Establishment And Application Of IMS-RPA-LFD Method For Detecting Vibrio Parahaemolyticus

Vibrio parahaemolyticus which can cause human food poisoning by infecting seafood has been found to be responsible for some foodborne bacterial infections.Detection of V.parahaemolyticus in seafood products and brine is of great significance for reducing V.parahaemolyticus infection.Conventional culture-based approach contains complex procedure,including liquid enrichment,selective isolation,biochemical identification and PCR,so this approach is time-consuming,low sensitive and labour-intensive.To reduce the possibility of V.parahaemolyticus infection,a more rapid and sensitive method of Vparahaemolyticus detection is required.In the present study,a cost-effective method named IMS-RPA-LFD for the detection of pathogenic V.parahaemolyticus was established.This method consists of immunomagnetic separation,recombinant enzyme polymerase amplification and laminar flow chromatography.Vparahaemolyticus was enriched into a smaller sample volume by using IMS and then RPA-LFD was used to detect target DNA.The developed method is relatively simple and has high sensitivity,at the same time,it can save a lot of time in bacterial enrichment and detection.1 Rapid enrichment of Vparahaemolyticus using immunomagnetic separationTo establish a rapid enrichment method for Vparahaemolyticus,the immunomagnetic beads were prepared from monoclonal antibody 1-1-D12 and superparamagnetic particles named PM3-050 which has a particle size of 750 nm.This process requires the assistance of corresponding chemical reagents.Immunomagnetic beads prepared with the assistance of relevant chemical reagents were used in the next experiment.The experiment included optimization of antibody coupling amount,coupling time,incubation time,etc,and then the method was evaluated by sensitivity and specificity.The results showed that the optimal antibody dose of the immunomagnetic beads was 300 μg/mg.At the optimal conditions of IMS,the detection line was as low as 70CFU/mL,the maximum number of captured bacteria was 9.95×104 CFU/mg,and the capture time was only 45 min.The specific results showed that except for the capture efficiency of V.parahaemolyticus exceeding 60%,the capture efficiency of other bacteria was less than 10%.This experiment showed that the newly established immunomagnetic separation technology of Vparahaemolyticus can effectively separate the target bacteria,and the enrichment time is short,and the sensitivity and the specificity is high,so it is suitable for field investigation and clinical examination.2 Recombinase polymerase amplification combined with lateral flow strip for V.parahaemolyticus detectionA new technology for the rapid detection of Vparahaemolyticus was established,which consists of recombinase polymerase amplification（RPA）and lateral flow dipstick（LFD）.Primers were designed according to the specific gene（Genbank:NC₀04605.1）of Vparahaemolyticus,and then the primers were simply screened by RPA Basic kit,after which the probes were designed according to the primers and the RPA-LFD method was established.The experiment included optimization of the reaction temperature,reaction time,and etc,and then the method was evaluated by sensitivity and specificity.Finally,the method was combined with IMS to detect Vparahaemolyticus in the mussel model.The results showed that the total reaction time in this method was 25 min,and the optimal reaction temperature was 37℃.The detection limit of this method was lOpg/μL and the method can specifically detect Vparahaemolyticus,which proves that the method is sensitive and specific,so it is suitable for rapid detection of V parahaemolyticus.3 Rapid detection of Vparahaemolyticus in raw mussel using immunomagnetic separation combined with RPA-LFDThe purpose of this study was to establish an IMS-RPA-LFD method for rapid enrichment and detection of Vparahaemolyticus,which combines IMS with RPA-LFD.In this experiment,a serially diluted Vparahaemolyticus solution was added to the muscle sample to prepare a simulated sample,and the immunomagnetic beads were used to capture the samples at optimal conditions.After enrichment,the DNA extract from the bacteria was used to RPA-LFD,and IMS-RPA-LFD was used to compared with this method.The results showed that the newly established IMS-RPA-LFD method could effectively enrich and separate Vparahaemolyticus,with rapid reaction,high sensitivity and excellent specificity.For low concentrations（2.0×10-1 to 2.0×102CFU/mL）of Vparahaemolyticus samples,IMS-RPA-LFD could also detect Vparahaemolyticus within 5 to 6 h,which proves that this method is suitable for field investigation and clinical examination.Compared with IMS-PCR-AGE,IMS-RPA-LFD took shorter time and was faster than the traditional detection procedure.Therefore,IMS-RPA-LFD is suitable for the detection of Vparahaemolyticus in seawater and seafood.