猪圆环病毒3型TaqMan-MGB荧光定量PCR方法的建立及应用

Establishment and application of TaqMan-MGB real-time PCR assay for detection of porcine circovirus 3

为建立猪圆环病毒3型(PCV3)荧光定量PCR检测方法,本研究根据GenBank中PCV3基因序列设计特异性引物和探针,经过反应体系和条件优化,建立了特异性检测PCV3的TaqMan-MGB荧光定量PCR方法。该检测方法在4.78×10~1拷贝/μL～4.78×10~9拷贝/μL质粒标准品范围内均有良好的线性关系;该方法特异性试验结果显示,其与多种常见猪病病毒均无交叉反应,特异性良好;本研究建立的方法敏感性是常规PCR方法的100倍,敏感性较高;批内批间重复性试验变异系数均小于2.3%,重复性良好。对临床样品的检测结果显示,该方法对PCV3的检出率高于常规PCR方法,并且PCV3阳性样品多存在混合感染情况。该方法的建立为PCV3的实验室诊断及流行病学调查提供了快速、准确的检测手段。

To established a sensitive detection method of porcine circovirus 3(PCV3), a TaqMan-MGB real-time PCR assay was established using specific primers and probe designed according to the genome sequence of PCV3. With the optimized reactions conditions, the established method was specific for PCV3 detection without cross-reaction with other common porcine viruses. Results showed that the method had linearity within the template ranges from 4.78×10~1 to 4.78×10~9 copies/μL and was100 times more sensitive than that of routine PCR assay. The coefficient of variation in the reproducible assays was less than 2.3%. The positive detection rate of PCV3 in clinical samples using the developed method was higher than that using the conventional PCR method and the PCV3 positive samples were mostly mixed infection. The establishment of the real-time PCR method provides a rapid and accurate detection means for the laboratory diagnosis and epidemiological investigation of PCV3.