新城疫病毒荧光RT-PCR检测方法的建立及应用

Establishment and application of fluorescent RT-PCR for detection of Newcastle disease virus

经对部分发表在NCBI里的新城疫病毒序列进行比对，在其F基因上找到1段相对保守序列，根据保守序列设计1对引物和1条TaqMan探针，以新城疫I系疫苗株（Mukteswar株）作为标准品，通过系列条件优化、特异性、灵敏性和重复性试验，建立了一种灵敏度高、特异性强和重复性好的荧光RT-PCR检测新城疫病毒的方法。该方法能检测最低初始病毒浓度为10^2.2ELD50／mL，且在10^8.2～10^2.2ELD50／mL内Ct值与病毒浓度的对数值（1gELD50／mL）呈很好的线性关系，R^2达到0．9975；该方法对禽流感、禽痘病毒和禽大肠杆菌等禽病病原核酸无交叉反应；批间和批内重复性良好；临床样品检测中荧光RT—PCR与鸡胚分毒的符合率、诊断灵敏度和诊断特异性分别为98．5％，50．0％和98．4％。因此，荧光RT—PCR检测方法的建立为新城疫病毒检测提供了一种快速有效的方法，为新城疫病毒的研究奠定了一定的基础。

A relatively conservative sequence was found in Newcastle disease virus F gene by align-menting Newcastle disease virus sequences published in NCBI. A pair of primers and a probe were designed based on the relatively conservative sequence. A high sensitivity,specificity and reproduc-ibility method of fluorescent RT-PCR for detecting Newcastle disease virus was established by u-sing Newcastle disease vaccine strain of system I （Mukteswar） as standard and a series of condi-tion optimizations, specific experiment, sensitivity experiment and repeated experiment. The lowest concentration of the initial virus detected by the method was 10^2.2 ELD50/mL,and in the range of 10^8.2-10^2.2 ELD50/mL,Ct value and the logarithm of the concentration of the virus （lgELD50/mL） showed a good linear relationship ,R2 was 0. 997 5. The method had no cross-reaction to avian in-fluenza virus,fowl pox virus,E, coli and other avian pathogenic nucleic ; and inter-and intra-assay reproducibility is good. In comparison to the method of virus isolation by chicken embryo, the a-greement,diagnostic sensitivity and diagnostic specificity of fluorescent RT-PCR were 98. 5%, 50.0% and 98.4%, respectively, in clinical sample detection. For this reason, the development of fluorescent RT-PCR method provided a rapid and effective method for Newcastle disease virus de- tection and lay a foundation for the study of Newcastle disease virus.