DIRECT RECOMBINASE POLYMERASE AMPLIFICATION ASSAY FOR ACCURATE AND RAPID DETECTION OF LISTERIA MONOCYTOGENES IN FOOD

Abstract

Listeria monocytogenes is one of the most common types of food poisoning bacteria which can cause serious foodborne diseases or even lethality. Generally, L. monocytogenes can be detected using traditional microbiology or molecular biology techniques, notably PCR. However, the application of these methods at the field is restricted due to the strict requirement of equipment and skilled personnel. In this study, recombinase polymerase amplification (RPA), an isothermal PCR assay was developed to rapidly detect L. monocytogenes in crude samples. The results showed that the RPA reaction, without requiring complex thermal cycles, was well-performed in the optimal conditions of 39°C within only 25 minutes. The limit of detection was identified as 310 fg of L. monocytogenes genomic DNA, which was 1000-fold more sensitive than the conventional PCR. RPA also succeeded to directly detect L. monocytogenes cells at a concentration as low as 2.5 × 10¹ Colony Forming Unit (CFU)/mL in pure cultures. In addition, RPA could accurately detect L. monocytogenes at 2.5 × 10² CFU/mL in milk without sample extraction or processing. Therefore, RPA established in this study could be an alternative standard method to confirm the presence of L. monocytogenes in food. Accordingly, this rapid and sensitive method could be further applied to clinical testing for the diagnosis of L. monocytogenes infection, especially in areas with limited settings.