Study On Loop-mediated Isothermal Amplification (LAMP) Method For Detection Of Three Foodborne Pathogens

Abstract:Evens about food poisoning were caused by food-borne pathogens were reported more and more in recent years. Listeria monocytogenes, Sakazakii bacteria and Shigella bacteria are all belong to foodborne pathogens. In order to ensure food security, we need to strengthen food inspection, and to establish rapid and sensitive detection methods.This paper to set up LAMP method for rapid detection of three kinds of pathogenic bacteria for research purposes, provide technical support for the rapid detection of food security.Our study focus on Enterobacter sakazakii （16S/23S rDNA IGS sequence,GB:AY748354.1）,Listeria monocytogenes （iap gene,GB: NC₀03210.1）, and Shigella bacteria （ipaH gene,GB:HE616529.1）to design two set of LAMP primers respectively.At the same zone,the specific primers were chosed for Real-time PCR. Optimizeing the reaction condition to determine its reaction system:10mmol/LFIP&BIP,10mmol/LF3&B3,10mmol/LdNTPs,5M Betaine,16mmol/LMgSO4,20mmol/LKCl,20mmol/L （NH4）2SO4,0.2%Triton X-100,8U Bst DNAPolymerase,1μL DNAtemplate,add ddwater to25μL。After incubation at67℃for60min. Identification of experimental results by three ways,On the other hand, the resulting reaction products were visible due to the turbidity （or white precipitate） form; in addition, the products could be detected by adding fluorescent dye（SYBR GREEN I,0.51L/251L volume, Takara）, reaction products after gel electrophoresis, is a trapezoid.To determine specificity of the LAMP assay, Escherichia coli, Listeria, Shigella, Salmonella and Staphylococcus were used as non-Enterobacter sakazakii,negative controls. To determine the sensitivity of the assay, a ten-fold serial dilution of genomic DNA, Enterobacter sakazakii’s detection limits for91fg/uL, Listeria detection limits for9.6fg/uL, Shigella detection limit for27.5fg/uL. Detection of Enterobacter sakazakii, Listeria monocytogenes, Shigella bacteria from artificially contaminated infant formula to examine the detection efficacy and sensitivity of this assay, compare whith Real-time PCR.The results show that LAMP assay have the same effect in specificity with Real-time PCR, The sensitivity assay showed that the LAMP method exhibits higher sensitivity than Real-time PCR. Enterobacter sakazakii in infant formula detected by LAMP and Real-time PCR assays’limites are101CFU/mL and102CFU/mL respectively. Listeria monocytogene in infant formula detected by LAMP and Real-time PCR assays’limites are3×100CFU/mL and3X101CFU/mL respectively. Shigella bacteria in infant formula detected by LAMP and Real-time PCR assays’limites are4×100CFU/mL and4×101CFU/mL respectively. LAMP was much more sensitive than Real-time PCR.During the actual sample detection, use LAMP, Real-time PCR and Industry standard method （sN/T1632.1-2005[v6]）, we find that:Enterobacter sakazakii in76pieces milk powder, LAMP method’s positive detection rate of3.95%, and take time about1.5h. Shigella bacteria in76pieces milk powder, LAMP method’s positive detection rate of3.95%, and takeing time about1.5h. Listeria monocytogenes in54pieces meat products, LAMP method’s positive detection rate of13.0%, and take time about2.0h.In this study, we have established the LAMP detection method of Enterobacter sakazakii, Listeria monocytogenes, and Shigella bacteria separately, getting Listeria monocytogenes LAMP detection kits, mastering three kinds of methods in naked eyes to judge LAMP result.Make a good method for food-borne pathogens detection.Provide more technical reference for testing organizations.