DEVELOPMENT OF MULTIPLEX PCR ASSAY FOR THE DETECTION OF PATHOGENIC STRAINS OF AEROMONAS SPP. FROM FISH AND FISHERY PRODUCTS
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Date
2012
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Fisheries College and Research Institute, Thoothukudi, Tamil Nadu Fisheries University
Abstract
Three multiplex PCR assays for the detection of pathogenic strains of Aeromonas spp. using different toxin genes have been developed and the sensitivity of the developed assays was studied. To validate the developed multiplex PCR assays, 138 samples of fresh, frozen, dried and fermented fish and shellfishes were screened for the incidence of Aeromonas spp. Standard type cultures of A. hydrophila (ATCC 7966), A. sobria (MTCC 1608), A. liquefaciens (MTCC 2654), A. caviae (MTCC 7725) was used for developing and optimizing the MPCR assays. The specificity of the developed MPCR assays were tested by performing the PCR reaction with non-Aeromonas organisms like Salmonella typhi (ATCC 122235), Salmonella paratyphi A (MTCC 735), Vibrio cholerae (NICED 16582), V. parahaemolyticus, V. vulnificus and V. alginolyticus. The first assay was developed to simultaneously detect hemolytic strains of A. hydrophila and A. sobria along with other Aeromonas spp. from fish and fishery products. The primers used in this assay were A16S, ASA1 and AHH1. The assay was specific, as it amplified only the target genes in the respective Aeromonas spp. This assay was so sensitive that it detected 1 pg of genomic DNA and 7 and 9 cells of A. hydrophila and A. sobria after 12 h of enrichment. The validation of the MPCR assay showed that 71 of 138 samples of commercial samples of fresh, frozen, dried, and fermented fish and shellfish were positive for Aeromonas spp. with an incidence of A. hydrophila and A. sobria at a level of 17% and 11%, respectively. The second assay was developed to simultaneously detect A. jandaei, A. sobria and A. hydrophila from fish and fishery products. The primers used in this assay were Ajan, ASA1 and AHH1. This assay was also specific and sensitive as it detected 11, 9 cells and 8 cells of A. jandaei, A. sobria and A. hydrophila and 100 pg of genomic DNA in 12 h of enrichment. On validation, it showed that 11 were A. jandaei, 17 were A. sobria and 21 were A. hydrophila from 71 isolates examined from 138 samples. The third assay was developed to detect the hemolytic and enterotoxigenic strains of Aeromonas spp. from fish and fishery products. The primers used in this assay were A16S1, AHS, act/hlyA/aer complex and ALT. This assay was also specific and sensitive as it detected 6 cells of A. hydrophila and 100 pg for genomic DNA in 8 h of enrichment. This MPCR assay was validated by examining 71 Aeromonas spp. isolates including 23 A. hydrophila, 17 A. sobria, 8 A. caviae, 11 A. jandaei, 6 A. trota, 4 A. schubertii and 2 A. veronii. It also showed the presence of one or more genes either individually or in combination in the isolates tested. The four gene pattern was observed in the isolates such as alt; act/hlyA/aer; alt and act/hlyA/aer; ast, alt and act/hlyA/aer. The results showed that the prevalence of pathogenic Aeromonas spp. in fish and fishery products was 44%. As the conventional method for isolation of Aeromonas spp. is a laborious process and further biochemical confirmation lacks specificity, MPCR assays could be a powerful tool for the rapid, reliable and simultaneous detection of different strains of Aeromonas spp. from fish and fishery product samples with more specificity and sensitivity.
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