猪瘟病毒实时荧光定量PCR检测方法的建立与应用

doi:10.16431/j.cnki.1671-7236.2017.07.029

《中国畜牧兽医》 ›› 2017, Vol. 44 ›› Issue (7): 2112-2118.doi: 10.16431/j.cnki.1671-7236.2017.07.029

猪瘟病毒实时荧光定量PCR检测方法的建立与应用

王淑娟, 刘梅芬, 闫若潜, 班付国, 赵雪丽, 马震原, 王华俊, 王翠, 赵明军

河南省动物疫病预防控制中心, 郑州 450008

收稿日期:2017-01-13 出版日期:2017-07-20 发布日期:2017-07-22
通讯作者: 闫若潜 E-mail:yrq1688@126.com
作者简介:王淑娟(1985-),女,河南鹤壁人,硕士,兽医师,研究方向:动物疫病病原分子生物学,E-mail:snowangel517@163.com;刘梅芬(1971-),女,湖南茶陵人,学士,兽医师,研究方向:动物疫病病原分子生物学,E-mail:mfen2008@163.com
基金资助:
河南省科技创新人才计划项目"猪重要疫病快速检测集成技术研究"（174200510003）

Establishment and Application of Real-time Quantitative PCR Assay for Detection of Classical Swine Fever Virus

WANG Shu-juan, LIU Mei-fen, YAN Ruo-qian, BAN Fu-guo, ZHAO Xue-li, MA Zhen-yuan, WANG Hua-jun, WANG Cui, ZHAO Ming-jun

Henan Centre for Animal Disease Control & Prevention, Zhengzhou 450008, China

Received:2017-01-13 Online:2017-07-20 Published:2017-07-22

摘要/Abstract

摘要：

为建立一种快速、敏感、特异的猪瘟病毒（classical swine fever virus，CSFV）实时荧光定量PCR检测方法，本研究根据GenBank中CSFV E2基因保守区域序列，设计了一对特异性引物和一条特异性探针，以CSFV总RNA为反转录模板，经优化反应条件，建立CSFV实时荧光定量PCR检测方法，并对其进行了特异性、敏感性、重复性试验；利用所建立的方法对35份临床疑似CSFV感染样品进行了检测。结果表明，本研究建立的CSFV实时荧光定量PCR检测方法在10¹~10⁶拷贝/μL范围内有很好的线性关系，相关系数为0.999；CSFV细胞培养物出现阳性扩增信号，但ST正常细胞对照和其他8种病原对照未出现扩增，特异性良好；该方法重复性好、敏感性高，最低检测模板浓度为10拷贝/μL，并且CSFV的最低检测限为1 TCID₅₀/mL；自35份疑似CSFV感染样品中检出19份阳性样品，与本课题组建立的CSFV Nested RT-PCR检测结果和克隆测序结果一致。本研究成功建立了CSFV实时荧光定量PCR检测方法，可用于CSFV的快速检测。

关键词: 猪瘟病毒; 实时荧光定量PCR; 特异性; 重复性; 敏感性; 应用

Abstract:

A Real-time quantitative PCR assay for detection of classical swine fever virus (CSFV) was developed using the specific probe and primers designed basing on the E2 gene of CSFV. The Real-time quantitative PCR assay was established using the total RNA of CSFV as template. The specificity, sensitivity and repeatability of the assay were tested, and samples taken from clinic suspicious CSFV infected pigs had been testified by the established assay. The results indicated that the Real-time quantitative PCR assay was successfully established, and showed a good linear relationship at a template range of 10¹ to 10⁶ copies/μL with a coefficient correlation of 0.999; The specificity of the assay revealed that amplifications were showed on CSFV samples, but other pathogens had no amplifications; The sensitivity of the assay was 10 copies/μL nucleic acid and 1 TCID₅₀/mL virus; Meanwhile,19 positive samples were detected, which were consistent with results of CSFV detected by Nested RT-PCR, cloning and sequencing. The eatablished Real-time quantitative PCR assay was specific, sensitive rapid and suitable for early detection and epidemiological study of CSFV.

Key words: classical swine fever virus; Real-time quantitative PCR; specificity; repeatability; sensitivity; application

中图分类号:

S852.65⁺1

王淑娟, 刘梅芬, 闫若潜, 班付国, 赵雪丽, 马震原, 王华俊, 王翠, 赵明军. 猪瘟病毒实时荧光定量PCR检测方法的建立与应用[J]. 《中国畜牧兽医》, 2017, 44(7): 2112-2118.

WANG Shu-juan, LIU Mei-fen, YAN Ruo-qian, BAN Fu-guo, ZHAO Xue-li, MA Zhen-yuan, WANG Hua-jun, WANG Cui, ZHAO Ming-jun. Establishment and Application of Real-time Quantitative PCR Assay for Detection of Classical Swine Fever Virus[J]. , 2017, 44(7): 2112-2118.

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猪瘟病毒实时荧光定量PCR检测方法的建立与应用

Establishment and Application of Real-time Quantitative PCR Assay for Detection of Classical Swine Fever Virus

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