Establishment and Application of a duplex real-time qPCR method for detection ofSalmonellaspp. andSerratia fonticolain imported feedstuffs

Abstract

AbstractSalmonellaspp. is a high-risk bacterial pathogen that is monitored in imported animal-derived feedstuffs.Serratia fonticolais the bacterial species most frequently confused withSalmonellaspp. in traditional identification methods based on biochemical characteristics, which are time-consuming and labor-intensive, and thus unsuitable for daily inspection and quarantine work. In this study, we established a duplex real-time qPCR method withinvA-andgyrB-specific primers and probes corresponding toSalmonellaspp. andS. fonticola. The method could simultaneously detect both pathogens in imported feedstuffs, with a minimum limit of detection forSalmonellaspp. andS. fonticolaof 197 copies/μL and 145 copies/μL, respectively (correlation coefficient R2= 0.999 in both cases). The amplification efficiency forSalmonellaspp. andS. fonticolawas 98.346% and 96.49%, respectively. Detection of clinical samples was consistent with method GB/T 13091-2002, and all 20 artificially contaminated imported feed samples were positively identified. Thus, the developed duplex real-time qPCR assay displays high specificity and sensitivity, and can be used for the rapid and accurate detection of genomic DNA fromSalmonellaspp. andS. fonticolawithin hours. This represents a significant improvement in the efficiency of detection of both pathogens in imported feedstuffs.ImportanceImported feedstuffs must be tested for pathogenicSalmonellaspecies that represent a biological hazard. Variousnon-Salmonellacolony-forming species belong toEnterobacteriaceae,andSerratia fonticolaforms colonies of similar color and morphology toSalmonellaspp., leading to confusion in daily quarantine tests. Traditional methods based on biochemical and serological characteristics are cumbersome and labor-intensive, and unable to fully support current quarantine testing demands. Thus, there is an urgent need to develop a rapid and accurate method for the effective identification of these pathogens. The duplex real-time qPCR method established herein can rapidly identifySalmonellaspp. andS. fonticola, and has great potential for application to feed safety and prevention of exterior pathogens.