猪瘟病毒荧光定量RT-PCR检测方法的建立及其初步应用

Development and preliminary application of fluorescence quantitative RT-PCR for detection of classical swine fever virus

针对CSFV基因组5′端非编码区序列设计并合成了高度特异的一对引物和一条探针,用于猪瘟病毒实时荧光定量PCR检测方法的建立。将提取的病毒的总RNA做为模板进行反转录和PCR,将PCR产物克隆到pMD18-T载体后进行大肠杆菌转化,提取阳性质粒做为标准品绘制标准曲线,成功地建立了特异性检测CSFV的荧光定量RT-PCR方法,其灵敏度达到100拷贝/μL。将猪瘟活疫苗(细胞源)采用不同剂量免疫猪后,取全血及组织,用所建立的方法进行检测,发现病毒主要分布在猪的血液、脾脏、淋巴结、扁桃体中。

A set of primers and a TaqMan probe directed to the 5＇ NTR of CSFV genome sequences were designed for the PCR detection of CSFV. The total RNA extracted from the virus was used as the template for reverse transcription and PCR. The PCR products were cloned into pMD 18-T vector and the plasmid isolated from the positive clone was used for the standard material for the standard curve. So the specific RT-PCR method for detection of CSFV was established. The sensitivity of the assay was 10~ copies/tJ L. The viruses were detected in blood, spleen, lymph nodes and tonsils of the pigs immunized with different doses of classical swine fever cell vaccine.