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

TITLE=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

JOURNAL=Frontiers in Microbiology

VOLUME=11

YEAR=2020

URL=https://www.frontiersin.org/journals/microbiology/articles/10.3389/fmicb.2020.569105

DOI=10.3389/fmicb.2020.569105

ISSN=1664-302X

ABSTRACT=<p>O157 <italic>Escherichia coli</italic> 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 <italic>E. coli</italic> 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 <italic>E. coli</italic> O157:H7 in VBNC state. Five primers (2a/1s, 2a, 3a, 4s, and 5a) were specially designed for recognizing three targets, which were <italic>rfbE</italic>, <italic>stx1</italic>, and <italic>stx2</italic>, and evaluated for its effectiveness in detecting VBNC cell of <italic>E. coli</italic> O157:H7 with detection limits of pure VBNC culture at 10<sup>3</sup>, 10<sup>5</sup>, and 10<sup>5</sup> colony-forming units (CFUs)/ml for <italic>rfbE</italic>, <italic>stx1</italic>, and <italic>stx2</italic>, respectively, whereas those of food samples (frozen pastry and steamed bread) were 10<sup>3</sup>, 10<sup>5</sup>, and 10<sup>5</sup> CFUs/ml. The application of the PMA-CPA assay was successfully used on detecting <italic>E. coli</italic> 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 <italic>E. coli</italic> 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.</p>