Research On A Combined IMS,PAMxx And QPCR Assay For Rapid And Accurate Detection Of Viable But Nonculturable Vibrio Parahaemolyticus

Vibrio parahaemolyticus is a gram-negative and halophilic microorganism naturally present in aquatic environment and is one of the leading causative agents of acute bacterial gastroenteritis associated with consumption of raw or undercooked seafood.The bacterium may enter viable but nonculturable(VBNC)under adverse environmental stress.However,the existing detection methods has defects in missed and false detection of pathogenic bacteria.Therefore,the development of rapid,simple,high sensitive and specific detection protocol is of great significance for prevention and control of VBNC food-borne pathogens.In this study,we systematically investigated the induction mechanism of VBNC V.parahaemolyticus during frozen storage of raw shrimp,and creatively combined the immunomagnetic separation assay(IMS),improved propidium monoazide(improved PMA dye,PMAxx)and real-time quantitative PCR for rapid detection of VBNC V.parahaemolyticus in seafood without any pre-enrichment.The main contents and results are as follows.(1)Induction of V.parahaemolyticus into VBNC state.V.parahaemolyticus ATCC 17802 with an initial concentration of 10~6 CFU/m L was induced by-20°C and 3%Na Cl(frozen storage).By monitoring the number of total cells,viable cells and culturable cells,it was easily found that V.parahaemolyticus entered into the VBNC state completely on the 9st day.And V.arahaemolyticus in VBNC state still remained intact cell membrane and metabolic activity by fluorescence microscopy and flow cytometry,but showed some differences in morphological and physiological characteristics by scanning electron microscopy.The VBNC V.parahaemolyticus provided experimental subjects for subsequent detection method.(2)PMAxx combined qPCR and qLAMP assays for quantitative detection of V.parahaemolyticus in VBNC state were established and compared.DNA dye was combined with qPCR and qLAMP by optimizing the reaction conditions of PMAxx.The optimal concentration and exposure time of PMAxx dye for qPCR method were 16?M and 10 min,while q ALMP was 4?M and 2 min.The combined PMAxx-qPCR method,requiring 100 min,demonstrated a quantification limit of 10.5 colony-forming units(CFU)/m L in pure culture and 28 CFU/g in raw shrimp respectively,which were 10-fold lower than the PMAxx-qLAMP method.When testing mixtures containing different ratios of VBNC to dead V.parahaemolyticus,especially in the case of low VBNC cell concentration ratios,both specificity and sensitivity of PMAxx-based methods were notably superior to PMA-based methods at distinguishing between VBNC and dead bacteria.Therefore,PMAxx dye is a more effective means for achieving the detection and quantification of VBNC V.parahaemolyticus combined with qPCR and qLAMP methods.(3)Biotinylated polyclonal antibody against V.parahaemolyticus was prepared.The V.parahaemolyticus cells treated with 0.4%formaldehyde incubating at 37?for 24 h were used as experimental antigen,and two Japanese healthy female rabbits were immunized to obtain polyclonal antibody against V.parahaemolyticus.By indirect ELISA,SDS-PAGE and Western blot methods,the results indicated that polyclonal antibody against V.parahaemolyticus we prepared had high titer,95%purity and high specificity.(4)Establishment of IMS-PMAxx-qPCR quantitative detection method.The IMBs of V.parahaemolyticus were prepared by conjugated streptavidin magnetic nanobeads with biotinylated V.parahaemolyticus polyclonal antibody,and the coupling and enrichment conditions for VBNC V.parahaemolyticus were optimized.Then based on the specific tlh gene of V.parahaemolyticus,the IMS-PMAxx-qPCR assay was established and the specificity and sensitivity were evaluated for rapid and high efficient detection of VBNC V.parahaemolyticus remaining in seafood.The results showed that the capture efficiency of IMS was more than 88%,the amount of antibody during coupling was 30?g,the optimum amount of immunomagnetic beads was 150?L,the incubation time was 45 min,the magnetic separation time was 4 min and the reaction temperature was room temperature.In addition,IMS-PMAxx-qPCR can only detect V.parahaemolyticus specifically,while other bacteria cannot be detected.IMS-PMAxx-qPCR can detect VBNC V.parahaemolyticus in raw shrimps as low as 1.05 CFU/g without any preenrichment.It has high sensitivity,good selectivity,simplicity and can be accomplished within 5 hours.It can be concluded that the IMS-PMAxx-qPCR quantitative detection method established in this study has some technical advantages of high sensitivity,specificity,simplicity and rapidity compared with the existing methods.