猫杯状病毒荧光定量PCR检测方法的建立及初步应用

根据GenBank中编码猫杯状病毒（FCV）衣壳蛋白ORF2的保守序列, 设计并合成了一对引物和相应的TaqMan探针, 建立了快速检测FCV的荧光定量PCR
方法. 通过对该方法的反应体系和反应条件进行优化, 建立了标准曲线, 给出了特异性、 敏感性和重复性实验, 并对临床24份疑似样品（其中阳性3份、 阴性21份）进行检测.  结果表明: 该方法检测cDNA的线性关系为0.992, 线性范围为2.26×10¹¹~2.26×10¹拷贝/μL; 可检测出样品中的FCV, 其他猫相关病毒检测为阴性; 批内重复性实验变异系数为2.158%， 与病毒分离结果相符.

Development and Preliminary Application of Real TimePCR Assayfor Detection and Quantization of Feline Calicivirus

To detect feline calicivirus (FCV), a real time quantitative PCR (qPCR) assay was established by using a pair of primers and TaqMan probe designed and synthesized according to the conserved capsid protein gene ORF2sequence of FCV in the GenBank. The reaction system and conditions of the qPCR assay were optimized and the standard curve was established. Further, the specificity, sensitivity and repeatability test were also assessed. The established quantitative PCR assay was applied to detecting clinical samples infected by FCV. The results show that the correlation rate of the standard curve for the qPCR was 0.992. The detected quantity was from 2.26×10¹ to 2.26×10¹¹ copies/μL of FCV cDNA. With the qPCR method, a reliable diagnostic result was obtained fordetecting FCV samples. But detection of other feline pathogenic agents was negative. In addition, reproducible assay showed a good performance of repeatabilityand the variation coefficient of intragroup was 2.158%.