Abstract:This experiment aimed to investigate the effectiveness and evaluation of the reverse transcription-loop-mediated isothermal amplification(RT-LAMP) in detecting Listeria monocytogenes infection in meat products. The assay was designed to target the iap gene of Listeria monocytogenes for detection of meat samples based on the principle of RT-LAMP. The specificity, sensitivity and differentiation between living and dead bacteria of this identification were compared to Chinese national standards. This experiment optimized the RT-LAMP conditions and obtained the optimum amplification at 63 ℃ for 60 min, the optimal concentrations of the primers were 0.4 μmol/L outer primer and 0.8 μmol/L inner primer, 2.0 mmol/L MgSO4, 1.0 mol/L betaine, 2.0 mmol/L dNTPs, 10 U/μL AMV reverse transcriptase, 320 U/mL Bst DNA polymerase. The assay has a sensitivity of 7.3×101 CFU/per reaction, which was twice as sensitive as LAMP. The specificity test demonstrated that only the target genes of Listeria monocytogenes were detected without false positives. The ability of RT-LAMP to detect Listeria monocytogenes in commercially available meat samples(n=100) was comparable to the Chinese National Standard GB 4789.30-2016, and RT-LAMP only amplified live microorganisms. Differentiation experiments between living and dead bacteria revealed that this method could eliminate the interference from dead bacteria in meat samples. In comparison with the method of national standards, RT-LAMP proves to be an effective detection method.
