Development And Application Of Microfluidic LAMP For The Simultaneous Detection Of Foodborne Pathogens

Foodborne pathogenic bacteria are one of the main causes of foodborne diseases.Food safety problems caused by foodborne pathogenic bacteria not only seriously threaten people's health,but also cause huge economic losses to consumers and food related industries,and adversely affect the social,economic and political stability of the country.EHEC O157:H7,S.typhimurium,S.aureus,L.monocytogenes,Y.enterocolitica,C.perfringens and C.jejuni are the most common foodborne pathogens in nature.High-throughput rapid detection of foodborne pathogens is one of the key to establish monitoring network.To effectively prevent and control food safety problems and establish a foodborne pathogenic bacteria monitoring network,this study established 5LAMP detection methods for detecting foodborne pathogenic bacteria,and a multiple microfluidic LAMP detection method for detecting foodborne pathogens.The main elements are as follows:1.Establish a LAMP method for the detection of E.coli O157:H7,L.monocytogenes and C.perfringens.Design and screening the LAMP primers of stx1,stx2,eaeA genes,actA gene,cpe enterotoxin.Through primer screening experiment,stx1-1?stx2-2?eaeA-2 three pairs of primers were selected as the primers for the LAMP method of EHEC O157:H7.The actA-3 primer was selected as the primer for the LAMP method of L.monocytogenes.The cpe-2 primer was selected as the primer for the LAMP method of C.perfringens.The results of specific experiments showed that only the corresponding positive strain were amplified in each method,and the other strain templates and blank controls had no obvious amplification bands.The sensitivity test results showed that the LAMP methods limit of stx1?stx2?eaeA gene were 4.3×10~2 cfu/mL,which were100 to 1000 times higher than that of conventional PCR methods;the LAMP method limit of actA gene was 4.7×10~1 cfu/mL,which was 100 times higher than that of conventional PCR method;the LAMP method limit of cpe enterotoxin was 5.2×10~1cfu/mL,which was 100 times higher than that of conventional PCR method.2.Establish a multiple microfluidic LAMP method of foodborne pathogensThe LAMP methods of EHEC O157:H7?L.monocytogenes and C.perfringens established in this study and the LAMP methods of S.typhimurium,S.aureus,Y.enterocolitica and C.jejuni(A total of 10 LAMP methods and 10 target genes)established in our laboratory were combined with microfluidic technology to establish a microfluidic LAMP method for detection of the above 7 foodborne pathogens.The results of data analysis showed that the specific primers in each reaction hole only produced good amplification with the corresponding genomic DNA of positive strain,and there was no obvious amplification curve between the other genomic DNA of non-positive strain and the blank control reaction hole,and it showed that its specificity was consistent with LAMP methods.The minimum detection limits of EHEC O157:H7,S.typhimurium,S.aureus,L.monocytogenes,Y.enterocolitica,C.perfringens and C.jejuni were 4.3×10~2cfu/mL,3.8×10~1 cfu/mL,4.7×10~1 cfu/mL,5.6×10~1 cfu/mL,3.9×10~2cfu/mL,5.2×10~1 cfu/mL,respectively.It's basically consistent with LAMP test results.Meanwhile,the optimal preamplification time of LAMP for 5 non-anaerobic detection target bacteria mixture solution was more than 10 h.Analysis of the sensitivity results of microfluidic LAMP methodof foodborne pathogens,there were great linear relationships between the logarithm of bacterial concentration and the Tt value.Moreover,118 pork clinical samples were detected by microfluidic LAMP method.There was only 1 positive sample of EHEC O157:H7 that was detected,and the detection rate was 0.847%.There were 9 positive samples of S.typhimurium,and the detection rate was 7.63%.There were 6 positive samples of S.aureus,and the detection rate was 5.08%;There were 3 positive samples of L.monocytogenes,and the detection rate was 2.54%;The Y.enterocolitica,C.perfringens and C.jejuni were not detected.There's 1 sample that was detected with both S.typhimurium and L.monocytogenes in all the positive samples detected.The clinical samples were also tested by the national standard assay and conventional PCR assay,and only samples 2 and 15 of the national standard assay results for S.typhimurium were different from the microfluidic-LAMP assay and showed negative results,while the rest of the assay results were consistent with each other.A multiple microfluidic LAMP method of foodborne pathogens established in this study has the advantages of fast,good specificity and high sensitivity,which is suitable for rapid detection with foodborne samples in laboratories and food safety regulatory departments.