Establishment Of LAMP Method For Detecting Six Food-borne Pathogens

In recent years,food-borne diseases have erupted in more and more areas all over the word.Food safety problems are increasingly in prominent way in our daily life.Enterohemorrhagic E.coli(EHEC)0157:H7,Salmonella,Staphyloccocus aureus,Listeria monocytogenes,Yersinia enterocolitica,Campylobacter jejuni,the six pathogens are common food-borne pathogens.In order to strengthen the detection and management of food safety,it is necessary to establish a rapid and sensitive detection method to improve the risk assessment system.In this study,we established a loop-mediated isothermal amplification method for rapid detection of the six food-borne pathogenic bacteria to provide technical support for the rapid detection of these food-borne pathogenic bacteria.Firstly,the detection target genes for each pathogen and LAMP detection primers were designed.Two LAMP-specific primers were designed for rfbE gene,stxl gene,Stx2 gene,Z3276 gene and eaeA gene of enterohemorrhagic Escherichia coli,Two groups of LAMP specific primers were designed for fimY gene and one group of LAMP specific primers for bcfD gene of Salmonella;two groups of LAMP specific primers for nuc gene of Staphylococcus aureus;and four groups of LAMP specific primers for hly gene of Listeria monocytogenes.Two groups of LAMP specific primers were designed for the ail gene of Yersinia enterocolitica.Two LAMP specific primers were designed for Campylobacter jejuni specific hipO gene.A total of 25 LAMP specific primers were identified.Then,the LAMP method for detecting target genes of food-borne pathogenic bacteria was established.LAMP primers were used to detect target genes of food-borne pathogenic bacteria and LAMP detection methods were established.The LAMP reaction products were analyzed by agarose gel electrophoresis with 4 non target bacteria DNA and distilled water as negative template to determine whether the primers designed by various target genes of food-borne pathogens were feasible.The results showed that the LAMP detection primers of rjbE,stx2 and Z3276 genes of 0157 Enterohemorrhagic Escherichia coli were successfully identified;the LAMP detection primers of Salmonella fimY gene;the LAMP detection primer of Staphylococcus aureus nuc gene;the LAMP detection primer of the Listeria monocytogenes hly gene;the LAMP detection primer of Yersinia enterocolitica ail gene;the LAMP detection primer of Campylobacter jejuni hipO gene.The LAMP assay established by the above primers showed that only positive samples could amplify the bands,while no bands were found in other bacterial samples also in negative controls.Finally,using the non-target bacteria as a reference,the eight groups of primers selected above were used to determine the six target bacteria by LAMP method,and the specificity of the method was tested,and the DNA template of the target strain was diluted by a 10-fold gradient to conduct a sensitivity test.The results showed that the target genes of the corresponding target bacteria could be specifically amplified,and the non-target bacteria were not significantly amplified,indicating good specificity;the sensitivity test results showed that the detection limit of E.coli was 1.45×10-1 fg/?L,Salmonella was 1.72×10-6 fg/?L,Staphylococcus aureus was 3.48 fg/?L,Listeria monocytogenes was 3.31 fg/?L,Yersinia enterocolitica was 6.10 fg/?L,and Campylobacter enterica was 62.7 fg/?L.The sensitivity of LAMP assay for six food-borne pathogenic bacteria constructed in this study was 100-1000 times higher than that of ordinary PCR,with lower detection limit and higher sensitivity.In summary,this study screened for rapid detection of Enterohemorrhagic Escherichia coli,Salmonella,Staphylococcus aureus,Listeria monocytogenes,Yersinia enterocolitica and Campylobacter jejuni target gene-specific LAMP method.which lays a foundation for the next step of rapid detection of high-throughput food-borne pathogens combined with microfluidic chip.