Reverse transcription - loop-mediated isothermal amplification assay for the rapid detection of pathogenic Listeria monocytogenes in meat products.

This study reports the use of reverse transcription - loop-mediated isothermal amplification (RT-LAMP) to detect Listeria monocytogenes in meat. The assay was designed to target the iap gene of L. monocytogenes , to which four primers, recognizing six distinct iap sites, were designed. We optimized the RT-LAMP conditions and established the following optimal systems: 60 min, 63 °C, 2.0 mmol/L MgSO ₄ , 1.0 mol/L betaine, 2.0 mmol/L dNTPs, 320 U/mL Bst DNA polymerase, 0.4 μmol/L outer primers, and 0.8 μmol/L inner primers. The RT-LAMP amplification products were identified by a visible white Mg ₂ P ₂ O ₇ precipitate or electrophoresis on a 2% agarose gel. RT-LAMP has a sensitivity of 7.3 × 10 ¹ CFU/mL, which is 2-fold higher than that of LAMP. When commercially available raw meat samples (including beef, pork, mutton, and rabbit) were analyzed simultaneously with RT-LAMP and the Chinese National Standard GB 4789.30-2016, their abilities to detect L. monocytogenes were the same. Samples containing L. monocytogenes killed by 15 psi at 121 °C for 15 min were used to confirm the specificity of RT-LAMP for live microorganisms. Thus, we used RT-LAMP to efficiently detect L. monocytogenes in meat products.