Establishment of real-time PCR for direct detection of IBRV in semen and relation between semen carrying virus and serum antibody to IBRV

• Published in: Xi bei nong lin ke ji da xue xue bao.Zi ran ke xue ban Vol. 38; no. 5
• Main Authors: Ji Xincheng, Northwest Agriculture and Forestry University, Yangling (China), College of Veterinary Medicine, Niu Guohui, Xinjiang University, Urumqi (China), College of Life Science, Yuan Lijuan, Xinjiang Exit-import Inspection and Qurantin Bureau, Urumqi (China)
• Format: Journal Article
• Language: Chinese
• Published: 01.05.2010
• Subjects: BOVIN; CATTLE; GANADO BOVINO; http://www.fao.org/aos/agrovoc#c_1391; http://www.fao.org/aos/agrovoc#c_34079; http://www.fao.org/aos/agrovoc#c_6961; PCR; SEMEN; SPERME
• ISSN: 1671-9387

建立牛精液中牛传染性鼻气管炎病毒（IBRV）的荧光PCR检测方法，并分析精液带毒与血清抗体的关系，为牛精液中IBRV的荧光PCR诊断和判定提供技术依据。采用Sephycral S-400凝胶过滤法和蛋白酶K预消化法，分别对新鲜牛精液和冻存牛精液进行处理，用DNA Zol方法提取病毒核酸，通过PCR反应条件的优化，建立了牛精液中直接检测IBRV的荧光PCR方法，对该方法进行特异性和重复性检验；最后用该方法分别对120份新鲜牛精液和40份冻存牛精液进行检测，同时用普通PCR方法和病毒分离方法加以对比；并对其中的10头牛定期采集精液、鼻腔拭子和血清样品，用该荧光PCR方法进行病原检测，对血清样品进行IBRV抗体检测。建立的荧光PCR方法具有较好的特异性，重复性和稳定性很好。用该方法可检测到新鲜牛精液中0.002 TCID50的病毒粒子，检测到冻存牛精液中0.02 TCID50的病毒粒子，检测时间在3 h之内，是OIE已报道方法灵敏度的40～400倍，是病毒分离方法灵敏度的100倍。120份新鲜牛精液和40份冻存牛精液中，检测到阳性样品25份，用普通PCR检测得到阳性样品15份，病毒分离检测得到阳性样品12份。检测结果表明，精液为阳性的牛血清抗体不一定为阳性，血清抗体阳性的牛精液中也不一定携带病毒。建立了牛精液中IBRV的荧光PCR检测方法；通过分析精液带毒与血清抗体的关系，进一步表明不能简单地以血清抗体阴阳性作为精液是否带毒的依据。鼻腔拭子带毒具有间歇性。 The study was to develop a real-time PCR for direct detection of infectious bovine rhinotracheitis virus (IBRV) inbovine semen and analyze the relation between semen carrying virus and serum antibody to IBRV, providing technical support for IBRV detection and determination in bovine semen. The semen was treated with Sephacryl S-400 chromatography to remove seminal inhibitors or the Pro