Establishment Of Rapid Detection Methods For Three Common Food-borne Pathogens In Milk

Foodborne illness is one of the the serious public health problems in the world.The foodborne illness is caused by pathogenic microorganisms,parasites and their metabolites,biotoxins,and chemical toxic and harmful substances.Foodborne diseases caused by microorganisms are the most important challenges of the global food safety.About 70%?80%of bacterial food poisoning diseases are caused by Salmonella in developed countries,and even 90%in China.Among the food caused Salmonella poisoning diseases,animal products such as eggs and milk account for about 90%.Salmonella has a strong survival ability and can survive in dairy products for several months.Listeria monocytogenes is widely found in nature and can grow at 4? refrigerated temperature.Listeria monocytogenes can cause serious infectious diseases in high-risk populations.The foods causing food poisoning of Listeria monocytogenes mainly include milk and dairy products,meat products,aquatic products,vegetables and fruits,in which dairy products are the most common product.Shigella,also known as dysentery bacilli,is the most common foodborne pathogen causing bacterial dysentery in humans.It is also one of the food hygiene detection indicators required by the Ministry of Health and the Ministry of Agriculture in China.In this paper,multiple LAMP detection system and multiple HAND-HRM detection system were established through loop-mediated isothermal amplification and high-resolution melting curve technique,taking the Listeria monocytogenes,Salmonella and Shigella as the target strains,which are the common foodbone pathogenic bacteria in milk.The results are as follows:1.Primers were designed for the highly conserved sequences of Listeria monocytogenes hemolysin protein(hly)gene,Salmonella invasive protein A(invA)gene and Shigella invasive plasmid antigen H(ipaH)sequence and three single LAMP reaction systems for these foodborne pathogenic bacteria were established through primer screening.A multiple LAMP detection system was established after the optimization of magnesium ion,betaine and primer which have impact on reaction efficiency and specificity.The specificity and sensitivity of multiple LAMP reaction system were tested.In the specific test,a specific amplification was observed in the template containing target bacteria while it was not shown in the blank control and non-target bacteria control,which indicated a strong specificity of the multiple LAMP reaction system.In the sensitivity test,the sensitivity of Listeria monocytogenes was 23 fg/?l,while that of Salmonella and Shigella was 250 fg/?l,which was improved by an order of magnitude compared with that of PCR.The three pathogenic bacteria liquids were diluted to different concentrations and contaminated milk samples.Genomic DNA was extracted through boiling,and multiple LAMP tests were performed.The minimum detection limit of Listeria monocytogenes,Salmonella and Shigella were 9.47×101 CFU/ml,4.1×102 CFU/ml and 5.33×101 CFU/ml,respectively.2.Primers were designed for the highly conserved sequences of the Listeria monocytogenes hly gene,the Salmonella fim gene and the Shigella ipaH gene.Multiple HAND-HRM reaction system for the three foodborne pathogenic bacteria was established through the optimization of the concentration and temperature of the primers,which were selected based on the dissolution temperature of the amplified products.The specificity,sensitivity and stability of multiple HAND-HRM reaction system were tested.In the specific test,a melting curve peak was observed in the template containing target bacteria while it was shown in the blank control and non-target bacteria control,which indicated the strong specificity of the multiple HAND-HRM reaction system.In the sensitivity test,the sensitivity of single pathogen of Listeria monocytogenes and Salmonella was 10 fg/?l,while that of Shigella was 100 fg/?l.The sensitivity of three pathogenic bacteria in the same system was 100 fg/?l.In the stability evaluation,the intra-group CV value of Tm was:0.10%-0.25%,0.04%-0.22%and 0.18%-0.69%for Listeria monocytogenes,Salmonella and Shigella respectively,and the inter-group CV values of Tm of the Listeria monocytogenes,Salmonella and Shigella was 0.16%-0.34%,0.27%-0.38%and 0.41%-1.18%,respectively.The small intra-and inter-group CV values of the three pathogenic bacteria indicated the strong stability of the multiple HAND-HRM system.It can be seen from the evaluation result of the effects of different concentration ratios of the three pathogens on the detection results of multiple HAND-HRM systems that the template in high concentration has a certain inhibitory effect on the amplification of the template in low concentration under the condition that the concentration of the templates is at a low level while have a large difference.However,the inhibition can be eliminated by bacteria enrichment.A simulated contaminated milk model was established by the same method as LAMP for multiple HAND-HRM detection.The minimum detection limits of Listeria monocytogenes,Salmonella and Shigella are 102 CFU/ml.Detection on 73 actual samples showed that the sensitivity of the multiple LAMP detection system is consistent with that of the national standard method,and the sensitivity of the multiple HAND-HRM system is even higher than that of the national standard method.In the practical test,especially for large amount sample test,multiple LAMP system can be used as primary screening to check the presence of pathogenic bacteria,and then determine the type of pathogenic bacteria through multiple HAND-HRM system.The quick detection system established in this study has the advantages of strong specificity,high sensitivity,strong stability,low cost,easy operation and great applicable value.