Development of isothermal amplification based Recombinase Polymerase Amplification (RPA) assay for rapid detection of hly gene of Listeria monocytogenes

Abstract

    Listeria monocytogenes is one of the most common agents of food poisoning which can cause serious foodborne diseases or even lethality. L. monocytogenes can be detected using traditional microbiology or molecular biology techniques, notably PCR. However, the application of these methods for detecting L. monocytogenes is limited due to the time-consuming, and strict requirement of equipment and skilled personnel. In this study, Recombinase polymerase amplification (RPA), an isothermal amplification method was established to rapidly and specifically detect hly gene from L. monocytogenes. Results showed that the hly gene was successfully amplified by the designed RPA primers. The method was well-performed in the optimal conditions of 39 ⁰C within 25 minutes. The limit of RPA detection was 310 fg of genomic DNA of L. monocytogenes, which was 1000 - fold more sensitive than the conventional PCR.  The RPA with the advantages of rapidity and sensitivity has the potential of alternating traditional methods and being developed into a rapid test kit applied for detection of L. monocytogenes.