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

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. Sensitivity of S‐CPA (a1) and D‐CPA (a2) for L. monocytogenes detection was monitored by real‐time measurement of turbidity and the corresponding curves of concentrations of DNA were marked in the figure. The threshold value was 0.1 and the tur...
Source: FEMS Microbiology Letters - Category: Microbiology Authors: Tags: Research Letter Source Type: research