产气荚膜梭菌荧光PCR和RPA检测方法的建立和比较

摘要/Abstract

摘要： 本研究根据产气荚膜梭菌（Clostridium perfringens）高度保守的plc基因，设计实时荧光PCR和聚合酶重组酶扩增 (recombinase polymerase amplification，RPA)的特异性引物和探针，建立了针对产气荚膜梭菌的实时荧光PCR和RPA检测方法，并对两种方法的特异性、灵敏性以及人工污染样品的检出限进行了分析和比较。结果表明，建立的两种方法特异性强，仅对产气荚膜梭菌产生特异性扩增，对其它细菌均无扩增；灵敏性高，检出限均为1.3 pg/μL。在人工污染产气荚膜梭菌的模拟样品检测中，两种方法的检出限均为1.0×102 CFU/mL； real-time RPA需要3～13min即可实现对所有阳性样品的检测，而real-time PCR则需要24～46 min（Ct值为17.45～33.65）。Real-time RPA方法在检测时间、操作方便性和仪器便携性方面，明显优于real-time PCR方法。本研究建立的实时荧光PCR和RPA检测方法特异性强，灵敏性高，操作方便，为实验设备装备较差的基层检测实验室和疫情突发现场对产气荚膜梭菌的快速检测提供了有效的技术手段。

关键词: 产气荚膜梭菌, plc基因, real-time RPA, real-time PCR

Abstract: To develop a rapid method for Clostridium perfringens, The study was designed specific primer and probe , and based on the conserved sequence of plc gene of Clostridium perfringens by real-time PCR and real-time recombinase polymerase amplification. Analyzed and compared real-time PCR with RPA through specificity、sensitivity and detection limits of artificial contaminated samples. The results showed that two established detection methods were specifically and were not detected other bacterial, and the same sensitivities of the two methods were 1.3 pg/μL. For artificially contaminated chicken and milk samples with the Clostridium perfringens, the detection limits were 1.0×102 cfu/mL. The positive samles could be detected after 3～13 min by real-time RPA, and could be detected at least 24～46 min by real-time PCR(the Ct was between 17.45 and 33.65). Real-time RPA detected clinical samples, detected cycles、isothermal amplification and instrument portable were better than real-time PCR. In this study, real-time RPA and real-time PCR which was provided an effective technical for the rapid detected Clostridium perfringens

Key words: Clostridium perfringens, plc gene, real-time RPA, real-time PCR

中图分类号:

R155.5
Q93-3

刘立兵. 产气荚膜梭菌荧光PCR和RPA检测方法的建立和比较[J]. 食品科学, 0, (): 0-0.

Li-Bing LIU. Development and Comparison of the Real-time PCR and RPA Assay for Detection of Clostridium perfringens in Food[J]. FOOD SCIENCE, 0, (): 0-0.

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