Detection Of Three Foodborne Pathogenic Bacteria By Multiplex Fluorescent Probe-Based LAMP

Foodborne pathogenic bacteria pose a serious threat to human health and food safety,which greatly hinder social progress and economic development.Seafood is the most traded food in the world,and ensuring the microbial safety of seafood is a key step to solve the problem of foodborne pathogenic bacteria pollution.Vibrio parahaemolyticus,Staphylococcus aureus and Salmonella Enteritidis are three major foodborne pathogenic bacteria that cause foodborne diseases in seafood.They can all cause gastroenteritis in humans and may exist in seafood at the same time,posing a greater potential threat.The rapid and sensitive multiplex detection of foodborne pathogenic bacteria in seafood is of great significance for ensuring food safety.The traditional cultivation method usually takes 3-7 days to complete the detection,which is time-consuming.Immunological methods based on protein analysis need to prepare specific detection antigens or antibodies to complete detection,and their sensitivity are usually lower than that of molecular detection methods.Polymerase chain reaction needs to be cycled among several temperatures to realize amplification,which is expensive and time-consuming.Loop-mediated isothermal amplification technology is the most widely used isothermal amplification technology,which can achieve exponential amplification of target DNA by using 4-6 primers and Bst DNA polymerase with strand displacement activity at 65℃,eliminating the need for precision thermal cycler.Traditional loop-mediated isothermal technology usually uses endpoint detection,turbidity detection and real-time detection of fluorescent DNA dyes to detect amplification products,which can not achieve multiple analysis.In contrast,the loop-mediated isothermal amplification technology based on fluorescent probes is a feasible tool to realize multi-target detection of foodborne pathogenic bacteria in a short time.In this study,a MFP-LAMP detection technology was established,which could detect Vibrio parahaemolyticus,Staphylococcus aureus and Salmonella Enteritidis simultaneously in a single reaction.The corresponding LAMP primers and probes were designed based on the tox R gene of Vibrio parahaemolyticus,the nuc gene of Staphylococcus aureus and the fim Y gene of Salmonella Enteritidis,and the probes for each foodborne pathogenic bacteria were tried to use different fluorophore labeling.Singleplex probe-based LAMP method and dual probe-based LAMP method were used to screen the best fluorophore combination,which provided the basis for the initial establishment of the MFP-LAMP system.Finally,the fluorophore combination of HEX fluorophore labeled Vibrio parahaemolyticus’s probe,Texas Red fluorophore labeled Staphylococcus aureus’s probe and CY5 fluorophore labeled Salmonella Enteritidis’s probe was established and applied to the detection of MFP-LAMP.After the MFP-LAMP reaction system was initially established,the optimal MFP-LAMP detection system was established by optimizing the reaction temperature,enzyme type,enzyme concentration and probe ratio.The optimized MFP-LAMP could detect three foodborne pathogenic bacteria within 25 min.In the sensitivity experiment,the detection sensitivity of MFP-LAMP to three foodborne pathogenic bacteria were as low as 1×10~1CFU/m L,which indicated that the established MFP-LAMP method had high sensitivity.In the specific experiment,only Vibrio parahaemolyticus,Staphylococcus aureus and Salmonella Enteritidis were detected,other 18 foodborne pathogenic bacteria had no detection signal and no cross-reaction was generated,which indicated that the established MFP-LAMP had high specificity.In the detection of spiked seafood samples,the recovery rates of Vibrio parahaemolyticus,Staphylococcus aureus and Salmonella Enteritidis were95.71%-104.52%,95.64%-103.94%and 94.16%-109.17%,respectively,which proved the reliability of MFP-LAMP detection in seafood samples.In the detection of actual samples,the detection results of MFP-LAMP were compared with the traditional bacterial culture method.The results showed that two detection methods had the same positive detection rate,which proved the accuracy of MFP-LAMP detection in seafood samples.In a word,the MFP-LAMP assay established and optimized in this study can detect Vibrio parahaemolyticus,Staphylococcus aureus and Salmonella Enteritidis in seafood rapidly,sensitively,specifically,reliably and accurately at the same time,which has a broad market application prospect.