Reverse transcription loop‐mediated isothermal amplification assays allow the rapid detection of Listeria monocytogenes in fresh‐cut fruits and vegetables

Abstract

Abstract Listeria monocytogenes (L. monocytogenes) is a major concern in human food safety. Here, the development of a reverse transcription‐loop‐mediated isothermal amplification (RT‐LAMP) method, targeting the hly A gene of L. monocytogenes, to rapidly detect this important pathogen in food is described. The RT‐LAMP conditions were optimized and the following parameters established: 60 min, 63°C, 0.2 M betaine, 8.0 mM MgSO4, 0.8 mM dNTPs, 0.4 μM outer primers, and 1.6 μM inner primers. RT‐LAMP products were identified by the visualization of white precipitates and bands on a 2% agarose gel after electrophoresis. Results revealed the specificity of RT‐LAMP with no false‐positives or false‐negatives observed. RT‐LAMP detection of L. monocytogenes was highly sensitive at levels of one colony forming unit (CFU) per reaction. Samples (up to 100) of fresh‐cut fruits and vegetables were simultaneously assayed using RT‐LAMP and Chinese national standard (GB) methods to further confirm its accuracy and reliability. Data revealed that the RT‐LAMP results were comparable to those obtained with standard GB methods. The method presents the first example of the use of RT‐LAMP for the detection of L. monocytogenes. This work provides a new and simple detection method for identifying L. monocytogenes in infected food products and the method should be considered in future testing protocols.Practical ApplicationsSuccessful detection of live pathogens is the key to food safety testing. However, the inability to distinguish between live and dead cells is a major drawback of almost all DNA‐based assays. This study has developed a RNA‐based detection method for the detection of L. monocytogenes and this method using RT‐LAMP addresses these deficiencies. RT‐LAMP could detect L. monocytogenes at levels of 1 CFU/reaction, which was 10‐fold higher than the sensitivity of PMA‐LAMP methods. The testing of fresh‐out foods using RT‐LAMP further demonstrated the practicality and reliability of the method. Results show that this method could become a diagnostic tool for accurately detecting the presence of live L. monocytogenes in food.