Development and Application of a Simple "Easy To Operate" Propidium Monoazide-Crossing Priming Amplification on Detection of Viable and Viable But Non-culturable Cells of O157 Escherichia coli.

Wenqu Zhou,Kan Wang,Xin Fu,Wei Hong,Caiying Bai,Junyan Liu,Tengyi Huang,Ling Chen

doi:10.3389/fmicb.2020.569105

Abstract

O157 Escherichia coli is one of the most important foodborne pathogens causing disease even at low cellular numbers. Thus, the early and accurate detection of this pathogen is important. However, due to the formation of viable but non-culturable (VBNC) status, the golden standard culturing methodology fails to identify O157 E. coli once it enters VBNC status. Crossing priming amplification (CPA) is a novel, simple, easy-to-operate detection technology that amplifies DNA with high speed, efficiency, and specificity under isothermal conditions. The objective of this study was to firstly develop and apply a CPA assay with propidium monoazide (PMA) for the rapid detection of the foodborne E. coli O157:H7 in VBNC state. Five primers (2a/1s, 2a, 3a, 4s, and 5a) were specially designed for recognizing three targets, which were rfbE, stx1, and stx2, and evaluated for its effectiveness in detecting VBNC cell of E. coli O157:H7 with detection limits of pure VBNC culture at 103, 105, and 105 colony-forming units (CFUs)/ml for rfbE, stx1, and stx2, respectively, whereas those of food samples (frozen pastry and steamed bread) were 103, 105, and 105 CFUs/ml. The application of the PMA-CPA assay was successfully used on detecting E. coli O157:H7 in VBNC state from food samples. In conclusion, this is the first development of PMA-CPA assay on the detection of VBNC cell, which was found to be useful and a powerful tool for the rapid detection of E. coli O157:H7 in VBNC state. Undoubtedly, the PMA-CPA method can be of high value to the food industry owing to its various advantages such as speed, specificity, sensitivity, and cost-effectiveness.

Highlights

In recent years, the outbreak of foodborne diseases caused by pathogens and its related virulence factors is a major threat for many countries, much attention had been paid to food safety issues
The objective of this study was to firstly develop and apply a Crossing priming amplification (CPA) assay with propidium monoazide (PMA) for the rapid detection of the foodborne E. coli O157:H7 in viable but non-culturable (VBNC) state
This study aimed to develop a rapid PMA-CPA assay to detect E. coli O157:H7 in VBNC state targeting on rfbE, stx1, and stx2 genes combining visual methods with the addition of calcein and applying this assay to detect the E. coli O157:H7 strains from food samples

Summary

Introduction

The outbreak of foodborne diseases caused by pathogens and its related virulence factors is a major threat for many countries, much attention had been paid to food safety issues. E. coli O157:H7 is the main EHEC serotype that causes the majority of EHEC human infections. Developing a rapid, sensitive, and accurate method to detect E. coli O157:H7 in various foods with a complex matrix is crucial in preventing disastrous E. coli O157:H7 outbreaks and associated human infections

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