猪瘟病毒巢式RT-PCR检测方法的建立与应用

doi:10.16431/j.cnki.1671-7236.2016.09.010

《中国畜牧兽医》 ›› 2016, Vol. 43 ›› Issue (9): 2285-2290.doi: 10.16431/j.cnki.1671-7236.2016.09.010

猪瘟病毒巢式RT-PCR检测方法的建立与应用

刘梅芬¹, 王淑娟¹, 闫若潜¹, 王东方¹, 曹伟伟¹, 赵雪丽¹, 李勤楠², 李宁²

1. 河南省动物疫病预防与控制中心, 郑州 450008;
2. 河南科技大学动物科技学院, 洛阳 471003

收稿日期:2016-01-25 出版日期:2016-09-20 发布日期:2016-09-20
通讯作者: 闫若潜 E-mail:yrq1688@126.com
作者简介:刘梅芬(1971-),女,兽医师,研究方向:动物疫病病原分子生物学,E-mail:mfen2008@163.com;王淑娟(1985-),女,兽医师,研究方向:动物疫病病原分子生物学,E-mail:snowangel517@163.com
基金资助:
河南省科技攻关项目“病死动物无害化处理技术及管理机制研究”（142102310224）

Establishment and Application of Nested RT-PCR Assay for Detection of Classical Swine Fever Virus

LIU Mei-fen¹, WANG Shu-juan¹, YAN Ruo-qian¹, WANG Dong-fang¹, CAO Wei-wei¹, ZHAO Xue-li¹, LI Qin-nan², LI Ning²

1. Henan Centre for Animal Disease Control & Prevention, Zhengzhou 450008, China;
2. College of Animal Science and Technology, Henan University of Science and Technology, Luoyang 471003, China

Received:2016-01-25 Online:2016-09-20 Published:2016-09-20

摘要/Abstract

摘要：

为建立一种快速、特异、灵敏的检测猪瘟病毒（classical swine fever virus，CSFV）巢式RT-PCR（nested RT-PCR）方法，本研究参照GenBank中公布的CSFV E2基因保守区域序列设计2对特异性引物，以CSFV总RNA为模板，优化反应条件，建立CSFV nested RT-PCR方法，对其进行特异性、敏感性和重复性试验；利用所建立的方法对35份临床疑似CSFV感染样品进行了应用检测，并对阳性PCR产物进行克隆测序鉴定。结果表明，本研究成功建立了CSFV nested RT-PCR检测方法，能够特异性地扩增CSFV，但对ST正常细胞对照和其他8种病原对照未扩增出任何条带；稳定性和重复性好；敏感性高，最低检测病毒含量为1 TCID₅₀；自35份疑似CSFV感染样品中检出19份阳性样品，PCR阳性产物克隆测序结果表明均为CSFV E2基因片段。本研究成功建立了CSFV nested RT-PCR检测方法，可用于CSFV的快速检测，为CSF的早期检测诊断提供了特异、灵敏的方法。

关键词: 猪瘟病毒; 巢式RT-PCR; 检测; 建立; 应用

Abstract:

A nested RT-PCR for detection of classical swine fever virus (CSFV) was developed using the total RNA of CSFV as a reverse transcription template,and two pairs of specific primers were designed based on the conserved region of CSFV E2 gene.The specificity,sensitivity and repetition of CSFV nested RT-PCR were tested,and 35 samples taken from clinical suspicious CSFV infected pigs were tested by the established nested RT-PCR assay.The results indicated that the nested RT-PCR assay was successfully established.The sensitivity and specificity of the nested RT-PCR assay revealed that the minimum detectable virus content limit was 1 TCID₅₀,and no products were amplified from the nucleic acid of ST normal cell or other 8 pathogenic viral or bacterial microorganisms.The repetition test indicated the nested RT-PCR assay showed a good repeatability.Meanwhile,19 positive samples were detected in 35 suspected samples,which was consistent with the results tested by sequencing.The study suggested that the established nested RT-PCR assay was specific,sensitive and suitable for early detection of CSFV.

Key words: classical swine fever virus; nested RT-PCR; detection; establishment; application

中图分类号:

S852.65⁺1

刘梅芬, 王淑娟, 闫若潜, 王东方, 曹伟伟, 赵雪丽, 李勤楠, 李宁. 猪瘟病毒巢式RT-PCR检测方法的建立与应用[J]. 《中国畜牧兽医》, 2016, 43(9): 2285-2290.

LIU Mei-fen, WANG Shu-juan, YAN Ruo-qian, WANG Dong-fang, CAO Wei-wei, ZHAO Xue-li, LI Qin-nan, LI Ning. Establishment and Application of Nested RT-PCR Assay for Detection of Classical Swine Fever Virus[J]. , 2016, 43(9): 2285-2290.

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猪瘟病毒巢式RT-PCR检测方法的建立与应用

Establishment and Application of Nested RT-PCR Assay for Detection of Classical Swine Fever Virus

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