Rapid and sensitive detection of Listeria monocytogenes by cross-priming amplification of lmo0733 gene.

Listeria monocytogenes is a food-borne pathogen that causes severe opportunistic infection in humans and animals. This study reports the development of single cross-priming amplification (S-CPA) and double CPA (D-CPA) assays targeting species-specific gene lmo0733 for identifying L. monocytogenes strains. The CPA assays were performed at a constant temperature 64 °C using seven specific primers and evaluated for specificity and sensitivity. The color change of positive amplification was directly observed by Loopamp® Fluorescent Detection Reagent (FD), and the DNA products were visualized as a ladder-like banding pattern on 2.5% gel electrophoresis. Moreover, the positive reactions were also detected by real-time measurement of turbidity. 50 L. monocytogenes and 46 non-L. monocytogenes strains were used for the method verification, and the specificity was 100%. The limit of detection (LoD) of the S-CPA and D-CPA assays was 2.5 pg DNA per reaction and 10-fold more sensitive than PCR. A total of 60 pork samples were tested for L. monocytogenes using the S-CPA assay developed in the study, and the accuracy of the S-CPA and the culture-biotechnical method was 100% identical. The results suggested that the S-CPA assay was a rapid, sensitive, and valuable tool for detection of L. monocytogenes in food products.