Establishment and application of isothermal amplification techniques for the detection of heat-stable I enterotoxin of enterotoxigenic Escherichia coli.

Abstract

The aim of this study was to establish isothermal multiple self-matching initiated amplification (IMSA) and cross-priming amplification (CPA) methods to detect heat-stable I enterotoxin (STa)-producing enterotoxigenic Escherichia coli (ETEC). To avoid cross-contamination of aerosols, a closed independent isothermal amplification tube (IAT) was used to perform the assays. Optimal amplification conditions for IMSA and CPA were selected for specificity and sensitivity, respectively, and for clinical relevance. Both IMSA and CPA assays could specifically recognize all 3-STa positive strains in which they fluoresced green under UV light, but not in the 11 non-STa strains. The results of the sensitivity analysis indicated that the detection limit of the IMSA assay was 1.5 ×102 CFU, comparable to real-time PCR, but 10-fold more sensitive than CPA and LAMP. Further evaluation of the detection methods of swine diarrhea samples demonstrated that both could successfully identify the DNA of STa-producing ETEC in clinical specimens, consistent with LAMP and qPCR methods. The results demonstrated that the IMSA and CPA methods had high specificity and sensitivity with rapid detection of ETEC, so having great potential in clinical applications.