Real-time Fluorescence Loop Mediated Isothermal Amplification For The Diagnosis Of Yersinia Enterocolitica

Yersinia enterocolitica can infect animals and humans, and it is an extensivepathogenic bacteria and diffusely distributed in nature. Yersinia enterocolitica has becomea conventional test item of the import and export food. Conventional detection of Yersiniaenterocolitica is complex and time consuming. It is generally required4to6days, and cannot achieve the requirements for rapid detection of Yersinia enterocolitica bacteria in food.So, to establish a fast and effective detection method of Yersinia bacteria in food isurgently needed.In2000, a new type of nucleic acid detection technology developed by Notomi T. andhis colleagues. It was named as Loop-mediated Isothermal Amplification (LAMP).Real-time fluorescence loop mediated isothermal amplification used in this study can beconnected to the computer directly to observe the reaction, which does not require the gelelectrophoresis.In this study, we choose ail gene of Yersinia enterocolitic to be the target gene anddesign a pair of specific primers. the real-time fluorescence LAMP reaction systemTheLAMP reaction was ultimately determined a reaction system with containing3μmol/L eachof inner primers FIP and BIP,0.5μmol/L each of outer primers F3and B3,2.5μLdeoxynucleoside triphosphates,0.5mM MgSO4,2μL10×BstDNA buffer,1μL8U of BstDNA polymerase，1μL the specified amounts of target DNA,1mmol/L Betaine,0.5μLSYBR Green Ⅰ, and sterilized double-distilled water.Controls for method specificity were made with1Yersinia enterocolitica strains, and19non-Yersinia enterocolitica strains, the result shows that Yersinia enterocolitica waspositive and other strains were negative. Compared the results of3methods of extractingDNA template from Yersinia enterocolitica in chicken, it shows that the method ofextracting DNA did not affect the reaction. When testing10-fold serial dilutions of Yersiniaenterocolitica CMCC52302pure culture, the detection limit was61CFU/ml, up to10-foldmore sensitive than that of PCR.From the results of this study, it shows that the ail-based LAMP assay is an effectivemethod for the rapid detection of Yersinia enterocolitica in food.