牛传染性鼻气管炎病毒和牛病毒性腹泻病毒分子生物学检测技术研究
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摘要
牛传染性鼻气管炎(IBR)和牛病毒性腹泻(BVD)是危害养牛业的两种重要病毒性传染病,给养牛业造成巨大的经济损失,我国进出境牛及其产品大多有对这两种疫病检疫的要求。牛精液中可携带这两种病毒,其在人工受精技术中的广泛应用是将该病传播给未感染牛群及地区的重要途径之一。目前对牛精液中这两种病的检测大多采用病毒分离的方法,因精液自身的细胞毒性及抗病毒活性,致使检测灵敏度极大的降低。虽然PCR方法已应用于这两种病毒的检测,但因精液中抑制PCR扩增成分的存在,致使检测灵敏度降低或操作复杂,抑制成分的存在也常造成假阴性结果的发生。结合进出境检验检疫工作,本研究完成了以下内容:
1.采用Institue pourquier公司的IBRV血清抗体检测试剂盒完成了新疆地区684份牛血清IBRV抗体检测,检出阳性血清370份,最高阳性率达97.6%,最低为5.0%,平均为54.1%。在近3年具有流产史记录的四个牛群中,有3个牛场牛IBRV抗体阳性率高达100.0%,1个牛场为12.5%,平均为90.5%。结果显示该病已在新疆普遍存在,感染没有明确的地域分布。
2.根据牛疱疹病毒1型(BHV1)gB基因序列,设计引物和荧光探针,通过搭桥法PCR扩增,构建了BHV1内标法荧光定量PCR共扩增模板,进一步构建了共扩增荧光PCR检测体系,该体系可以检测到10个拷贝/PCR反应的核酸分子。经对牛精液中抑制荧光PCR扩增成分进行分析和去除方法的研究,将该方法直接用于牛精液中IBRV的检测,可检测到牛精液中0.02 TCID50病毒,检测时间在2.5h之内。检测灵敏度比不含内标的荧光PCR检测方法低10倍,比病毒分离方法高500倍,比普通PCR方法高50倍,比OIE已报道的方法灵敏度高40倍。通过对120份新鲜牛精液、40份冷冻牛精液和39份牛鼻腔拭子检测,得到阳性样品27份。含有共扩增内标模板的检测体系可对反应体系进行监测,指示并校正假阴性结果,从而达到提高PCR检测准确率的目的。通过对定期采集的10头牛血清样品IBRV抗体检测,发现精液阳性牛血清抗体不一定为阳性,血清抗体阳性牛精液中也不一定带毒,本试验结果初步表明不能以血清抗体阴阳性做为精液是否带毒的依据。鼻腔拭子带毒也只是间歇性的。
3.采用Idexx公司BVDV血清抗体检测试剂盒完成了新疆地区875份牛血清BVDV抗体检测,共检出抗体阳性血清759份,最高阳性率为100.0%,最低为55.0%,平均为86.7%,从已有的数据来看,近3年有过流产史的牛血清BVDV抗体阳性率普遍偏高。感染牛没有明显的年龄差别。结果显示该病已在新疆普遍存在,且有不断增强的趋势。与病毒中和试验相比较,ELISA操作简便,并具有较好的敏感性和特异性,两者符合率为80.0%。
4.采用Idexx公司BVDV抗原检测试剂盒从160份牛血清中检测出6份阳性血清,对这6分血清和随机选择的20份肛门拭子进行了病毒分离,成功分离出8株BVDV,8株分离毒株TCID50为4.38×103～5.82×106。对BVDV阳性血清的中和效价为1:23～1:26。8株分离株之间的同源性为87.5%～99.7%;与NADL株核苷酸序列同源性为82.0%～94.8%;与KE9株核苷酸序列同源性为81.0%～82.0%;分离株之间具有较高的同源性,表明该8株病毒与NADL株为同一基因型。
5.建立了牛精液中BVDV荧光RT-PCR检测方法,该方法可检测到新鲜牛精液和冷冻牛精液中0.0125TCID50的病毒。灵敏度比常规RT-PCR高100倍,比病毒分离法高200～2000倍,研究发现,新鲜精液本身对RT-PCR扩增具有一定的抑制性,精液冻存液中的卵黄是抑制RT-PCR扩增的主要成分。用该方法对120份新鲜精液40份冻存精液进行检测,没有检测到阳性样品。对定期采集的10头牛血清BVDV抗体进行了检测,结果表明不能以血清抗体结果作为精液是否带毒的判定标准,血清抗体效价至少持续半年以上。
6.根据IBRV gB基因、BVDV5’UTR区和犬新孢子虫Nc-5基因,通过PCR扩增各目的基因的特异性片段,成功构建了各特异性基因的重组质粒,在此基础上,建立了上述3种病原的多重PCR检测方法。将标有荧光染料的PCR产物变性后与芯片上寡核苷酸探针杂交,经系列反应条件的优化,建立了IBRV、BVDV和N. caninum 3种病原的基因芯片检测方法,本研究对三种病原重组DNA的检测灵敏度均为103拷贝/反应。对牛精液中IBRV和BVDV两种病毒的检测灵敏度约为0.1TCID50,且具有较好的特异性和可重复性。经检测应用,进一步证明了该方法的可行性。
7.建立了IBRV、BVDV和N. caninum xMAP悬浮液态芯片检测技术。对基因组DNA分别进行xMAP悬浮芯片单重和多重目标物的检测试验,建立的方法具有较好的特异性和可重复性。对BVDV重组质粒可检测到102拷贝/反应,对NCP和IBRV的检测灵敏度为103拷贝/反应。比PCR灵敏度高10~100倍。经检测应用,进一步证明了该方法的可行性。根据检测项目的不同,对临床样品分别采用了病毒分离、普通PCR、荧光PCR、基因芯片和悬浮芯片等方法进行了检测,结果表明,荧光PCR具有较高的灵敏度,悬浮芯片和基因芯片灵敏度次之。目前虽然有采用荧光PCR法、基因芯片法和悬浮芯片法等技术对动物疫病检测的研究,但尚未见将这些技术作为一个整体进行研究的报道,也没有以共扩增内标模板对检测结果进行质量控制的研究。本研究搭建了DNA病毒、RNA病毒和寄生虫病芯片检测技术平台,为今后更多动物疫病检测技术的研究开展奠定了基础。并同步完成了精液带毒与血清抗体关系进行了分析。结果的取得为出入境检验检疫条款的制定和检测方法的选择提供了依据,为进出境动物检验检疫提供了必要的技术储备。
Infectious bovine rhinotracheitis(IBR)and Bovine viral diarrhea(BVD) are two mainly infection illness that lead to cow propagation and cause huge economic losses. The two illness should be inspected to import and exit bovine base on the inspection order. Bovine semen can carry these virus, that can infect other health bovine through inseminating. Virus isolation is the main detection method to IBRV and BVDV in bovine semen, but the method has some difficulty such as lower sensitivy, operation complex and cost a long time, and so on. It is important to know the relation of semen carrying virus and serum antibody in semen produce center, that decide wether the bovine is valuble. There were not report about IBRV and BDVD detection used molocole biology technology. Based on the inspection and quarantine of entry and exit, following results were achieved in the study:
1. 684 bovine serum samples antibody against IBRV coming Xinjiang 17 different regions were examined used Institute pourquiers company ELISA kit, 370 serum samples were conformed positive. The postive rate was between 5% and 97.6%, the mean postive rate was 54.1%. There were four farms that had a clear record about abortion in the past three years, in of them, IBRV serum antibody postive rate were 100% in three farms and one farm was 12.5%, the mean postive rate was 90.5%. The results showed that IBRV had an extensive exist in Xinjiang province. The infection rate had no a clear region distribution.
2. According to the gB gene sequence of Bovine Herpesvirus 1(BHV1), primers and fluorescent probe were designed to amplify the nucleotide fragment containing the real-time PCR amplification region. An Internal Amplification Contro(lIAC)template of real-time PCR was achieved by amplifying with bridge-building PCR method, and the real-time PCR detection system with IAC was established. The detective limit was about 10 copies/PCR reaction. The real-time PCR with IAC was used directly detection IBRV in raw semen and extended semen samples.The detective limit was about 0.02 TCID50. Compared with OIE reference method, the real-time PCR was 40 folds more sensitive,and that was 500 folds and 50 more sensitivity compared virus isolation and PCR assay respectively. 120 bovine fresh semen samples and 40 extended semen samples and 39 nose swabs were detected with the real-time PCR with IAC,27 samples were positive. These results were almost coincident with real-time PCR without IAC. What is more, the IAC in the real-time PCR system can indicate and proofread false negative results. Serum antibody against IBRV of 10 bulls collected regularly were detected at the same time, and the results showed the bull can not be confirmed whether the semen carrying virus through serum antibody, vice verse. Nose swabs carrying virus was intermittent.
3. 875 bovine serum samples antibody against BVDV coming Xinjiang different region were detected used Idexx company indirect ELISA kit, 759 serum samples were conformed positive. The postive rate was between 55.0% and 100%, and the mean postive rate was 86.7%. The results showed that the positive rate was clearly heigher to aborted bovine and the infection had no a clear different to different age bovine. IBRV had an extensive exist in Xinjiang province and is becoming more and more. Compared with VN method, the ELISA was convenient for operation, and was sensitive and specific to BVDV antibody detection. The agreement was 80.0% to VN.
4. 6 bovine serum were conformed postive by Idexx company antigen-capture ELISA kit from 160 bovine serum and 20 anus swabs were inoculated by MDBK cells. The 6 serum and 2 anus swabs caused cell pathological. TCID50 of the 8 virus strains were 4.38×103 to 5.82×106. The Serum neutralization titer was 1:23 to1:26. The homology of the 8 islolated strains were 82.0 % to 94.8% and 81.0% to 82.0% compared with NADL strain and KE9 strain respectively. The results showed that the gene type were samely between the 8 isolated virus and NADL strain.
5. The fluorescent quantitative RT-PCR (FQ-RT-PCR) method was established for direct detection BVDV in raw semen and extended semen samples. The level of sensitivity was about 0.0125TCID50 for semen samples. The FQ-RT-PCR assay in this study was 200 to 2000 times more sensitive than virus isolation, and that was 100 folds more sensitive compared with conventional RT-PCR. 120 bovine raw semen samples and 40 extended semen samples were collected within 6 months from a bull farm and detected with the FQ-RT-PCR. All the samples were negative. In of them, serum antibodies against BVDV of 10 bulls collected regularly were detected with VN at the same time. The results showed the bull can not be confirmed whether the semen carrying virus through serum antibodies and the antibody can maintain at least half a year.
6. According to the published N. caninum Nc-5 gene and BHV-1 gB gene and BVDV 5’UTR, primers and probes were designed. After PCR amplication and gene clone, recombiant plasmids were achieved and mult-PCR method was developed of the three illness. The denaturated PCR products marked fluorescent dye were hybridized to the gene chip, after the optimization of reaction condition, the gene chip diagnostic technique detecting IBRV and BVDV and N. caninum. was established. The sensitivity of the gene chip method was 103 copies each reaction to recombiant plasmids of IBRV and BVDV and N. canimum. The sensitivity to BVDV and IBRV in bovine semen was 0.1TCID50. The application of detection proved the method was repecific and reliable.
7. Suspension array of detection IBRV and BVDV and N. caninum was establishe in the study. The single test and mult-test of the suspension array both showed the method was repecific and reliable, and the sensitivity was 102 copies each reaction to recombiant plasmids of BVDV, and that was 103 copies each reaction to N. canimum. and IBRV. The sensitivity to BVDV and IBRV in bovine semen was 0.01TCID50.That was 100 folds more sensivity compared to PCR. The application of detection proved the method was viable.
Some samples were detected with virus isolation, PCR, real-time PCR, gene chip and suspension array, and so on. The results showed the real-time PCR had the highest sensitivity compared with gene chip and suspension array, but gene chip and suspension array can detection illness at the same time. There were some report about animal illness detection used real-time PCR, gene chip and suspension array by now. But without study on these different method as a whole, and no a further study report on quality control of animal illness detection. The built of technology platform of DNA virus, RNA virus and parasites illness detection was contributed to more animal illness study in the future. The analysis of semen carrying virus and serum antibody was finished at the same time. All these were helpful to constitution of inspection and quantine policy and to choice of detection method and to afford necessary technology store to animal entry and exit inspecction and quarantine.
1.采用Institue pourquier公司的IBRV血清抗体检测试剂盒完成了新疆地区684份牛血清IBRV抗体检测,检出阳性血清370份,最高阳性率达97.6%,最低为5.0%,平均为54.1%。在近3年具有流产史记录的四个牛群中,有3个牛场牛IBRV抗体阳性率高达100.0%,1个牛场为12.5%,平均为90.5%。结果显示该病已在新疆普遍存在,感染没有明确的地域分布。
2.根据牛疱疹病毒1型(BHV1)gB基因序列,设计引物和荧光探针,通过搭桥法PCR扩增,构建了BHV1内标法荧光定量PCR共扩增模板,进一步构建了共扩增荧光PCR检测体系,该体系可以检测到10个拷贝/PCR反应的核酸分子。经对牛精液中抑制荧光PCR扩增成分进行分析和去除方法的研究,将该方法直接用于牛精液中IBRV的检测,可检测到牛精液中0.02 TCID50病毒,检测时间在2.5h之内。检测灵敏度比不含内标的荧光PCR检测方法低10倍,比病毒分离方法高500倍,比普通PCR方法高50倍,比OIE已报道的方法灵敏度高40倍。通过对120份新鲜牛精液、40份冷冻牛精液和39份牛鼻腔拭子检测,得到阳性样品27份。含有共扩增内标模板的检测体系可对反应体系进行监测,指示并校正假阴性结果,从而达到提高PCR检测准确率的目的。通过对定期采集的10头牛血清样品IBRV抗体检测,发现精液阳性牛血清抗体不一定为阳性,血清抗体阳性牛精液中也不一定带毒,本试验结果初步表明不能以血清抗体阴阳性做为精液是否带毒的依据。鼻腔拭子带毒也只是间歇性的。
3.采用Idexx公司BVDV血清抗体检测试剂盒完成了新疆地区875份牛血清BVDV抗体检测,共检出抗体阳性血清759份,最高阳性率为100.0%,最低为55.0%,平均为86.7%,从已有的数据来看,近3年有过流产史的牛血清BVDV抗体阳性率普遍偏高。感染牛没有明显的年龄差别。结果显示该病已在新疆普遍存在,且有不断增强的趋势。与病毒中和试验相比较,ELISA操作简便,并具有较好的敏感性和特异性,两者符合率为80.0%。
4.采用Idexx公司BVDV抗原检测试剂盒从160份牛血清中检测出6份阳性血清,对这6分血清和随机选择的20份肛门拭子进行了病毒分离,成功分离出8株BVDV,8株分离毒株TCID50为4.38×103～5.82×106。对BVDV阳性血清的中和效价为1:23～1:26。8株分离株之间的同源性为87.5%～99.7%;与NADL株核苷酸序列同源性为82.0%～94.8%;与KE9株核苷酸序列同源性为81.0%～82.0%;分离株之间具有较高的同源性,表明该8株病毒与NADL株为同一基因型。
5.建立了牛精液中BVDV荧光RT-PCR检测方法,该方法可检测到新鲜牛精液和冷冻牛精液中0.0125TCID50的病毒。灵敏度比常规RT-PCR高100倍,比病毒分离法高200～2000倍,研究发现,新鲜精液本身对RT-PCR扩增具有一定的抑制性,精液冻存液中的卵黄是抑制RT-PCR扩增的主要成分。用该方法对120份新鲜精液40份冻存精液进行检测,没有检测到阳性样品。对定期采集的10头牛血清BVDV抗体进行了检测,结果表明不能以血清抗体结果作为精液是否带毒的判定标准,血清抗体效价至少持续半年以上。
6.根据IBRV gB基因、BVDV5’UTR区和犬新孢子虫Nc-5基因,通过PCR扩增各目的基因的特异性片段,成功构建了各特异性基因的重组质粒,在此基础上,建立了上述3种病原的多重PCR检测方法。将标有荧光染料的PCR产物变性后与芯片上寡核苷酸探针杂交,经系列反应条件的优化,建立了IBRV、BVDV和N. caninum 3种病原的基因芯片检测方法,本研究对三种病原重组DNA的检测灵敏度均为103拷贝/反应。对牛精液中IBRV和BVDV两种病毒的检测灵敏度约为0.1TCID50,且具有较好的特异性和可重复性。经检测应用,进一步证明了该方法的可行性。
7.建立了IBRV、BVDV和N. caninum xMAP悬浮液态芯片检测技术。对基因组DNA分别进行xMAP悬浮芯片单重和多重目标物的检测试验,建立的方法具有较好的特异性和可重复性。对BVDV重组质粒可检测到102拷贝/反应,对NCP和IBRV的检测灵敏度为103拷贝/反应。比PCR灵敏度高10~100倍。经检测应用,进一步证明了该方法的可行性。根据检测项目的不同,对临床样品分别采用了病毒分离、普通PCR、荧光PCR、基因芯片和悬浮芯片等方法进行了检测,结果表明,荧光PCR具有较高的灵敏度,悬浮芯片和基因芯片灵敏度次之。目前虽然有采用荧光PCR法、基因芯片法和悬浮芯片法等技术对动物疫病检测的研究,但尚未见将这些技术作为一个整体进行研究的报道,也没有以共扩增内标模板对检测结果进行质量控制的研究。本研究搭建了DNA病毒、RNA病毒和寄生虫病芯片检测技术平台,为今后更多动物疫病检测技术的研究开展奠定了基础。并同步完成了精液带毒与血清抗体关系进行了分析。结果的取得为出入境检验检疫条款的制定和检测方法的选择提供了依据,为进出境动物检验检疫提供了必要的技术储备。
Infectious bovine rhinotracheitis(IBR)and Bovine viral diarrhea(BVD) are two mainly infection illness that lead to cow propagation and cause huge economic losses. The two illness should be inspected to import and exit bovine base on the inspection order. Bovine semen can carry these virus, that can infect other health bovine through inseminating. Virus isolation is the main detection method to IBRV and BVDV in bovine semen, but the method has some difficulty such as lower sensitivy, operation complex and cost a long time, and so on. It is important to know the relation of semen carrying virus and serum antibody in semen produce center, that decide wether the bovine is valuble. There were not report about IBRV and BDVD detection used molocole biology technology. Based on the inspection and quarantine of entry and exit, following results were achieved in the study:
1. 684 bovine serum samples antibody against IBRV coming Xinjiang 17 different regions were examined used Institute pourquiers company ELISA kit, 370 serum samples were conformed positive. The postive rate was between 5% and 97.6%, the mean postive rate was 54.1%. There were four farms that had a clear record about abortion in the past three years, in of them, IBRV serum antibody postive rate were 100% in three farms and one farm was 12.5%, the mean postive rate was 90.5%. The results showed that IBRV had an extensive exist in Xinjiang province. The infection rate had no a clear region distribution.
2. According to the gB gene sequence of Bovine Herpesvirus 1(BHV1), primers and fluorescent probe were designed to amplify the nucleotide fragment containing the real-time PCR amplification region. An Internal Amplification Contro(lIAC)template of real-time PCR was achieved by amplifying with bridge-building PCR method, and the real-time PCR detection system with IAC was established. The detective limit was about 10 copies/PCR reaction. The real-time PCR with IAC was used directly detection IBRV in raw semen and extended semen samples.The detective limit was about 0.02 TCID50. Compared with OIE reference method, the real-time PCR was 40 folds more sensitive,and that was 500 folds and 50 more sensitivity compared virus isolation and PCR assay respectively. 120 bovine fresh semen samples and 40 extended semen samples and 39 nose swabs were detected with the real-time PCR with IAC,27 samples were positive. These results were almost coincident with real-time PCR without IAC. What is more, the IAC in the real-time PCR system can indicate and proofread false negative results. Serum antibody against IBRV of 10 bulls collected regularly were detected at the same time, and the results showed the bull can not be confirmed whether the semen carrying virus through serum antibody, vice verse. Nose swabs carrying virus was intermittent.
3. 875 bovine serum samples antibody against BVDV coming Xinjiang different region were detected used Idexx company indirect ELISA kit, 759 serum samples were conformed positive. The postive rate was between 55.0% and 100%, and the mean postive rate was 86.7%. The results showed that the positive rate was clearly heigher to aborted bovine and the infection had no a clear different to different age bovine. IBRV had an extensive exist in Xinjiang province and is becoming more and more. Compared with VN method, the ELISA was convenient for operation, and was sensitive and specific to BVDV antibody detection. The agreement was 80.0% to VN.
4. 6 bovine serum were conformed postive by Idexx company antigen-capture ELISA kit from 160 bovine serum and 20 anus swabs were inoculated by MDBK cells. The 6 serum and 2 anus swabs caused cell pathological. TCID50 of the 8 virus strains were 4.38×103 to 5.82×106. The Serum neutralization titer was 1:23 to1:26. The homology of the 8 islolated strains were 82.0 % to 94.8% and 81.0% to 82.0% compared with NADL strain and KE9 strain respectively. The results showed that the gene type were samely between the 8 isolated virus and NADL strain.
5. The fluorescent quantitative RT-PCR (FQ-RT-PCR) method was established for direct detection BVDV in raw semen and extended semen samples. The level of sensitivity was about 0.0125TCID50 for semen samples. The FQ-RT-PCR assay in this study was 200 to 2000 times more sensitive than virus isolation, and that was 100 folds more sensitive compared with conventional RT-PCR. 120 bovine raw semen samples and 40 extended semen samples were collected within 6 months from a bull farm and detected with the FQ-RT-PCR. All the samples were negative. In of them, serum antibodies against BVDV of 10 bulls collected regularly were detected with VN at the same time. The results showed the bull can not be confirmed whether the semen carrying virus through serum antibodies and the antibody can maintain at least half a year.
6. According to the published N. caninum Nc-5 gene and BHV-1 gB gene and BVDV 5’UTR, primers and probes were designed. After PCR amplication and gene clone, recombiant plasmids were achieved and mult-PCR method was developed of the three illness. The denaturated PCR products marked fluorescent dye were hybridized to the gene chip, after the optimization of reaction condition, the gene chip diagnostic technique detecting IBRV and BVDV and N. caninum. was established. The sensitivity of the gene chip method was 103 copies each reaction to recombiant plasmids of IBRV and BVDV and N. canimum. The sensitivity to BVDV and IBRV in bovine semen was 0.1TCID50. The application of detection proved the method was repecific and reliable.
7. Suspension array of detection IBRV and BVDV and N. caninum was establishe in the study. The single test and mult-test of the suspension array both showed the method was repecific and reliable, and the sensitivity was 102 copies each reaction to recombiant plasmids of BVDV, and that was 103 copies each reaction to N. canimum. and IBRV. The sensitivity to BVDV and IBRV in bovine semen was 0.01TCID50.That was 100 folds more sensivity compared to PCR. The application of detection proved the method was viable.
Some samples were detected with virus isolation, PCR, real-time PCR, gene chip and suspension array, and so on. The results showed the real-time PCR had the highest sensitivity compared with gene chip and suspension array, but gene chip and suspension array can detection illness at the same time. There were some report about animal illness detection used real-time PCR, gene chip and suspension array by now. But without study on these different method as a whole, and no a further study report on quality control of animal illness detection. The built of technology platform of DNA virus, RNA virus and parasites illness detection was contributed to more animal illness study in the future. The analysis of semen carrying virus and serum antibody was finished at the same time. All these were helpful to constitution of inspection and quantine policy and to choice of detection method and to afford necessary technology store to animal entry and exit inspecction and quarantine.
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