基于M基因的新城疫病毒实时荧光定量RT-PCR的建立及其对临床样品中新城疫病毒检测的研究

Development of a Rea1-Time Fluorescent Quantitative RT-PCR Based on M Gene of Newcastle Disease Virus (NDV) to Detect NDV in Clinical Samples

根据新城疫病毒M基因的保守序列，设计合成1对引物和TaqMan探针，以作者实验室构建并保存的鹅源新城疫病毒ZJ1株M基因阳性重组质粒为标准品模板建立荧光定量PCR标准曲线，结合ABI公司的7300型荧光定量PCR仪，建立了一种敏感、特异、重复性好的快速检测新城疫病毒核酸载量的TaqMan荧光定量RT-PCR方法。该方法在106~101拷贝范围内具有良好的线性关系，可检测到初始模板中3拷贝.μL-1的病毒核酸，与传统的病毒分离方法具有相近的敏感性，二者对500份临床禽泄殖腔棉拭样品检测结果阳性数？阴性数符合率分别为90.0%、99.8%。经临床应用表明：荧光定量PCR的建立为早期诊断禽新城疫病毒、定量分析禽新城疫病毒感染程度奠定了基础。

Pair of primers and a TaqMan probe were synthesized according to M gene conservative sequence of Newcastle disease virus. The positive recombinant plasmid containing M gene of NDV ZJ1 strain isolated from goose was used as a positive quantitative template to establish a standard curve. And then a real-time fluorescent quantitative RT-PCR assay was established. The method has a good linear relationship within the 10^6 to 10^1copies, with which 3 copies ·uL^-1 of the virus nucleic acid can be detected in the initial template, and has similar sensitivity with traditional virus isolation methods. The conforming rate of positive sum and negative sum with traditional virus isolation method was 90.0% and 99.8% respectively in detecting 500 clinic cloacal swab samples. The result showed that the constructed method paved the way for the early and rapid detection of NDV as well as quantitative analysis for the infection degree of NDV.