CONGXIU YE*, JINLING YI*, SONGCHAO YIN*, MEIRONG LI*, YANMEI LI**
Department of Dermatology and Venerology, the Third Affiliated Hospital of Sun Yat-sen University, Guangzhou 510630, China -
**College of Light Industry and Food Sciences, South China University of Technology, Guangzhou 510640, China
Introduction: A loop-mediated isothermal amplification (LAMP) method based on color change for rapid detection of the food-borne E. coli O157:H7 had been designed by targeting rfbE, stx1 and stx2 genes and evaluated in this study.
Materials and methods: Amplification of DNA was monitored through visual inspection of calcein pre-addition and 2% agarose gel electrophoresis (AGE). The optimal reaction system was found to be composed of 150 mM Mg2 +, 50 μM calcein as well as the 1:8 ratio of Mn2 + and calcein. Orange to green color change and ladder like multiple bands of different sizes could be inspected in only E. coli O157:H7.
Results: The sensitivity of LAMP assays was approximately 10 pg/μL and significantly prior to 10 ng/μL of polymerase chain reaction (PCR). In the parallel diagnosis of 66 E. coli samples, 36 (54.55%) strains were positive for rfbE. Of these 36 O157:H7, 31 (86.11%) and 29 (80.56%) were positive for stx1 and stx2, respectively.
Conclusion: the colorimetric LAMP assay based on rfbE gene was established and optimized. All the results indicated that the improved LAMP assay satisfied the basic demand for gene diagnose and could be used in the rapid detection of E. coli O157:H7.
colorimetric LAMP, visual detection, calcein, E. coli O157:H7, food pathogenic organisms.