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

Abstract

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 MgSO₄, 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 Mg₂P₂O₇ precipitate or electrophoresis on a 2% agarose gel. RT-LAMP has a sensitivity of 7.3 × 10¹ 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.

Keywords: Listeria monocytogenes; RT–LAMP; amplification isotherme à médiation par boucles et transcription inverse; détection rapide; meat; rapid detection; reverse transcription – loop-mediated isothermal amplification; viande.