摘要
将叠氮溴化丙锭(propidium monoazide,PMA)、羟基萘酚蓝(hydroxynaphthol blue,HNB)与环介导等温扩增(loop-mediated isothermal amplification,LAMP)技术相结合,建立一种可视化PMA-HNB-LAMP方法快速检测畜禽肉中单核细胞李斯特菌(Listeria monocytogenes)。结果表明,当PMA终质量浓度为5.0μg/mL、孵育和曝光时间均为15 min时,可有效抑制10~5 CFU/m L的单核细胞李斯特菌死菌DNA的聚合酶链式反应扩增,而对活菌DNA的扩增不具有抑制作用。LAMP反应体系中HNB终浓度为210μmol/L时,检测显色结果肉眼可见。该方法的纯菌液灵敏度为2.4×10~2 CFU/m L,人工污染肉制品的检出限为2.3×10~4 CFU/m L。分别采用国标法、PMA-HNB-LAMP法和HNB-LAMP法检测20份市售畜禽肉样,3种方法的单核细胞李斯特菌检出率分别为5%、10%和20%。PMA-HNB-LAMP法在一定程度上可解决LAMP法的假阳性问题,并且具有操作简单、可视性强的优点。
Using propidium monoazide(PMA) treatment and hydroxynaphthol blue(HNB) staining combined with loopmediated isothermal amplification, a rapid and visual detection method(PMA-HNB-LAMP) for Listeria monocytogenes was established in this study. The optimization of experimental parameters showed that a final PMA concentration of 5.0 μg/m L, incubation time of 15 min and exposure time of 15 min could restrain DNA amplification of 10~5 CFU/m L dead cells but not of live cells. A final HNB concentration of 210 μmol/L could give results visible to the naked eyes. The sensitivity of the PMAHNB-LAMP method was 2.4 × 10~2 CFU/m L for pure bacterial culture. The detection limit for artificially contaminated meat was 2.3 × 10~4 CFU/m L. Using the national standard method, PMA-HNB-LAMP and HNB-LAMP, the detection rates of L. monocytogenes in 20 meat products available on the market were 5%, 10% and 20%, respectively. PMA-HNB-LAMP is superior to traditional LAMP in avoiding false-positive results to a certain extent, and it has the advantages of simple operation and strong visibility.
作者
戴小芳
陈琼
董华夏
向春燕
虞伟明
郦娟
DAI Xiaofang;CHEN Qiong;DONG Huaxia;XIANG Chunyan;YU Weiming;LI Juan(Wuhan Institute for Food and Cosmetic Control,Wuhan 430012,China)
出处
《肉类研究》
北大核心
2020年第5期48-52,共5页
Meat Research
基金
湖北省自然科学基金计划项目(2017CFB366)
江苏省海洋药物活性分子筛选重点实验室开放基金项目(HY201702)。
关键词
单核细胞李斯特菌
叠氮溴化丙锭
羟基萘酚蓝
环介导等温扩增
畜禽肉
Listeria monocytogenes
propidium monoazide
hydroxynaphthol blue
loop-mediated isothermal amplification
poultry and livestock meat
