作 者: ; ; ; ; ; ; ; ; ; ; ; ; ;
机构地区: 深圳出入境检验检疫局
出 处: 《中国预防兽医学报》 2012年第9期719-723,共5页
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。 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-I-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.