Study On Multiplex Recombinase Polymerase Amplification Method For Rapid Detection Of Foodborne Pathogens

Food is the material basis for human survival and development.Food safety is a major issue related to people’s health and national economy and people’s livelihood.At present,various food safety problems are emerging,especially the pollution of food-borne pathogens,which has caused great harm to human health.Worldwide,the three most common prevalent foodborne pathogens were Staphylococcus aureus,Vibrio parahaemolyticus and Salmonella.Since these three pathogens might coexist in one food sample with relatively low concentration it was crucial to development a rapid,accurate,highly sensitive and specific detection method for detection simultaneously on multi-objective pathogens.With the development of biotechnology,in addition to traditional culture methods,many novel techniques have been applied to the detection of pathogenic bacteria.Among them,the detection techniques of Nucleic acid amplification technology have the advantages of rapid amplification,high sensitivity and strong specificity.It is an important technical means for the detection of foodborne pathogens.However,traditional nucleic acid amplification techniques such as polymerase chain reaction(PCR)usually require complicated instruments and professional training,and take a relatively long time,which is not suitable for rapid on-site detection.In this study,RPA technology was applied to multiple nucleic acid detection technology,and combined with lateral flow dipstick to establish a multiplex LFD-RPA detection assay to achieve multi-target simultaneous rapid detection of foodborne pathogens.Based on the nuc gene(Gen Bank accession:EF529607.1)of Staphylococcus aureus,tox R gene(Gen Bank accession:GQ228073.1)of Vibrio parahaemolyticus and fim Y gene(Gen Bank accession:JQ665438.1)of Salmonella,three sets of RPA primers were designed by using software(Primer Premier 5.0).Through the preliminary experiment,it was determined that the primers with target-specific labels were tagged with biotin-and digoxin-(primers for nuc gene),FAM-and digoxin-(primers for tox R gene)or Cy5-and digoxin-(primers for fim Y gene).After a series of optimization experiments,the final multiple RPA reaction system was determined as follows:25μL 2x reaction buffer(with d NTPs),2μL of each Vibrio parahaemolyticus and Salmonella specific primer(10μM),0.75μL of each Staphylococcus aureus specific primer(10μM),10μL enzymes,1μL of each template,and 2.5μL Magnesium acetate.The optimal reaction temperature was 37℃,and the optimal reaction time was 10 min.Specific experimental results showed that only Staphylococcus aureus,Vibrio parahaemolyticus and Salmonella strains had positive results,while other strains had negative results.The specificity results also indicated that there was no cross-reactivity among nuc gene for Staphylococcus aureus,tox R gene for Vibrio parahaemolyticus and fim Y gene for Salmonella.The results of sensitivity experiments showed that the visual detection limit of multiplex LFD-RPA for simultaneously detection of Staphylococcus aureus,Vibrio parahaemolyticus and Salmonella was 2.6×10~1CFU/m L,7.6×10~1CFU/m L,and1.29×10~1CFU/m L,respectively.Moreover,the standard linear curve plotted according to the data of test strip reader have correlation coefficients of determination for Staphylococcus aureus(R~2=0.9903),Vibrio parahaemolyticus(R~2=0.9928)and Salmonella(R~2=0.9945).In artificial contaminated experiments,the detection limits of LFD-RPA assay for Staphylococcus aureus,Vibrio parahaemolyticus and Salmonella were 4.5×10~1CFU/m L,8.3×10~1CFU/m L and 2.7×10~1CFU/m L,respectively.What’s more,the recoveries of Staphylococcus aureus in fresh sleeve-fish,shrimp and cod were92%-106.22%.The recoveries of Vibrio parahaemolyticus in fresh sleeve-fish,shrimp and cod were 93.73%-107.59%.And,the recoveries of Salmonella in fresh sleeve-fish,shrimp and cod were 92.96%-106.66%.For practical sample testing,123 samples were analyzed.Staphylococcus aureus,Vibrio parahaemolyticus,and Salmonella were tested in all samples using multiplex LFD-RPA assay and traditional culture method.The positive detection rates for Staphylococcus aureus of samples from the local market and the Zhoushan Entry-Exit Inspection and Quarantine Bureau were both 0%.The positive detection rates for Vibrio parahaemolyticus and Salmonella of samples from the local market were both 1.9%,and the positive detection rates of samples from the Zhoushan Entry-Exit Inspection and Quarantine Bureau were both0%.For simultaneously detection of Staphylococcus aureus,Vibrio parahaemolyticus and Salmonella,a multi-objective recombinase polymerase amplification(RPA)combined with lateral flow dipstick(LFD)have been developed in this paper.Furthermore,to solve the bottleneck of unquantifiable,we employed a test strip reader(TSR-200)to scan and evaluate the colored signals of test lines on the lateral flow dipsticks.Such a reader can also decrease the man-made influence and increase the correctness of diagnosis.In addition,the multiplex LFD-RPA assay has been successfully applied to the detection of actual food samples.Multiplex LFD-RPA detection method is a rapid,portable,highly sensitive and specific technology,and it is simple to operate,not need specialized instruments.This assay provides a new idea for the rapid detection of foodborne pathogens in resource-poor areas and has a wide range of applications.