Study On The Rapid Detection Technology Of Two Kinds Of Foodborne Pathogens In Aquatic Products

Vibrio parahaemolyticus(Vp)and Grimontia hollisae(Gh)are two kinds of foodborne pathogens existed in aquatic products,which both can produce thermostable direct hemolysin.In most cases,infection causes diarrhea,nausea,vomiting and other symptoms of gastroenteritis.Up to now,the existing national standard for detecting V.parahaemolyticus and G.hollisae was based on the culture method,and it has the disadvantages of complex operations,poor repeatability and time-consuming process.In this study,combining with immunological and molecular biology techniques,the rapid detection technologies for two foodborn pathogens in aquatic products were developed,which was great significance to improving the efficiency of food safety monitoring and ensuring food safety.In this study,having taken V.parahaemolyticus and G.hollisae as the research objects,the detecting technologies were developed including immunomagnetic separation,multiple PCR,real-time quantitative PCR,LAMP,and digital PCR.The main results are as follows:1.Four pairs of primers were designed based on toxR,tdh,trh and tlh genes sequences of V.parahaemolyticus.Optimized the four pairs of primers concentration and annealing temperature to obtain the best ratio of primer and amplification conditions,the rapid quadruple PCR detection of pathogenic V.parahaemolyticus was established.The detection limits of the multiplex PCR for toxR,tdh,trh and tlh were each 50 pg DNA/?L.Detection sensitivity of V.parahaemolyticus in pure culture condition was 6.7×10~3 CFU/mL.Four targeted fragments were detected in artificially contaminated seafood with 1.36 CFU/g V.parahaemolyticus after 6 h enrichment.The multiplex-PCR method can simultaneously detect V.parahaemolyticus with toxR,tdh,trh and/or tlh.It is of great significance to carry out the detection of pathogenic V.parahaemolyticus and virulence genes.2.The preparation of Vp-immunomagnetic beads was optimized,and a Taqman triplex realtime PCR was developed for detecting toxR,tdh and tlh of V.parahaemolyticus.There was a good linear relationship between in 10~3~10~8 CFU/mL.Detection sensitivity of toxR,tlh and tdh in artificial seafood by IMS-realtime mPCR were 1.6×10~3,1.6×10~4,1.6×10~3 CFU/mL,respectively.3.A duplex ddPCR system was established for detecting toxR and tdh gene of V.parahaemolyticus.The optimalizing PMA treatment,10?M and exposure for 15 min,was evaluated by realtime PCR.Combining PMA whith ddPCR,the method achieved the quantification for viable V.parahaemolyticus cell.There was a good linear relationship between copies concentration and 10~2~10~6 CFU/mL strain concentration.This method can provide an important foundation to achieve absolute quantification of viable V.parahaemolyticus.4.The preparation of Gh-immunomagnetic beads was optimized.Then,fur specific primers of G.hollisae was designed for establishing successfully LAMP assay.The reaction is completed within 40 min,and the detection limits for G.hollisae in artificial seafood was each 1.10×10~4CFU/mL by IMS-LAMP,which was less one magnitude than centrifugation-LAMP.5.Three pairs of primers were designed for toxR species-specific genes of V.parahaemolyticus,recA species-specific gene of G.hollisae and a high homology virulence gene(tdh).After optimizing reaction system,the detection limits of the multiplex PCR for both bacteria genomic DNA were each 0.5 pg DNA/?L,and for both bacteria solution were 10~3 CFU/mL.