C型禽偏肺病毒SYBR Green Ⅰ实时荧光定量PCR方法的建立及应用

Development and Application of a SYBR Green Ⅰ based Real-time Quantitative PCR Method for Avian Metapneumovirus Subtype C

根据GenBank发表的禽偏肺病毒(aMPV)F基因序列,设计一对特异性引物,建立C亚型aMPV的SYBR GreenⅠ实时荧光定量PCR方法。对该反应体系进行条件优化,建立了标准曲线,并进行特异性、敏感性及重复性试验,然后将建立的方法应用于临床样品和攻毒样品的检测。结果显示:标准曲线循环阈值与模板浓度呈现良好的线性关系;建立的方法只能检测出C亚型aMPV,最低可以检测到0.8×10~1拷贝/μL的核酸模板,重复性试验的变异系数小于4%。应用建立的方法对43份临床样品检测,结果显示均为阴性;对42份35日龄SPF鸡人工感染后1～21 d的气管和肺脏样品进行检测,结果显示攻毒样品均为阳性。因此,研究建立的特异、灵敏、重复性好的C型aMPV实时荧光定量PCR方法,既可适合于该病的早期诊断和流行病学调查,又为该病毒的致病机制研究提供技术支撑。

In order to establish a SYBR Green Ⅰ real-time quantitative PCR method for avian metapneumovirus （aMPV） subtype C, a pair of specific primers was designed based on F gene sequence of aMPV subtype C in CenBank. The reaction system was optimized and the standard curve was established. Specificity, sensitivity, and reproducibility tests were performed. The clinical samples and challenged samples were detected by the developed real-time PCR. The results showed that the standard curve showed a good linear relationship between threshold cycle and template concentration. The established method could only detect aMPV subtype C and had a detection limit of 0.8×10t copies/μL of initial templates. The method had a coefficient of variations less than 4% for both intra-and inter-assay. 43 clinical samples were detected by the established method and the results showed that all of clinical samples were negative for aMPV subtype C. 42 trachea and lung samples collected at 1 to 21 days post infection with aMPV subtype C in 35-day-age SPF chickens were detected and the results showed that all the challenged samples were positive. Therefore,a specific, sensitive and reproducible real-time quantitative PCR method for aMPV subtype C was developed in this study. The method can be used for early diagnosis and epidemiological investigation of the disease, and provide technical support for study of the pathogenesis of aMPV.