基于iap基因的RT-LAMP技术检测肉类食品中单核细胞增生李斯特菌的效果评价

Detection and Evaluation of Listeria monocytogenes in Meat Products by RT-LAMP Based on iap Gene

摘要:

本研究旨在探讨逆转录环介导等温扩增(reverse transcription–loop-mediated isothermal amplification，RT-LAMP) 技术在检测单核细胞增生李斯特菌感染的肉类食品中的效果和评价。根据RT-LAMP 原理，以iap基因为靶基因设计引物，检测肉制品中的单核细胞增生李斯特菌，并将该方法的特异性、灵敏度、死菌活菌鉴别效果与国标进行对比。实验获得的RT-LAMP体系最佳参数为63 ℃扩增60 min，反应体系中引物的最佳浓度为外引物0.4 μmol/L和内引物0.8 μmol/L，以及MgSO₄ 2.0 mmol/L、甜菜碱1.0 mol/L、dNTPs 2.0 mmol/L、AMV逆转录酶10 U/μL、Bst DNA聚合酶320 U/mL。该试验的灵敏度为每管7.3×101 CFU，是LAMP的2倍。特异性实验显示该方法仅检测单增李斯特菌的靶基因，无假阳性。用RT-LAMP 和中国国家标准GB 4789.30—2016检测市售肉样品(n=100)中单核细胞增生李斯特菌的能力相似，且RT-LAMP 仅对活菌有扩增作用。死菌活菌鉴别实验显示该法可以排除肉样中死菌的干扰。经与国标方法做对比，可知RT-LAMP可作为一种有效的检测手段。

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.