| 摘要: |
| 【目的】 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。【方法】 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。【结果】 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。【结论】 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。 |
| 关键词: 牛传染性鼻气管炎病毒 荧光PCR 牛精液病毒 血清抗体 |
| DOI： |
| 分类号: |
| 基金项目:国家质量监督检验检疫总局科研项目（2007IK023） |
|
| Establishment of real time PCR for direct detection of IBRV in semen and relation between semen carrying virus and serum antibody to IBRV |
|
|
| Abstract: |
| 【Objective】 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.【Method】 The semen was treated with Sephacryl S-400 chromatography to remove seminal inhibitors or the Proteinase K to liberate IBRV prior to DNA extraction.The primers and probe were designed,Mg2+ concentration,primers concentration and probe concentration were optimized,the real-time PCR method for direct detection of IBRV in bovine semen was developed.120 bovine raw semen samples and 40 extended semen samples were detected with the real-time PCR，traditional PCR and virus isoaltion method respectively.Semen samples and nose swabs samples of 10 bulls were detected regularly with the real-time PCR,and the serum antibody against IBRV were detected at the same time.【Result】 The detective limit was about 0.002 TCID50 to fresh semen samples and 0.02 TCID50 to extended semen samples.Compared with OIE reference method,the real-time PCR was 40 to 400 folds more sensitive in the detection of IBRV in semen samples,and that was 100 folds compared with conventional PCR and virus isolation.120 bovine fresh semen samples and 40 extended semen samples were detected with the real-time PCR,25 semen samples were positive,15 of them were positive by PCR and 12 positive samples by virus isolation.And the relation between bovine semen carrying virus and serum antibody against IBRV was not clear.【Conclusion】 A real-time PCR was developed for direct detection of IBRV in bovine semen and the relation between semen carrying virus and serum antibody against IBRV was analyzed.The results showed that the bull can not be confirmed whether the semen carrying virus through serum antibody.The nose swabs carried virus was intermittent. |
| Key words: IBRV real-time PCR bovine semen virus serum antibody |
