| 基于锁核酸探针的双重荧光实时RT-PCR检测方法鉴别新城疫中强毒株与弱毒株 |
| |
| 引用本文： | 秦智锋,刘建利,卢体康,林庆燕,吕建强,曹琛福,阮周曦,曾少灵,陈书琨,廖立珊,孙洁,张彩虹,花群义.基于锁核酸探针的双重荧光实时RT-PCR检测方法鉴别新城疫中强毒株与弱毒株[J].中国预防兽医学报,2012(9):719-723. |
| |
| 作者姓名： | 秦智锋 刘建利 卢体康 林庆燕 吕建强 曹琛福 阮周曦 曾少灵 陈书琨 廖立珊 孙洁 张彩虹 花群义 |
| |
| 作者单位： | 深圳出入境检验检疫局;深圳市外来有害生物质检测技术研发重点实验室 |
| |
| 基金项目： | 深圳市基础研究科研项目(JC2009031907784) |
| |
| 摘 要： | 为鉴别新城疫病毒(NDV)强毒株和弱毒株,本研究建立了基于新型锁核酸(LNA)探针的实时荧光RT-PCR检测方法(Duplex LNA rRT-PCR)。该方法针对NDVF基因裂解位点设计了两条新型LNA探针,通过对11株NDV株进行大将军脂duplex LNA rRT-PCR检测方法检测,验证该方法的特异性;通过对副粘病毒I型(APMV-1)和NDV中强毒株(vNDV)不同浓度病毒液进行检测,确定该方法的灵敏度,并与TaqMan实时荧光RT-PCR检测方法进行比较。结果显示本研究所建立的方法对11株NDV检测的特异性为100%(11/11),优于TaqMan实时荧光RT-PCR检测方法(10/11);所建立的duplex LNA rRT-PCR方法检测中强毒株F48E9和弱毒株LaSota的灵敏度分别为10个EID50和0.1个EID50,比美国农业部推荐的TaqMan实时荧光RT-PCR检测方法低10倍。本研究利用新型LNA探针技术,建立了鉴别NDV中强毒株与弱毒株的duplex LNA rRT-PCR检测方法,可以特异性检测NDV并有效区分中强毒株与弱毒株,适合用于鸡场和进出境动物产品中NDV的快速检测。
|
| 关 键 词： | 新城疫病毒 中强毒株 弱毒株 LNART-PCR 鉴别检测 |
Development of duplex locked nucleic acid real-time RT-PCR to differentiate the pathovars of Newcastle disease virus |
| |
| QIN Zhi-feng,LIU Jian-li,LU Ti-kang,LIN Qing-yan,LV Jian-qiang,CAO Chen-fu,RUAN Zhou-xi,ZENG Shao-ling,CHEN Shu-kun,LIAO Li-shan,SUN Jie,ZHANG Cai-hong,HUA Qun-yi.Development of duplex locked nucleic acid real-time RT-PCR to differentiate the pathovars of Newcastle disease virus[J].Chinese Journal of Preventive Veterinary Medicine,2012(9):719-723. |
| |
| Authors: | QIN Zhi-feng LIU Jian-li LU Ti-kang LIN Qing-yan LV Jian-qiang CAO Chen-fu RUAN Zhou-xi ZENG Shao-ling CHEN Shu-kun LIAO Li-shan SUN Jie ZHANG Cai-hong HUA Qun-yi |
| |
| Institution: | 1(1.Shenzhen Entry-Exit Inspection & Quarantine Bureau,Shenzhen 518001,China; 2.Shenzhen Key Laboratory of Inspection Research & Development of Alien Pests,Shenzhen 518045,China) |
| |
| Abstract: | To differentiate velogenic(highly virulent),mesogenic(intermediate virulence) from lentogenic(nonvirulent) NDV,a duplex real-time RT-PCR(duplex LNA rRT-PCR) was developed based on locked nucleic acid(LNA) directed at the fusion-cleavage site of NDV.The sensitivity and specificity of the duplex LNA rRT-PCR was evaluated with the avian paramyxovirus-1(APMV-1) TaqMan real time RT-PCR(TaqMan-APMV-1-rRT-PCR) assay and virulent(velogenic and mesogenic) NDV TaqMan real time RT-PCR assay(TaqMan-vNDV-rRT-PCR) validated by Unite State department agriculture(USDA).The specificity of the the duplex LNA rRT-PCR was superior to APMV-1-rRT-PCR.The sensitivity of F48E9 strain to LNA assay was 10 times less than TaqMan real time RT-PCR assay,but the same for lentogenic strain(LaSota).These results showed that the duplex LNA-rRT-PCR is a promising method for routine quarantine of NDV and clinic monitoring of Newcastle disease. |
| |
| Keywords: | Newcastle disease virus velogenic and mesogenic strains lentogenic strains LNA RT-PCR pathetyping |
| 本文献已被 CNKI 等数据库收录！ |
|
