鹅源新城疫病毒实时荧光定量PCR检测方法的建立

Development of a real-time PCR assay for detection of Newcastle disease virus in goose

根据GenBank上鹅源新城疫病毒NA-1株M基因的序列，在保守区域设计并合成了1对引物，采用荧光嵌合法（SYBRGreenI）建立了检测鹅源新城疫病毒的实时荧光定量PCR（real—time PCR）。以鹅源新城疫病毒NA-1株反转录产物cDNA为标准品，通过优化反应条件，建立了标准曲线，并进行了融解曲线分析。结果，标准曲线的C1值检测范围为23～36，相关系数（r^2）为0．992；无引物二聚体及非特异性产物，且Tm=（83±0）℃。结果表明，建立的检测鹅源新城疫病毒实时荧光定量PCR方法特异性强、灵敏性高，能为以后快速诊断鹅源新城疫病毒提供有效保障。

According to the M gene sequence of Newcastle disease virus（NDV） strain NA-1 from goose available in GenBank,a pair of primers was designed for establishing a SYBR Green Ⅰ real-time PCR for M gene of NDV. To establish the standard curve,the cDNA of the NDV strain NA-1 was served as a standard. The analysis of melting curve was also carried out. The results indicated that the linear range of C, values ranged from 23 to 36 with a good correlation coefficient（r^2 = 0. 992） and the melting curve also showed a single peak with an Tm value of （83±0） ℃. It was concluded that the established real-time PCR assay was highly sensitive and specific, which could provide a tool for rapid diagnosis of NDV in clinical samples from geese.