猪瘟病毒实时荧光定量RT-PCR的建立及其对人工感染猪体内猪瘟病毒的检测

Detection of Classical Swine Fever Virus in Experimentally Infected Pigs Using a Newly Developed Real-time Fluorescent Quantitative RT-PCR

本研究应用猪瘟病毒通用引物和野毒特异性TaqMan探针,并以质粒作为阳性标准品,结合Corbett公司的RotorGene3000荧光定量PCR仪,建立了一种敏感、特异、重复性好的快速检测猪瘟病毒核酸载量的TaqMan荧光定量RT-PCR方法。该方法在10^8-10^1范围内具有良好的线性关系,可检测到初始模板中10拷贝/μL的病毒核酸,与本实验室建立的RT-套式PCR（RT-nPCR）具有相近的敏感性,二者对152份不同样品检测符合率达94.7%。应用本方法对人工感染10^6TCID50猪瘟石门强毒的猪只感染后0-14 d全血中猪瘟病毒RNA进行了定量检测,揭示了猪瘟病毒在猪体内复制的动态变化,证实了感染猪临床表现与病毒滴度存在明显的时间相关性。此外发现,在同居感染猪出现猪瘟临床症状前3-4 d可从其全血中检出病毒RNA。

A real-time fluorescent quantitative RT-PCR assay for detecting classical swine fever virus （CSFV） was established and evaluated in experimentally infected pigs. The sensitivity of the assay was 10copies/μL viral RNA, with an agreement of 94. 7% with RT-nested PCR in detecting 152 different samples. The assay was used to detect CSFV in the whole blood samples of the pigs infected with 10^6TCID50 Shimen strain and contact pigs. The results showed that there was an excellent correlation between clinical symptoms and viral RNA loads in the blood. Viral RNA can be detected in the blood samples of contact pigs 3 to 4 days prior to the onset of clinical signs.