibrio cholerae,Vibrio parahaemolyticusandVibrio vulnificus

Development of agroELgene-based species-specific multiplex polymerase chain reaction assay for simultaneous detection ofVibrio cholerae,Vibrio parahaemolyticusandVibrio vulnificus

Detection of hemolytic strains of Aeromonas hydrophila and A. sobria along with other Aeromonas spp. from fish and fishery products by multiplex PCR

Detection of virulence and antibiotic resistance genes in environmental strains of Vibrio spp. from mussels along the coast of Rio de Janeiro State, Brazil

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