An Advanced Visual Qualitative and EVA Green‐Based Quantitative Isothermal Amplification Method to Detect Listeria Monocytogenes

Abstract

AbstractThe purpose of this study is to apply the visual qualitative (LFD‐LAMP) and advanced quantitative loop‐mediated isothermal amplification (EVA‐LAMP) methods to detect Listeria monocytogenes, which is an important pathogenic foodborne bacterium. This research designed a new set of primers to amplify the hlyA gene of L. monocytogenes. The LFD‐LAMP products were detected by lateral flow device (LFD) with the naked eyes, and the test results were consistent with agarose gel electrophoresis. Meanwhile, an advanced quantitative LAMP assay was established which depended on the EVA Green. The detection limit of the EVA‐LAMP method was 10 pg/μL, and a good linear relationship was obtained. The subsequent analysis of contaminated raw food samples showed that the primers were able to identify L. monocytogenes in food. The LFD‐LAMP and EVA‐LAMP reactions can be used as sensitive, rapid and simple methods for the detection of L. monocytogenes, especially on field detection.Practical ApplicationsThis work suggests a specific, sensitive, cost‐effective and nonpollution LAMP assay for the detection of Listeria monocytogenes. A new set of hlyA‐based primers was designed and successfully used to detect L. monocytogenes in raw food samples. Amplicon detection by visual LFD method proved to be as useful as the gel, but more convenient and clean. The LFD‐LAMP detection method in this study testifies as a significant advancement toward making LAMP a potential on‐site detection. Otherwise, in contrast to the traditional real‐time LAMP approach, this is a novel study to describe a real‐time quantitative LAMP assay based on EVA Green to detect L. monocytogenes in the fields of food safety.