DEVELOPMENT AND EVALUATION OF A LOOP-MEDIATED ISOTHERMAL AMPLIFICATION METHOD FOR DETECTING ESCHERICHIA COLI O157 IN RAW MILK

摘要

In the present study, we report the performance of a loop-mediated isothermal amplification (LAMP) assay detecting foodborne pathogen Escherichia coli O157. Three pairs of primers were specially designed for recognizing eight distinct sequences of rfbE gene. Time and temperature conditions for amplification of E. coli O157 were optimized to be 40 min at 65C. The LAMP assay gave artificially contaminated raw milk sample detection limit level of 410 cfu/mL which corresponds to three to five cells per reaction tube, while the detection level of conventional polymerase chain reaction was 4.1 x 10t cfu/mL. Data on naturally contaminated raw milk samples indicated that the LAMP method was highly specific and sensitive, giving 100% concordance with the ISO 16654: 2001 reference method. The loop-mediated isothermal amplification method reported here provides a powerful tool for the detection of Escherichia coli O157 in raw milk samples because of its specificity, sensitivity and rapidity.