Yang Fa-long, Jia Wen-xiang, Xie Yi, Zeng Wei, Yang Wei-qing, Yue Hua
Department of Microbiology, West China School of Preclinical and Forensic Medicine, Sichuan University, Chengdu 610041, China.
Sichuan Da Xue Xue Bao Yi Xue Ban. 2006 Sep;37(5):801-3, 813.
To develop a real-time quantitative PCR assay to detect duck plague virus (DPV) for the rapid diagnosis of DPV infection, the investigation of its nosogenesis and the screening of effective antiviral drugs.
The primer and probe were designed according to the gene sequence of DPV DNA polymerase gene. To establish a standard curve, a plasmid containing 125 bp PCR product was constructed and severed as a positive control. The TaqMan based real-time PCR method was adopted and compared with the traditional PCR approach in sensitivity, reliability and specificity.
A good linear correlation was demonstrated in the standard curve for the real-time PCR assay within the range from 2.3 x 10 to 2.3 x 10(5) copies. A minimum of 23 positive plasmids could be detected, indicating a good sensitivity of the assay. The coefficients of variance (CVs) were 1.22-6.69 and 2.09-8.84 for the intra-assay and inter-assay tests respectively, indicating a good reliability. No amplification products were found for DNA from other pathogens, indicating a good specificity. The comparative study proved that the TaqMan technology was much more sensitive than traditional PCR assay.
The real-time quantitative PCR assay for DPV DNA has good sensitivity, specificity and reliability.
建立一种实时定量PCR检测方法以检测鸭瘟病毒（DPV），用于DPV感染的快速诊断、发病机制研究及有效抗病毒药物的筛选。
根据DPV DNA聚合酶基因序列设计引物和探针。构建含125 bp PCR产物的质粒作为阳性对照建立标准曲线。采用基于TaqMan的实时PCR方法，并与传统PCR方法在灵敏度、可靠性和特异性方面进行比较。
实时PCR检测方法的标准曲线在2.3×10至2.3×10(5)拷贝范围内呈现良好的线性关系。最低可检测到23个阳性质粒，表明该检测方法具有良好的灵敏度。批内和批间检测的变异系数（CVs）分别为1.22 - 6.69和2.09 - 8.84，表明可靠性良好。未发现其他病原体DNA的扩增产物，表明特异性良好。对比研究证明TaqMan技术比传统PCR检测方法更灵敏。
DPV DNA的实时定量PCR检测方法具有良好的灵敏度、特异性和可靠性。
