荧光定量RT-PCR检测中、强毒力新城疫病毒方法的建立及验证

根据新城疫病毒F基因的裂解位点序列,设计合成一对引物和TaqMan探针,以本室构建并保存的鹅源新城疫病毒ZJ1株F基因阳性重组质粒作为中、强毒力新城疫病毒RNA定量检测的标准品,建立检测方法。结果表明本试验建立的标准曲线循环阈值（Ct值）与模板浓度具有良好的线性关系,相关系数为0.999,灵敏度约为3拷贝/μL,对禽流感病毒和其他禽病病毒无交叉反应,特异性好、重复性佳,为中、强毒力新城疫病毒检测提供了一种快速高效的定量检测方法。对28株标准分离株强弱毒力的检测与经典病毒分离方法符合率达100%,对187份临床样品的检测,二者结果符合率接近90.0%。在新城疫病毒临床样品快速检测、流行病学监测等方面显示良好的应用前景。

Development and Validation of Real-time RT-PCR Assay for Detection of Mesogenic and Virulent Newcastle Disease Virus

According to the cleavge site sequence of Newcastle disease virus（NDV） F gene, a pair of primers and a T aqMan probe were designed and synthesized. A serial 10 fold dilutions positive plasmid was prepared and used for standard. The standard curve revealed the linear relationship between CT （cycle threshold） and template concentration with a good correlation （R2=0.999）. The RRT-PCR method was highly specific and sensitive and could be used for rapid quantitative detection of mesogenic and virulent NDV. No cross-reaction was detected against other avian disease viruses. Sensitivity was 3 copies ofNDV genome. The total meet rate with traditional virus isolation method was about 90.0% in detecting 187 clinical samples as well as 100% in 28 standard isolated strains. It showed that a real-time RT- PCR method for quick, specific sensitive and quantitativedetection of mesogenic and virulent NDV had been established in this study and it displayed good prospects in the rapid screening of clinical samples and epidemiological monitoring of NDV.