Establishment Of Duplex Real-time PCR For Detection Of Serratia Fonticola And Salmonella Enterica In Imported Feed

Serratia fonticola is an unusual gram-negative bacterium that described as a new specie of Serratia in 1979,which distributed extensively in the environment,including drinking water,soil and sewage.The bacterium has long been considered harmless to humans,but because of its aggressive and resistant to many commonly used antibiotics,it has become an important pathogen.Serratia fonticola infection can lead to a large range of physical illness as a significant human pathogen,including respiratory tract infections,wound infections,septic arthritis,urinary tract infections,bloodstream infections,skin and soft tissue infections,and diarrhea.Salmonella enterica is a high-risk monitoring substance for the safety risk monitoring of imported animal-derived feed(fishmeal,meat powder,bone meal,meat and bone meal,feeding blood product,compound feed,etc.),which can cause a variety of animal infections and often cause human food poisoning and other groups of explosive events.Salmonella enterica including six different subspecies:Salmonella enterica subsp.enterica,Salmonella enterica subsp.salamae,Salmonella enterica subsp.arizonae,Salmonella enterica subsp.diarizonae,Salmonella enterica subsp.houtenae,Salmonella enterica subsp.indica,because of its pantropism,with a wide range of infected hosts,can cause people and a variety of animal Salmonella diseases,has important public health significance.During the process of daily quarantine of Salmonella in the imported feed stuffs,Serratia fonticola is the main interferon.The quantitative real-time PCR assays were developed for the detection of Salmonella enterica and Serratia fonticola respectively,which a species-specific primer pair and probe were designed for the quantitative real-time PCR detection of Serratia fonticola using gyrB gene as the target gene and a pair of primers and probe were designed for the quantitative real-time PCR detection of Salmonella enterica using invA gene as the target gene.In this study,we established a duplex real-time PCR method for the rapid diagnosis of Serratia fonticola and Salmonella enterica,which could decrease the following biochemical and serological identification steps to improve in the detection efficiency of Salmonella in imported feed.Based on the construction of positive recombinant plasmids,a simplex real-time quantitative PCR method was developed for the detection of Serratia fonticola and Salmonella enterica,and we also evaluated the two simplex real-time quantitative PCR methods,including specificity,sensitivity and reproducibility.The outcomes of specificity assay for detecting Serratia fonticola showed that the real-time quantitative PCR method was suitable for the detection of Serratia fonticola only,and the other eight kinds of Serratia species,ten Salmonella species and seven other kinds of Enterobacteria including Escherichia coli were negative.The outcomes of specificity test for detecting Salmonella enterica showed that the real-time quantitative PCR method was suitable for the detection of Salmonella enterica,and all other non-Salmonella strains were negative.The sensitivity of the simplex real-time quantitative PCR method for the detection of Serratia fonticola and Salmonella enterica were 145 copies/?L and 197 copies/?L,respectively.and the correlation coefficients of the standard curve was 0.999 and 0.998,respectively,The results of reproducibility test for the detection of Serratia fonticola and Salmonella enterica demonstrated that interclass coefficient of variation were no more than 0.8%and 0.6%,repectively.And then a duplex real-time quantitative PCR assay was developed on the foundation of real-time quantitative PCR assays detected Serratia fonticola and Salmonella enterica,which make it possible for simultaneous detection of the two pathogens.The detection limit of the established duplex real-time quantitative PCR method was determined by making a standard curve.The results showed that the slope of the two standard curves and the intercept on the Y-axis compared with the simplex real-time quantitative PCR method were very close,and the amplification efficiency does not change apparently.It can be seen that the simultaneous detection of two pathogens does not occur crossover phenomenon,the detection method we have established can be used for daily quarantine work.This study is of great theoretical and practical value for the effective prevention of exogenous pathogens and the effective quarantine treatment.