食品中单核细胞增生李斯特菌的重组酶聚合酶扩增检测方法的建立

Development of recombinase polymerase amplification detection method for Listeria monocytogenes in food

目的建立食品中单核细胞增生李斯特菌的重组酶聚合酶扩增(RPA)检测体系。方法针对单核细胞增生李斯特菌的特异性基因hlyA,筛选出一组特异性强且高效的引物,建立单核细胞增生李斯特菌的RPA检测体系,并进行特异性、灵敏度检测。结果建立的RPA方法在37℃恒温反应仅需30 min,能够特异地检测单核细胞增生李斯特菌,最低模板浓度可低至0.5 ng/μL。人工染菌试验显示,该RPA体系可以有效扩增出每2.5 g样品中人工染菌104 CFU样品中的单核细胞增生李斯特菌。结论 RPA等温扩增方法特异较强、灵敏度较高,具有操作简便、不需要昂贵仪器、常温进行扩增反应等优点,适用于现场检测以及在基层实验室推广应用。

Objective To establish a recombinase polymerase amplification(RPA) method for the detection of Listeria monocytogenes(L.monocytogenes) in food. Methods Based on the hlyA gene of L. monocytogenes, a set of RPA primers was selected for constructing RPA test, and its specificity and sensitivity were then tested.Results The RPA assay could be finished in 30 min at 37 ℃. The primers used in RPA were specific. Experiments confirmed a detection limit of DNA template as low as 0.5 ng/μL. L. monocytogenes in the artificially-contaminated meat could be detected at the limit of 104 CFU/2.5 g. Conclusion The RPA method for detecting L. monocytogenes has strong specificity and high sensitivity, which is easy to operate, and can be performed under normal temperature without expensive equipment. It is suitable for field detection and application in the basic laboratory.