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

Junjun Zhai,Xuelong Chen,Feng Ping,Zhang Yan,Yanping Qi,Qu Lei,Tianwen Wang

doi:10.1371/journal.pone.0230881

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.

Highlights

Infection by enterotoxigenic Escherichia coli (ETEC) is a major cause of acute diarrhea in animals and humans and plays an important role in the etiology of food-borne and water-borne outbreaks, especially in developing countries [1,2,3]
Previous studies have shown that the vast majority of piglet diarrhea is caused by enterotoxigenic Escherichia coli (ETEC), resulting in huge financial losses in the agricultural sector in developing countries [27]
With the continuous development of isothermal amplification technology in recent years [28], additional new methods have been proposed to detect diarrhea caused by ETEC due to the advantages of rapidity, accuracy and low cost

Summary

Introduction

Infection by enterotoxigenic Escherichia coli (ETEC) is a major cause of acute diarrhea in animals and humans and plays an important role in the etiology of food-borne and water-borne outbreaks, especially in developing countries [1,2,3]. Diarrhea caused by ETEC brings great economic loss on an annual basis to the global animal industry, among which the most conspicuous manifestation is disease in and death of suckling and weaning piglets [4, 5]. The factors that cause the foremost virulence in ETEC strains include adhesins and/or enterotoxins, allowing the bacteria to colonize the epithelial cells of the small intestines of animals after which heat-labile enterotoxin (LT, divided into LT-I and LT-II) and/or heat-stable enterotoxin. Isothermal amplification techniques for detection of heat-stable I enterotoxin.

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Conclusion