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

• Published in: Canadian journal of microbiology Vol. 65; no. 12; pp. 913 - 921
• Main Authors: Zhan, Ling-Zhi, Song, Da-Feng, Gu, Qing, Yan, Ting-Ting, Ma, Cong-Cong
• Format: Journal Article
• Language: English
• Published: Canada NRC Research Press 01.12.2019; Canadian Science Publishing NRC Research Press
• Subjects: amplification isotherme à médiation par boucles et transcription inverse; Animals; Bacterial Proteins - genetics; Betaine; Contamination; Deoxyribonucleic acid; DNA; DNA polymerase; DNA-directed DNA polymerase; détection rapide; Electrophoresis; Food Microbiology - methods; Food processing industry; Genetic transcription; Lipoproteins - genetics; Listeria; Listeria monocytogenes; Listeria monocytogenes - genetics; Listeria monocytogenes - isolation & purification; Meat; Meat - microbiology; Meat products; Meat Products - microbiology; Mediation; Microorganisms; Mutton; Nucleic Acid Amplification Techniques; Pork; Primers; rapid detection; Reverse transcription; reverse transcription – loop-mediated isothermal amplification; RT–LAMP; Sensitivity and Specificity; viande; China; viande; détection rapide; reverse transcription – loop-mediated isothermal amplification; Listeria monocytogenes; meat; amplification isotherme à médiation par boucles et transcription inverse; rapid detection; RT–LAMP
• ISSN: 0008-4166; 1480-3275
• DOI: 10.1139/cjm-2019-0114

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.