本研究针对单增李斯特菌的快速检测方法进行开发，首先根据单增李斯特菌hlyA基因序列保守区设计2对引物，在同一体系中对单增李斯特菌的RNA进行反转录及环介导等温扩增，同时使用羟基萘酚蓝(终浓度200 μM)作为反转录环介导等温扩增产物的指示剂，在不开盖进行凝胶电泳检测的情况下，可直接根据体系颜色变化判读结果，能够在20 h内(包括增菌时间)检测出样本中是否存在单增李斯特菌的RNA。本研究建立的RT-LAMP-HNB检测方法对单增李斯特菌RNA的检出限为5.8×10-3 μg/mL，起始接种浓度检出限为10 CFU/10 mL，灵敏度是RT-PCR的10倍，且能够检测出是牛奶中否存在的单增李斯特活菌，具有实际应用价值。本研究开发的检测方法具有快速、准确、便捷、灵敏度高等特点，适用于在基层或不便利地区推广使用。

A rapid detection method RT-LAMP-HNB for Listeria monocytogenes was developed. Two pairs of primers were designed according to the conserved region of the hlyA gene sequence. In the same reaction system, the RNA of Listeria monocytogenes was reverse transcripted to cDNA and then was amplified by loop mediated isothermal amplification. At the same time, hydroxyl naphthol blue (HNB, the final concentration of 200 μM) was used as an indicator for reverse transcription loop mediated isothermal amplification products (RT-LAMP). The amplified results can be interpreted according to the color change of the system, and the gel electrophoresis was not necessary. This detection can be done within 20 hours (including the time of enrichment) and the live Listeria monocytogenes in milk can be detected. The detection limit in this study for the RNA of Listeria monocytogenes is 5.8×10-3 μg /mL, the detection limit of the initial inoculation concentration is 10 CFU/10mL, which are 10 times more sensitive than the RT-PCR method. RT-LAMP-HNB detection method developed in this study has the characteristics of fast, accurate, convenient and high sensitivity, which is suitable for popularization and application in grass-root labs or inconvenient areas.

广东省科技计划项目(2017A020208002)