产气荚膜梭菌实时荧光PCR和实时荧光RPA检测方法的建立和比较

doi:10.7506/spkx1002-6630-20181026-307

食品科学 ›› 2020, Vol. 41 ›› Issue (4): 268-272.doi: 10.7506/spkx1002-6630-20181026-307

产气荚膜梭菌实时荧光PCR和实时荧光RPA检测方法的建立和比较

刘立兵，李睿文，陈志敏，王金凤，孙晓霞，袁万哲，王建昌

（1.石家庄海关，河北石家庄 050051；2.河北省检验检疫科学技术研究院，河北石家庄 050051；3.河北农业大学动物医学院，河北保定 071001）

出版日期:2020-02-25 发布日期:2020-03-02
基金资助:
国家质检总局科技计划项目（2016IK107）；河北省高校百名优秀创新人才支持计划项目（SLRC2017039）；河北省现代农业产业技术体系羊产业创新团队项目（HBCT2018140204）

Development and Comparison of Real-Time Polymerase Chain Reaction and Real-Time Recombinase Polymerase Amplification Assays for Detection of Clostridium perfringens in Food

LIU Libing, LI Ruiwen, CHEN Zhimin, WANG Jinfeng, SUN Xiaoxia, YUAN Wanzhe, WANG Jianchang

(1. Shijiazhuang Customs Distract, Shijiazhuang 050051, China; 2. Hebei Academy of Science and Technology for Inspection and Quarantine, Shijiazhuang 050051, China; 3. College of Veterinary Medicine, Agricultural University of Hebei, Baoding 071001, China)

Online:2020-02-25 Published:2020-03-02

摘要/Abstract

摘要： 根据产气荚膜梭菌（Clostridium perfringens）高度保守的plc基因，设计特异性引物和探针，建立产气荚膜梭菌的实时荧光聚合酶链式反应（polymerase chain reaction，PCR）和实时荧光聚合酶重组酶扩增（recombinase polymerase amplification，RPA）检测方法。特异性分析结果表明，建立的两种检测方法特异性强，仅对产气荚膜梭菌有特异性扩增，对其他细菌均无扩增；两种方法的检出限均为1.3 pg/μL。在人工污染模拟样品的检测中，两种方法的检出限均为1.0×102 CFU/mL；实时荧光RPA需要3～13 min即可实现对所有阳性样品的检测，而实时荧光PCR则需要24～46 min（Ct值为17.45～33.65）。实时荧光RPA方法在检测时间、操作方便性和仪器便携性方面，明显优于实时荧光PCR方法。本研究建立的实时荧光PCR和实时荧光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产气荚膜梭菌的快速检测提供有效技术手段。

关键词: 产气荚膜梭菌, plc基因, 实时荧光聚合酶重组酶扩增, 实时荧光聚合酶链式反应

Abstract: To develop a rapid detection method for Clostridium perfringens using real-time polymerase chain reaction (PCR) or real-time recombinase polymerase amplification (RPA), we designed specific primers and probe based on the conserved sequence of the plc gene of C. perfringens. The results of specificity analysis showed that the two established methods specifically detected C. perfringens but not other bacteria. The sensitivity of both methods was 1.3 pg/μL and the limit of detection for C. perfringens in artificially contaminated chicken and milk samples was 1.0 × 102 CFU/mL. The positive samples could be detected in 3–13 min by real-time RPA, and at least 24–46 min by real-time PCR (Ct = 17.45–33.65). Real-time RPA was better than real-time PCR with respect to analysis time, easiness of operation and portability. In conclusion, thanks to their high specificity and sensitivity and easy operation, real-time RPA and real-time PCR provided an effective technical approach for the rapid detection of C. perfringens.

Key words: Clostridium perfringens, plc gene, real-time recombinase polymerase amplification, real-time polymerase chain reaction

中图分类号:

R155.5
Q93.3

刘立兵，李睿文，陈志敏，王金凤，孙晓霞，袁万哲，王建昌. 产气荚膜梭菌实时荧光PCR和实时荧光RPA检测方法的建立和比较[J]. 食品科学, 2020, 41(4): 268-272.

LIU Libing, LI Ruiwen, CHEN Zhimin, WANG Jinfeng, SUN Xiaoxia, YUAN Wanzhe, WANG Jianchang. Development and Comparison of Real-Time Polymerase Chain Reaction and Real-Time Recombinase Polymerase Amplification Assays for Detection of Clostridium perfringens in Food[J]. FOOD SCIENCE, 2020, 41(4): 268-272.

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产气荚膜梭菌实时荧光PCR和实时荧光RPA检测方法的建立和比较

Development and Comparison of Real-Time Polymerase Chain Reaction and Real-Time Recombinase Polymerase Amplification Assays for Detection of Clostridium perfringens in Food

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