进口胎牛血清中牛病毒性腹泻病毒的分离及鉴定
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摘要
目的对进口胎牛血清中的牛病毒性腹泻病毒(bovine viral diarrhea virus,BVDV)进行分离及鉴定。方法Trizol法提取待检胎牛血清中的病毒RNA后,利用套式RT-PCR扩增BVDV,并利用生物信息学Lasergene 7.0软件对其核苷酸序列进行同源性分析。取生长状态良好的MDBK细胞,按1%比例加入待检胎牛血清,收获F1代BVDV FBS2014,反复冻融3次后,再次接种MDBK细胞,连传4代。采用荧光定量RT-PCR法、间接免疫荧光试验及抗原捕获ELISA法对收获的病毒进行鉴定。结果扩增产物大小均与预期相符,与已报道的BVDV-1 NADL株的核苷酸序列一致性为92.2%,属于BVDV-1a亚型毒株;分离的病毒经鉴定为BVDV,命名为BVDV FBS2014,病毒基因拷贝数为10~4拷贝/μl,感染MDBK细胞可见特异性绿色荧光;各代次的病毒收获液中BVDV的基因拷贝数均大于10~(4.92)拷贝/μl,BVDV抗原检测结果均为阳性。结论购买的进口胎牛血清中成功分离到可致MDBK细胞产生细胞病变的病毒,经鉴定所分离的病毒为BVDV,命名为FBS2014株,该病毒属于BVDV-1a亚型毒株。
Objective To isolate and identify bovine viral diarrhea(BVD) virus from imported fetal bovine serum.Methods Viral RNA was extracted from fetal bovine serum by Trizol method, with which bovine viral diarrhea virus(BVDV)gene was amplified by RT-PCR and analyzed for homology of nucleotide sequence by Lasergene 7. 0 software.Well-grown MDBK cells were inoculated with fetal bovine serum at a volume ratio of 1%. The BVDV of passage 1(F1)was harvested, subjected to three cycles of freezing and thawing, then inoculated to MDBK cells and subcultured for four passages. The harvested virus was identified by fluorescent quantitative RT-PCR, indirect IFA and antigen capture ELISA.Results The length of amplified product was consistent with those expected, while the homology of nucleotide sequence was 92. 2% to that of BVDV-1 NADL strain reported, indicating that the amplified product was of subtype BVDV-1a. The isolated virus was identified as BVDV and named as BVDV FBS2014, of which the gene copy number was 10~4 copies / μl.Specific green fluorescence was observed in MDBK cells infected with the isolated virus. All the gene copy numbers in harvested BVDV of various passages were more than 10~(4. 92) copies / μl, while all the test result for BVDV antigen were positive. Conclusion The virus causing CPE in MDBK cells was isolated from imported fetal bovine serum, which was identified as BVDV, named as FBS2014 strain, and was of subtype BVDV-1a.
Objective To isolate and identify bovine viral diarrhea(BVD) virus from imported fetal bovine serum.Methods Viral RNA was extracted from fetal bovine serum by Trizol method, with which bovine viral diarrhea virus(BVDV)gene was amplified by RT-PCR and analyzed for homology of nucleotide sequence by Lasergene 7. 0 software.Well-grown MDBK cells were inoculated with fetal bovine serum at a volume ratio of 1%. The BVDV of passage 1(F1)was harvested, subjected to three cycles of freezing and thawing, then inoculated to MDBK cells and subcultured for four passages. The harvested virus was identified by fluorescent quantitative RT-PCR, indirect IFA and antigen capture ELISA.Results The length of amplified product was consistent with those expected, while the homology of nucleotide sequence was 92. 2% to that of BVDV-1 NADL strain reported, indicating that the amplified product was of subtype BVDV-1a. The isolated virus was identified as BVDV and named as BVDV FBS2014, of which the gene copy number was 10~4 copies / μl.Specific green fluorescence was observed in MDBK cells infected with the isolated virus. All the gene copy numbers in harvested BVDV of various passages were more than 10~(4. 92) copies / μl, while all the test result for BVDV antigen were positive. Conclusion The virus causing CPE in MDBK cells was isolated from imported fetal bovine serum, which was identified as BVDV, named as FBS2014 strain, and was of subtype BVDV-1a.
引文
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[2]RIDPATH J F,BOLIN S R,DUBOVI E J.Segregation of bovine viral diarrhea virus into genotypes[J].Virology,1994,205(1):66-74.
[3]PETERHANS E,BACHOFEN C,STALDER H,et al.Cytopathic bovine viral diarrhea viruses(BVDV):emerging pestiviruses doomed to extinction[J].Vet Res,2010,41(6):44.
[4]淘洁,廖金虎,张倩,等.猪感染牛病毒性腹泻病毒研究进展[J].中国预防兽医学报,2014,36(5):410-413.
[5]YAO H W.Search of alternative method on potency test of hog cholera lapinzed virus and detective method of bovine viral diarrhea virus in vaccine[D].Chongqing:Southwest University,2011.(in Chinese)姚华伟.猪瘟兔化弱毒疫苗效检替代方法及疫苗中牛病毒性腹泻病毒检测方法的研究[D].重庆:西南大学,2011.
[6]SANDVIK T,PATON D J,LOWINGS P J.Detection and identification of ruminant and porcine pestiviruses by nested amplification of 5'untranslated c DNA regions[J].J Virol Methods,1997,64(1):43-56.
[7]ZHANG X J,HAN Q Y,SUN Y,et al.Development of a triplex Taq Man real-time RT-PCR assay for differential detection of wild-type and HCLV vaccine strains of classical swine fever virus and bovine viral diarrhea virus 1[J].Res Vet Sci,2012,92(3):512-518.
[8]The European Agency for the Evaluation of Medicinal Products.Note for guidance on the use of bovine serum in the manufacture of human biological medicinal product(CPMP/BWP/1793/02)[M].London:EMEA,2003:5-7.
[9]Chinese Pharmacopoeia Commission.Pharmacopoeia of People's Republic of China(VolⅢ)[S].Beijing:China Med Sci Press,2010:113.(in Chinese)国家药典委员会.中华人民共和国药典(三部)[S].北京:中国医药科技出版社,2010.