Development of loop-mediated isothermal amplification method for detection of Yersinia enterocolitica

Objective To detect Yersinia enterocolitica by developing loop-mediated isothermal amplification (LAMP) assay,which amplifies DNA with high specificity and rapidity under an isothermal condition. Methods A set of five primers,including two each for outer and inner primers and one loop primer,were designed specifically to recognize the thermolabile hemolysin gene(yruI/yruB) of Yersinia enterocolitica.The LAMP reaction mixture was optimized.Specificity and sensitivity were evaluated by Yersinia enterocolitica and 8 other associated bacteria strains. Results The optimal reaction temperature and time of the LAMP assay for the yruI/yruB gene were 60℃ and 60 min,respectively.Genomic DNAs from 8 bacterial strains were amplified using LAMP,and no amplification was observed in other bacterial strains.The detection limit of this LAMP assay was around 74 fg of Yersinia enterocolitica genomic DNA and 43 colonies forming units from pure cultures. In addition,this method was applied to detect artificially contaminated food samples,and the detection limit was 76 cfu/g for non-cultured artificially contaminated food samples. Conclusion The results suggested that detection of Yersinia enterocolitica by LAMP was an effective and low-cost procedure with high specificity and sensitivity. The assay does not need specialized equipment and is expected to become a valuable tool for rapid detection and identification of Yersinia enterocolitica.