为建立牛精液中基因I型牛病毒性腹泻病毒(BVDV-1)的快速检测方法,本研究采用Sephycral S-400凝胶对牛精液过滤处理后提取病毒核酸,根据BVDV-1 5'UTR保守区基因序列,设计特异性引物和荧光探针,通过反应条件的优化,建立了牛精液中BVDV-1荧光定量RT-PCR检测方法.该方法可以检测到牛精液中含量为0.0125 TCID50的病毒,灵敏度比病毒分离方法高200倍～2000倍,比常规RT-PCR方法高10倍.对从同一个牛场6个月内采集的120份新鲜牛精液和40份冷冻牛精液用该荧光RT-PCR方法检测,没有检测到BVDV-1阳性样品.对其中的10头牛定期检测精液中病毒的同时,并同步检测了全血中的病毒和血清抗体,结果血清抗体阳性牛精液中没有检测到病毒,本研究结果表明,不能以血清抗体阴阳性做为精液是否带毒的依据.%A fluorescent quantitative RT-PCR (FQ-RT-PCR) method was established for direct detection of genotype Ⅰ BVDV in bovine semen using specific primers and probe derived from the BVDV 5' UTR region. BVDV RNA was extracted from raw bovine semen or extended bovine semen samples by including a sephacryl S-400 chromatography filtration step to remove inhibitors. The FQ-RT-PCR assay could detect 0.0125 TCID50 BVDV which was 200 to 2,000 times more sensitive than virus isolation method, and 10-fold more sensitive than conventional RT-PCR. The assay was used to test 120 bovine raw semen samples and 40 extended semen samples collected from a bull farm within 6 months and all detected BVDV negative. The semen samples and serum samples from 10 bulls were detected with the FQ-RT-PCR and specific BVDV antibodies detected with VN method at the same time. The results showed that semen- carrying virus could not be confirmed by serum antibody detection.
展开▼
