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:2014-11-23
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:2014-11-23

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。

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.

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.

0
/ / 推荐

导出引用管理器 EndNote|Reference Manager|ProCite|BibTeX|RefWorks

链接本文: https://www.xmsyxb.com/CN/10.11843/j.issn.0366-6964.2014.11.012

https://www.xmsyxb.com/CN/Y2014/V45/I11/1824

参考文献

［1］RASMUSSEN L D，KRISTENSEN B，KIRKEBY C，et al.Culicoids as vectors of Schmallenberg virus［J］.Emerg Infect Dis，2012，18(7):1204-1206.
［2］VERONESI E，HENSTOCK M，GUBBINS S，et al.Implicating Culicoides biting midges as vectors of Schmallenberg virus using semi-quantitative RT-PCR［J］.PLoS One，2013，8(3):e57747.
［3］HOFFMANN B，SCHEUCH M，HÖPER D，et al.Novel orthobunyavirus in Cattle，Europe，2011［J］.Emerg Infect Dis，2012，18(3):469-472.
［4］BILK S，SCHULZE C，FISCHER M，et al.Organ distribution of Schmallenberg virus RNA in malformed newborns［J］.Vet Microbiol，2012，159(1-2):236-238.
［5］WERNIKE K，CONRATHS F，ZANELLA G，et al.Schmallenberg virus-Two years of experiences［J］.Prev Vet Med，2014，116（4）：423-434.
［6］FISCHER M，HOFFMANN B，GOLLER K V，et al.A mutation ‘hot spot’ in the Schmallenberg virus M segment［J］.J Gen Virol，2013，94(Pt6):1161-1167.
［7］BRÉARD E，LARA E，COMTET L，et al.Validation of a commercially available indirect ELISA using a nucleocapside recombinant protein for detection of Schmallenberg virus antibodies［J］.PLoS One，2013，8(1):e53446.
［8］HOFFMANN B，SCHULZ C，BEER M.First detection of Schmallenberg virus RNA in bovine semen，Germany，2012［J］.Vet Microbiol，2013，167(3-4):289-295.
［9］ZHANG Y，WU S，WANG J，et al.Expression and purification of the nucleocapsid protein of Schmallenberg virus，and preparation and characterization of a monoclonal antibody against this protein［J］.Protein Expr Purif，2013，92(1):1-8.
［10］张永宁，吴绍强，吕继洲，等.施马伦贝格病毒核衣壳蛋白的原核表达、纯化及多克隆抗体的制备［J］.畜牧兽医学报，2013，44（10）：1629-1636.
［11］张永宁，吴绍强，WERNIKE K，等.施马伦贝格病毒核衣壳蛋白单克隆抗体的制备及其特性分析［J］.细胞与分子免疫学杂志，2014，30（3）：289-293.
［12］张永宁，吴绍强，林祥梅.施马伦贝格病研究进展［J］.畜牧兽医学报，2014，45（7）：1029-1037.
［13］DOMINGUEZ M，HENDRIKX P，ZIENTARA S，et al.Preliminary estimate of Schmallenberg virus infection impact in sheep flocks-France［J］.Vet Rec，2012，171(17):426.
［14］MARTINELLE L，DAL POZZO F，GAUTHIER B，et al.Field veterinary survey on clinical and economic impact of Schmallenberg virus in Belgium［J］.Transbound Emerg Dis，2014，61(3):285-288.
［15］BLOMSTRÖM A L，STENBERG H，SCHARIN I，et al.Serological screening suggests presence of schmallenberg virus in cattle，sheep and goat in the zambezia province，mozambique［J］.Transbound Emerg Dis，2014，61(4):289-292.
［16］HOFFMANN B，BEER M，REID S M，et al.A review of RT-PCR technologies used in veterinary virology and disease control:sensitive and specific diagnosis of five livestock diseases notifiable to the World Organisation for Animal Health［J］.Vet Microbiol，2009，139(1-2):1-23.
［17］COUPEAU D，CLAINE F，WIGGERS L，et al.In vivo and in vitro identification of a hypervariable region in Schmallenberg virus［J］.J Gen Virol，2013，94(Pt6):1168-1174.
［18］FISCHER M，SCHIRRMEIER H，WERNIKE K，et al.Development of a pan-Simbu real-time reverse transcriptase PCR for the detection of Simbu serogroup viruses and comparison with SBV diagnostic PCR systems［J］.Virol J，2013，10:327.
［19］DE REGGE N，VAN DEN BERG T，GEORGES L，et al.Diagnosis of Schmallenberg virus infection in malformed lambs and calves and first indications for virus clearance in the fetus［J］.Vet Microbiol，2013，162(2-4):595-600.

[1]	孟令宅, 陈春丽, 于蒙蒙, 王占新, 王素艳, 刘鹏, 何塔娜, 郭茹, 陈运通, 刘长军, 祁小乐, 吴志强, 高玉龙. B亚型禽偏肺病毒对黄羽肉鸡致病性分析及其灭活疫苗免疫效果评价[J]. 畜牧兽医学报, 2023, 54(12): 5154-5161.
[2]	蒋盛强, 胡靖, 陈红英. H1N1亚型流感病毒感染A549细胞的环状RNA表达分析[J]. 畜牧兽医学报, 2023, 54(11): 4724-4734.
[3]	张傲, 谭斌, 刘可欣, 刘佳利, 张淑琴. 一株H1N1亚型猪流感病毒全基因组特征分析[J]. 畜牧兽医学报, 2023, 54(11): 4866-4871.
[4]	何辰香, 高闪电, 田占成, 独军政, 王锦明, 关贵全, 殷宏. 基于G_NS蛋白的牛流行热病毒感染与免疫鉴别诊断ELISA方法的建立[J]. 畜牧兽医学报, 2023, 54(10): 4320-4326.
[5]	常益铭, 汤承, 岳华. 牦牛源牛呼吸道合胞体病毒的分子流行病学调查[J]. 畜牧兽医学报, 2023, 54(5): 2030-2041.
[6]	方源, 侯巧弟, 项超辉, 赵红奕, 齐雪峰. IFITM3对小反刍兽疫病毒在山羊子宫内膜上皮细胞中增殖的调控效应[J]. 畜牧兽医学报, 2023, 54(5): 2200-2207.
[7]	李舜, 李桂珍, 原耀贤, 李康健, 马春全, 李守军, 黄良宗, 马骏. PA-X基因的长度变化对H3N2犬流感病毒复制能力和致病性的影响[J]. 畜牧兽医学报, 2023, 54(1): 272-280.
[8]	崔明仙, 王星博, 黄彦铭, 卞希一, 冯梦珂, 颜焰, 董伟仁, 周继勇. 3株H3N2亚型禽流感病毒的基因组特征与演化分析[J]. 畜牧兽医学报, 2022, 53(11): 4116-4122.
[9]	于蒙蒙, 包媛玲, 王素艳, 辛子琪, 刘鹏, 冯笑艳, 孟令宅, 郭茹, 张艳萍, 刘长军, 祁小乐, 李俊平, 王笑梅, 高玉龙. B亚型禽偏肺病毒的分离鉴定及致病性研究[J]. 畜牧兽医学报, 2022, 53(10): 3540-3549.
[10]	周勇, 李知新, 鲁宏伟, 孙燕, 李甜, 杜凡姝, 蒲娟. 我国H5和H7N9亚型高致病性禽流感的监测及疫情暴发分析[J]. 畜牧兽医学报, 2022, 53(9): 3093-3106.
[11]	潘春容, 邓瑞雪, 胡林杰, 朱学亮, 孙跃峰, 王桂荣, 蒙学莲. 组蛋白去乙酰化酶抑制剂对小反刍兽疫病毒复制的影响[J]. 畜牧兽医学报, 2022, 53(7): 2307-2316.
[12]	陈子轩, 张楠, 胡群, 全柯吉, 秦涛, 陈素娟, 彭大新, 刘秀梵. 我国H9N2亚型禽流感病毒血凝素蛋白145和153位抗原位点变异分析[J]. 畜牧兽医学报, 2022, 53(4): 1165-1172.
[13]	段志强, 谢玲玲, 陈佳琪, 唐宏, 王燕碧, 赵采芹, 赵佳福. 新城疫病毒M蛋白与宿主蛋白相互作用的研究进展[J]. 畜牧兽医学报, 2022, 53(1): 32-42.
[14]	严雅瑶, 顾敏, 刘秀梵. HA蛋白位点变异影响H7N9亚型流感病毒特性的研究进展[J]. 畜牧兽医学报, 2021, 52(8): 2093-2106.
[15]	蒋梅, 陈武, 翟俊琼, 卜婉迪, 谢逸伦, 刘灿彬, 单芬, 罗满林. 小熊猫源犬瘟热病毒株H、F基因的克隆及序列分析[J]. 畜牧兽医学报, 2021, 52(6): 1670-1676.

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

RichHTML

PDF

可视化

摘要/Abstract

引用本文

使用本文

参考文献

相关文章 15

编辑推荐

Metrics

本文评价