Rapid detection of Salmonella contamination in sea foods using Multiplex PCR

Abstract

Effective monitoring of Salmonella contamination in seafood processing to conform the requirements of HACCP is a great challenge today. Such challenges can be effectively addressed, if the conventional detection methods are replaced with DNA-based molecular methods. Accordingly, it was aimed to develop a robust PCR protocol for specific detection of Salmonella spp. Out of the different primers screened, one pair of primers developed in this study targeting invA gene demonstrated 100% inclusivity for a wide range of Salmonella serotypes and 100% exclusivity for wide range of non-target species. The in silico analysis of the nucleotide sequence obtained from the PCR product suggests its potential as a hybridization probe for genus specific detection of Salmonella spp. contamination. The PCR protocol was sensitive enough to detect 15 cells per reaction using crude DNA prepared within a short time directly from artificially contaminated shrimp tissue. The study demonstrated that the result of PCR reaction can come out on the same day of sample arrival. Incorporation of this pair of primers in a multiplex PCR designed for simultaneous detection of four common seafood-borne human pathogens yielded 147 bp, 302 bp, 403 bp, and 450 bp distinct DNA bands specifically targeting E. coli, toxigenic Vibrio cholerae, Salmonella spp., and V. parahaemolyticus, respectively in a single PCR tube. The PCR methods developed in this study has the potential to be used in the seafood processing plants for effective monitoring of CCPs required for implementation of HACCP-based quality assurance system.