鸭疱疹病毒1型实时荧光定量PCR方法的建立

Establishing a Real-Time PCR Assay for Anatid Herpesvirus 1

目的 建立检测鸭疱疹病毒1型（鸭瘟病毒）的实时荧光定量PCR方法，为快速诊断、致病机理研究及抗病毒药物筛选等奠定基础。方法 根据病毒DNA聚合酶基因的序列，设计引物和探针，采用TaqMan探针技术进行实时荧光定量PCR，用含有125bp扩增产物的pMD18-T载体质粒为阳性对照，构建标准曲线，对该方法的特异性、可重复性、敏感性进行评价，同时与传统PCR方法进行比较研究。结果 标准曲线表明在2．3×10^5～2．3×10拷贝数之间有很好的线性关系（r=0．999）；实时荧光定量PCR最少可检测到23个阳性质粒，说明有很好的敏感性；试验内及试验间变异系数分别为1．22～6．69以及2．09～8．84，说明有较好的重复性；对非鸭瘟病毒DNA无扩增，说明有很好的特异性；在对病毒DNA的检测方面，比传统PCR的敏感性高出10^4倍。结论 成功建立了针对鸭瘟病毒的特异、敏感、重复性强且可准确定量的实时荧光定量PCR方法。

Objectives To develop a real-time quantitative PCR assay to detect cluck plague virus （DPV） for the rapid diagnosis of DPV infection, the investigation of its nosogenesis and the screening of effective antiviral drugs. Methods 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. Results A good linear correlation was demonstrated in the standard curve for the real-time PCR assay within the range from 2.3× 10 to 2.3× 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. Conclusion The real-time quantitative PCR assay for DPV DNA has good sensitivity, specificity and reliability.