检测施马伦贝格病毒核酸的荧光定量RT-PCR方法

doi:10.11843/j.issn.0366-6964.2014.11.012

畜牧兽医学报 ›› 2014, Vol. 45 ›› Issue (11): 1824-1829.doi: 10.11843/j.issn.0366-6964.2014.11.012

检测施马伦贝格病毒核酸的荧光定量RT-PCR方法

张永宁，吴绍强，吕继洲，冯春燕，王彩霞，邓俊花，袁向芬，林祥梅^*

（中国检验检疫科学研究院动物检疫研究所，北京 100029）

收稿日期:2014-05-05 出版日期:2014-11-23 发布日期:2014-11-24
通讯作者: 林祥梅，E-mail：linxm@caiq.gov.cn
作者简介:张永宁（1980-），男，山东日照人，副研究员，博士，主要从事外来动物疫病检疫研究，E-mail:zhangyn@caiq.gov.cn
基金资助:
国家质检总局科技计划项目（2013IK054）；“十二五”国家科技支撑计划课题（2013BAD12B01）

A Real-time Quantitative Reverse Transcription-PCR for the Detection of Schmallenberg Virus Nucleic Acid

ZHANG Yong-ning，WU Shao-qiang，LYU Ji-zhou，FENG Chun-yan，WANG Cai-xia，DENG Jun-hua，YUAN Xiang-fen，LIN Xiang-mei^*

(Institute of Animal Quarantine，Chinese Academy of Inspection and Quarantine，Beijing 100029，China)

Received:2014-05-05 Online:2014-11-23 Published:2014-11-24

摘要/Abstract

摘要：

根据施马伦贝格病毒（Schmallenberg virus，SBV）S基因节段设计一对引物和TaqMan探针，建立了检测SBV核酸的荧光定量RT-PCR方法。通过优化反应体系和反应条件，该方法对10¹～10⁷半数组织培养感染量（TCID₅₀）的SBV核酸呈现良好的线性关系。利用德国FLI制备和保存的SBV核酸阳性（n=140）、SBV核酸阴性（n=132）和辛波血清群相关病毒核酸样品（n=16），对该方法的敏感性、特异性和重复性进行了验证。结果表明，所建立方法与FLI研制的SBV荧光定量RT-PCR具有相近的敏感性，两者对140份SBV核酸阳性样品的检测符合率达96.4%（135/140）；可灵敏地检测出1 TCID₅₀的SBV RNA，并具有良好的重复性；除道格拉斯病毒（Douglas virus）外，与辛波血清群相关病毒核酸无交叉反应。用该方法对采自内蒙古呼和浩特市某养殖场的绵羊血清（n=47）和澳大利亚进口绵羊血清（n=47）的RNA进行了检测，结果未发现SBV核酸阳性样品。SBV荧光定量RT-PCR检测方法的建立为应对施马伦贝格病提供了有效的检测手段。

关键词: 施马伦贝格病毒, 核酸, 引物, TaqMan探针, 荧光定量RT-PCR

Abstract:

In the present study，a pair of primers and a TaqMan probe were designed based on the S-segment sequence of Schmallenberg virus (SBV)，and a real-time quantitative reverse transcription-PCR (RT-qPCR) for the detection of SBV nucleic acid was established.After optimization of the reaction system and condition，the method presented a good linear relationship with the RNA template ranging from 10¹ to 10⁷ 50% tissue culture infectious dose (TCID₅₀) SBV.By using SBV nucleic acid-positive samples (n=140)，SBV nucleic acid-negative samples (n=132)，and RNA samples (n=16) of related viruses within the Simbu serogroup，the sensitivity，specificity and reproducibility of the RT-qPCR were validated.All the samples used for validation were prepared and preserved by the Friedrich-Loeffler-Institut (FLI) of Germany.The results demonstrated that the developed RT-qPCR had a similar sensitivity with that of FLI and the concordance rate between the two assays was 96.4% (135/140)，that the RT-qPCR could sensitively detect 1 TCID₅₀ SBV RNA and it had a good reproducibility，and that the RT-qPCR showed no cross-reactivity with related viruses within the Simbu serogroup except for Douglas virus.The RT-qPCR was further used to detect the RNA samples extracted from sheep sera which were collected from a breeding farm located in Inner Mongolia (n=47) and from the sheep imported from Australia (n=47)，respectively.However，no SBV nucleic acid-positive sample was detected.The successful development of SBV RT-qPCR provides us with an efficient detection tool for dealing with Schmallenberg disease.

Key words: Schmallenberg virus (SBV), nucleic acid；primer, TaqMan probe, real-time quantitative reverse transcription-PCR (RT-qPCR)

中图分类号:

S852.659.5

张永宁，吴绍强，吕继洲，冯春燕，王彩霞，邓俊花，袁向芬，林祥梅. 检测施马伦贝格病毒核酸的荧光定量RT-PCR方法[J]. 畜牧兽医学报, 2014, 45(11): 1824-1829.

ZHANG Yong-ning，WU Shao-qiang，LYU Ji-zhou，FENG Chun-yan，WANG Cai-xia，DENG Jun-hua，YUAN Xiang-fen，LIN Xiang-mei. A Real-time Quantitative Reverse Transcription-PCR for the Detection of Schmallenberg Virus Nucleic Acid[J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2014, 45(11): 1824-1829.

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检测施马伦贝格病毒核酸的荧光定量RT-PCR方法

A Real-time Quantitative Reverse Transcription-PCR for the Detection of Schmallenberg Virus Nucleic Acid

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