Real‐time loop‐mediated isothermal amplification assays combined with ethidium monoazide bromide and bentonite coated activated carbon for rapid and sensitive detection of viable Escherichia coli O157:H7 from milk without enrichment

Abstract

AbstractA real‐time loop‐mediated isothermal amplification (Rti‐LAMP) DNA assay was developed for rapid and sensitive detection of viable Escherichia coli O157:H7 in milk by combining with ethidium monoazide bromide (EMA) and bentonite coated activated carbon (BCAC) treatment without enrichment. The assay involved inoculating 25 mL portions of milk with various numbers of E. coli O157:H7. Then each of milk sample was adsorbed with 4 g BCAC to remove DNA amplification inhibitor. The recovered purified bacteria sample was further treated with EMA for discrimination viable from dead cells in Rti‐LAMP assay. The detection limit of viable E. coli O157:H7 was 10 CFU/mL in milk by the BCAC‐EMA‐Rti‐LAMP, and the entire assay could be completed in 5 hr. Twenty‐five raw milk samples were detected by the BCAC‐EMA‐Rti‐LAMP assay, using three methods as controls including E. coli O157:H7 plate culture, BCAC‐Rti‐LAMP, and Rti‐LAMP combined with enrichment at 37°C (E‐Rti‐LAMP). The results showed that the BCAC‐EMA‐Rti‐LAMP had similar accuracy and sensitivity as that of plate culture, as they both detected one sample of viable E. coli O157:H7 positive from 25 fresh raw milk samples. These data strongly suggested that the BCAC‐EMA‐Rti‐LAMP could rapidly and sensitively detect viable E. coli O157:H7 in milk without enrichment.Practical Applications Escherichia coli O157:H7 is one of the foodborne pathogens in milk, which has low dose of infection and high pathogenicity. In milk, components such as Ca2+, proteinase, fats, and milk proteins may block DNA and shield it from access by polymerase. This study was targeting VT2 and Z3276 genes for rapid and sensitive detection of viable E. coli O157:H7 in milk. BCAC was used to remove DNA amplification inhibitor and improve the detection sensitivity. EMA was used to discriminate viable from dead cells. The assay will be a potential tool in the rapidity, sensitivity, and specificity for detection of E. coli O157:H7 in milk without enrichment.