Rapid detection of Salmonella contamination in seafoods 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 DNAbased 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.

Early Detection of Contamination Vibrio parahaemolyticus and Escherichia coli in Fisheries Product Using Multiplex Polymerase Chain Reaction

“…Kontaminan bakteri patogen banyak ditemukan pada produk perikanan mentah dan produk olahan (Sahu et al, 2019). Penyakit kontaminasi bakteri pada makanan dan air minum di Uni Eropa tahun 2015 mencapai 4,362 kasus yang disebabkan oleh bakteri Vibrio parahaemolyticus dan Escherichia coli (Bintsis, 2017).…”

“…Pengembangan metode PCR terus dilakukan, salah satunya adalah metode multiplex-PCR mPCRyang memungkinkan pende teksian dua atau lebih jenis gen target dalam satu waktu pengujian (Jiang et al, 2017). Metode mPCR memberikan hasil cepat dan akurat dibandingkan dengan metode konvensional PCR dalam kegiatan pemeriksaan makanan produk perikanan (Sahu et al, 2019).…”

“…Stok bakteri dilakukan ekstraksi DNA dengan mengacu pada metode dari (Sahu et al, 2019) dengan sedikit modifikasi, yaitu 500 µL stok bakteri dimasukkan pada microtube 1,5 mL dan disentrifugasi selama 5 menit pada kecepatan 15.000 × rpm menggunakan alat sentrifugasi dingin (Cleaver Scientific Ltd., UK). Kemudian supernatan dibuang dan ditambahkan 200 µL Nuclease-free water (NFW; Qiagen, Germany) pada microtube dan dilakukan vortex.…”

Capture and identification of bacteria from fish muscle based on immunomagnetic beads and MALDI-TOF MS

“…Three foodborne pathogens in seafood were simultaneously detected by multiplex RPA assay ( Ma et al 2020 ). Multiplex polymerase chain reaction (PCR) was used to detect Salmonella contamination in seafoods ( Sahu et al 2019 ). Shiga-toxin-producing Escherichia coli (STEC) in shellfish was detected by real-time PCR ( Balière et al 2015 ).…”

Improvement of a new selective enrichment broth for culturing Salmonella in ready‐to‐eat fruits and vegetables

“…Most research has been focused on examining the presence of pathogens in enrichment samples by antibody‐, nucleic acid‐, and biosensor‐based methods (Cheng et al, 2019; Choi, Lee, Song, Jang, & Park, 2019; Hu et al, 2018; Sahu et al, 2019); however, little attention has been paid to the development of an enrichment procedure. Important considerations have to be taken for the successful recovery of low levels of bacteria in samples.…”