Establishment Of Rapid High-throughput Detection Methods For Five Pathogenic Vibrio

Vibrio,a type of halophilic bacterium,is Gram-negative and widely distributed in estuaries,seawater and marine animals.Pathogenic Vibrio mainly infects human through seafood,seawater,etc.It can cause infectious diarrheal disease,ear and wound infection and sepsis.With the improvement of the quality of life,the demand of people for seafood is increasing,and Vibrio has become one of the important pathogens of foodbome disease in China.To be able to quickly and efficiently detect pathogenic Vibrio in sample,in this study,immunomagnetic separation(IMS)was combined with multiplex PCR and Luminex xMAP to establish a IMS and multiplex PCR and Luminex xMAP detection method,which enrich five common pathogenic Vibrio,Vibrio parahaemolyticus,Vibrio cholerae,Vibrio vulnificus,Vibrio mimicus and Vibrio alginolyticus,simultaneously.This method has the advantages of fast,simple,high throughput and high sensitivity,and provides technical support for on-site detection of Vibrio.1.Rapid enrichment of five pathogenic Vibrio by immunomagnetic separationTo develope a rapid enrichment technique for immunomagnetic separation of five Vibrio species includng Vibrio cholerae,Vibrio parahaemolyticus,Vibrio vulnificus,Vibrio alginolyticus and Vibrio mimicus,one monoclonal antibody 2-1-D4 specific to the common epitope for the above five Vibrio was used to couple with magnetic beads to generate immunomagnetic beads.Briefly,the immunomagnetic beads were prepared by EDC/NHS chemical coupling method,and the preparation of immunomagnetic beads was optimized according to the actual capture efficiency.The dosage and capture time of the immunomagnetic beads during the enrichment process were also optimized.Moreover,the specificity,sensitivity and the capture efficiency of immunomagnetic beads in food samples were evaluated.The results showed that the optimal dosage of immunomagnetic beads in the 1mL system was 0.33 mg,and the optimal capture time was 60 min.The efficacy of this method for capturing the five Vibrio was more than 50%and it had no cross-reaction with common food-borne pathogens such as Salmonella,Escherichia coli and other pathogenic bacteria in the same genus during the test,highlighting its specificity.Therefore,as an important step for enriching targets,immunomagnetic bead can not only increase the sensitivity and specificity of the traditional medium based detection,but also reduce the error from the operation and improve the accuracy of the detection.2.Rapid detection of of five pathogenic Vibrio in aquatic products by multiplex PCRIn this study,multiplex PCR detection method for five pathogenic Vibrio were established by screening primers.The primers were designed based on the species-specific gene of five pathogenic Vibrio species,such as toxR gene of Vibrio parahaemolyticus,ompWgene of Vibrio cholerae,vvhA gene of Vibrio vulnificus,vm-toxR gene of Vibrio mimicus and va-coL gene of Vibrio alginolyticus.At the same time,IAC primer designed by I6S rRNA fragment was set as an internal amplification control(IAC).The annealing temperature and primer concentration in the multiplex PCR reaction were optimized.Data showed that the optimal annealing temperature was 57? and the optimal primer concentration was 0.1 ?mol/L.The limit of detection of Multiplex PCR was 1.8×105 CFU/mL.Moreover,the method has no cross-reaction with common food-borne pathogens such as Salmonella,Escherichia coli and other pathogenic bacteria in the same genus during the test.The existence of ICA excludes the false negatives that may occur in the detection.All these demonstrate that the simple and convenient Multiplex PCR detection method has excellent specificity,sensitivity and accuracy,laying the fundation for efficiently detecting and preventing the infection of aquatic pathogenic Vibrio.3.Rapid detection of of five pathogenic Vibrio in aquatic products by Multiplex Luminex xMAPIn this study,a Multiplex Luminex xMAP detection technique for simultaneous detection of five pathogenic Vibrio was established and was evaluated.The five Vibrio species were Vibrio parahaemolyticus,Vibrio cholerae,Vibrio vulnificus,Vibrio mimetic and Vibrio alginolyticus.Specific primers for biotin-labeled specific genes and virulence genes for these pathogenic Vibrio species were designed separately.The amino-labeled probe is coupled to a carboxyl microsphere that is used to hybridize the biotinylated PCR product.The Luminex 200 analysis system is capable of analyzing the fluorescence classification of encoded microspheres and the medium-strong fluorescence values of phycoerythrin.The hybridization time and hybridization temperature were optimized to determine that the optimal hybridization temperature for the reaction was 52? and the optimal hybridization time was 25 min.The multi-batch test could accurately identify 5 single strain samples and mixed strain samples without cross-reaction.The intra-assay coefficient of variation(CV%)was 2.71%-7.68%,indicating that the stability of the method are excellent.The sensitivity of the method was identified.The results showed that the sensitivity of the ten sets of probes was between 1.8×102 CFU/mL and 1.8×104 CFU/mL.The efficient method established in this study has the advantages of high sensitivity,excellent repeatability and controllability and time-saving,highlighting its excellent application prospect for the detection of the pathogens.4.Rapid detection of five pathogenic Vibrio in simulated samples using Multiplex IMS-Luminex xMAP and comparison with Multiplex IMS-PCRIn this experiment,five pathogenic Vibrio in simulated sample were captured by immunomagnetic beads under the optimal conditions,the DNA which was extracted from the captured sample was detected by Multiple PCR and Multiplex Luminex xMAP respectively.The results showed,when the concentration of five pathogenic Vibrio in the sample was 1.8 CFU/g,IMS-Multiple Luminex xMAP,but not IMS-multiplex PCR,could efficiently detect five Vibrio in 6-7 h;When the concentration of five pathogenic Vibrio in aquatic products was 1.8×103 CFU/g,IMS-multiplex PCR detection method could detect five Vibrio within 10 hours.All these demonstrate that the combination of IMS and Multiplex Luminex xMAP reduces the time and increases the sensitivity for detection of Vibrio,highlighting its high-throughput and high-sensitivity for detecting the five pathogenic Vibrio.