Development of isothermal amplification methods for rapid and sensitive detection of heat-labile enterotoxin producing Escherichia coli.

Abstract

The objective of this study was to establish a novel isothermal amplification method for detection of heat-labile enterotoxin (LT-I)-producing Escherichia coli. Loop-mediated isothermal amplification (LAMP), cross-priming amplification (CPA), and isothermal multiple-self-matching-initiated amplification (IMSA) were developed and evaluated. Optimal conditions, specificity, and sensitivity tests were performed and compared to qPCR findings. All three methods could produce ladder-like products with LT-I positive samples, while no products were generated with the negative controls. The amplified products could be directly visualized as negative or positive in the isothermal amplification (IAM) tube, which saved time and prevented the possibility of cross-contamination. The detection limits of each assay were similar, and all three assays could directly detect the DNA of Escherichia coli in clinical samples successfully. This is the first report on the application of CPA and IMSA methods for the detection of LT-I. The findings suggest that the three assays may be important tools for the rapid detection of enterotoxigenic Escherichia coli (ETEC) in the clinic.