猪圆环病毒DNA竞争定量PCR检测方法的建立及初步应用
摘要
本研究根据已发表的猪圆环病毒PCV-1和PCV-2的全基因序列，利用DNAstar软件对PCV-1和PCV-2的全基因序列进行同源性比较，应用Oligo6.0软件设计出两对引物，其中一对PCV特异性引物P1和P2，扩增出PCV-1和PCV-2上长938bp的片段；另一对为PCV-2型特异性引物P3和P4，扩增片段长490bp。复合PCR方法可以检测到1ng的病毒核酸样品。
建立了定量检测猪圆环病毒DNA的竞争PCR方法。PCR扩增PCV-2 ORF2上490bp片段P3P4，构建了目标模板。将P3P4片段应用BanⅡ酶切，再进行连接，之后克隆到pMD18-T载体，构建含694bp的c-P3p4 DNA片段的竞争模板，其携带同目标DNA和样本DNA相同的引物结合区。以定量的竞争模板同一系列稀释的竞争模板进行竞争PCR扩增，结果当反应体系中起始模板的量为1.0×10~7copies／ml，循环次数小于或等于30时，原始模板数以指数增长。以获得目标模板浓度和扩增产物的荧光强度(IOD)比值的回归曲线，将该曲线作为标准曲线。根据竞争模板和目标模板产物之间的荧光强度(IOD)差异，引进了校正系数(Lc／Lt)。将反应中的各种参数带回回归方程，获得根据样品量和PCR产物荧光强度计算浓度的公式。初步应用竞争定量PCR方法检测感染细胞内的PCV-2基因组的拷贝数增殖变化情况和临床样品中的PCV-2 DNA含量，这为猪圆环病毒核酸诊断和疫苗研究奠定了基础。
A multiplex polymerase chain reaction was developed to simultamneous detect type 1 and type 2 of porcine circovirus. The homology of the full sequences of PCV reported and registered in Genebank of different strains of PCV-1 and PCV-2 was respectively and compared with each other. Two sets of specific primers of PCV were designed. PCV can be amplified 938bp, PCV-2 can be amplified 490bp.The designed primers were used to PCR reaction. The result showed that the design of the two sets of primers was right. Then the optimal condition of PCR for PCV were determined.
Competitive quantitative PCR (cPCR) assay was developed for monitoring and detecting porcine circovirus (PCV-2) DNA. Using PCR assay, the cPCR was based on competitive co-amplification of a 490-bp region of the PCV-2 ORF2 gene, with a known concentration of competitor DNA, which produced a 692-bp fragment. The cPCR was validated by quantification of a known amount of competitor PCV DNA with one series of dilutions of target DNA. Using the regression equation, we protracted the standard curve and affirmed the quantity of the competitive template. According to the different of the IOD between the products of competitor and target, introduce Lc/Lt as the adjust coefficient. After all parameter regressed, got the concentration formulae that based on the quantity of the samples and the IOD of the PCR products.
Used this technique to determine PCV genome copy numbers in infected cells. Furthermore, we measured PCV DNA loads in clinical samples and found PCV-2 DNA content in the piglet with PMWS is higher than 1.0145 x 108copies/ml in lung, 0.8 x 107copies/ml in blood, and 9.698 x 108copies/ml in lymph. Our research might provide some hints to the study of the future work.
建立了定量检测猪圆环病毒DNA的竞争PCR方法。PCR扩增PCV-2 ORF2上490bp片段P3P4，构建了目标模板。将P3P4片段应用BanⅡ酶切，再进行连接，之后克隆到pMD18-T载体，构建含694bp的c-P3p4 DNA片段的竞争模板，其携带同目标DNA和样本DNA相同的引物结合区。以定量的竞争模板同一系列稀释的竞争模板进行竞争PCR扩增，结果当反应体系中起始模板的量为1.0×10~7copies／ml，循环次数小于或等于30时，原始模板数以指数增长。以获得目标模板浓度和扩增产物的荧光强度(IOD)比值的回归曲线，将该曲线作为标准曲线。根据竞争模板和目标模板产物之间的荧光强度(IOD)差异，引进了校正系数(Lc／Lt)。将反应中的各种参数带回回归方程，获得根据样品量和PCR产物荧光强度计算浓度的公式。初步应用竞争定量PCR方法检测感染细胞内的PCV-2基因组的拷贝数增殖变化情况和临床样品中的PCV-2 DNA含量，这为猪圆环病毒核酸诊断和疫苗研究奠定了基础。
A multiplex polymerase chain reaction was developed to simultamneous detect type 1 and type 2 of porcine circovirus. The homology of the full sequences of PCV reported and registered in Genebank of different strains of PCV-1 and PCV-2 was respectively and compared with each other. Two sets of specific primers of PCV were designed. PCV can be amplified 938bp, PCV-2 can be amplified 490bp.The designed primers were used to PCR reaction. The result showed that the design of the two sets of primers was right. Then the optimal condition of PCR for PCV were determined.
Competitive quantitative PCR (cPCR) assay was developed for monitoring and detecting porcine circovirus (PCV-2) DNA. Using PCR assay, the cPCR was based on competitive co-amplification of a 490-bp region of the PCV-2 ORF2 gene, with a known concentration of competitor DNA, which produced a 692-bp fragment. The cPCR was validated by quantification of a known amount of competitor PCV DNA with one series of dilutions of target DNA. Using the regression equation, we protracted the standard curve and affirmed the quantity of the competitive template. According to the different of the IOD between the products of competitor and target, introduce Lc/Lt as the adjust coefficient. After all parameter regressed, got the concentration formulae that based on the quantity of the samples and the IOD of the PCR products.
Used this technique to determine PCV genome copy numbers in infected cells. Furthermore, we measured PCV DNA loads in clinical samples and found PCV-2 DNA content in the piglet with PMWS is higher than 1.0145 x 108copies/ml in lung, 0.8 x 107copies/ml in blood, and 9.698 x 108copies/ml in lymph. Our research might provide some hints to the study of the future work.
引文
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[7] Choi Changsun, Chae Chanhee, Clark EG. Porcine postweaning multisystemic wasting syndrome in Korean pig: detection of porcine circovirus 2 infection by immunohistochemistry and polymerase chain reaction. Journal of Veterinary Diagnostic Investigation (2000) 12(2): 151-153.
[8] 曹胜波，陈涣春，猪环状病毒2型的PCR检测方法的建立及应用．华中农业大学学报．2001(20)1：53-56
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[11] Onuki, A.; Abe, K.; Togashi, K.; etc. Detection of porcine circovirus from lesions of a pig with wasting disease in Japan. Journal of Veterinary Medical Science. 1999; 61(10): 1119-1123.
[12] Allan, G. M.; McNeilly, E.; Kennedy, S.; etc. PCV-2-associated PDNS in Northern Ireland in 1990. Veterinary Record. 2000b; 146(24):711-712.
[13] Ramos-Vara, J.;. Duran, O.; Render, J. etc. Porcine dermatitis and nephropathy syndrome in the USA. Veterinary Record. 1997. 141: 18, 479-480.
[14] Segales, J.; Piella, J.; Marco, E.; Mateu-de-Antonio, E.; Espuna, E., and Domingo, M. Porcine dermatitis and nephropathy syndrome in Spain. Veterinary Record. 1998. 142: 18,
483-486.
[15] Bogdan, J.; West, K.; Clark, E.; etc. Association of porcine circovirus 2 with reproductive failure in pigs: a retrospective study, 1995-1998. Canadian Veterinary Journal. 2001; 42(7):548-550.
[16] Hines, R and Lukert, P. Porcine circovirus as a cause of congential tremors in newborn piglets. Proceedings of the American Association of Swine Practitioners. 1994; 344-345.
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[18] Dulac GC, Afshar A: Porcine circovirus antigens in PK-15 cell line (ATCC CCL-33) and evidence of antibodies in Canadian pigs. Can J Vet Res 53:431-433, 1989
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[20] Ellis, J.; Hassard, L.; Clark, E.; etc. Isolation of circovirus from lesions of pigs with postweaning multisystemic wasting syndrome. Canadian Veterinary Journal. 1998; 39(1):44-51.
[21] Allan, G. M.; Kennedy, S.; McNeilly, F; etc. Experimental reproduction of severe wasting disease by co-infection of pigs with porcine circovirus and porcine parvovirus. Journal of Comparative Pathology. 1999; 121(1):1-11.
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[27] Ellis, J., S. Krakowka, M. Lairmore, D. etc. Reproduction of lesions of postweaning multisystemic wasting syndrome in gnotobiotic piglets. J. Vet. Diagn. Investig. 1999,11:3-14.
[28] Ellis, J. A., A. Bratanich, E. G. Clark, G.ect. Coinfection by porcine circoviruses and porcine parvovirus in pigs with naturally acquired postweaning multisystemic wasting syndrome. J. Vet. Diagn. Investig. 2000, 12:21-27.
[29] Larochelle, R.; Bielanski, A., Muller, P., and Magar, R. PCR detection and evidence of shedding of porcine circovirus type-2 in boar semen. Journal of Clinical Microbiology. 2000; 38:4629-4632.
[30] Stevenson. GW, Kiupel, M,Mittal S.K. Ultrastructure of porcine circovirus in persistently infected PK-15 cell. Vet Path.(1999)36(5):368-378
[31] Tischer, I.; Mields, W.; Wolff, D.; etc. Studies on epidemiology and pathogenicity of porcine circovirus. Archives of Virology. 1986. 91: 3/4, 271-276.
[32] Magar, R.; Larochelle, R.; Thibault, S., etc. Experimental transmission of porcine circovirus type 2 (PCV-2) in weaned pigs: a sequential study. Journal of Comparative Pathology. 2000; 123(4):258-69.
[33] 郎洪武，王力，张广川等．猪圆环病毒分离鉴定及猪断奶多系统衰弱综合征的诊断．中国兽医科技．2001，31(3)：3-5．
[34] 曹胜波，陈涣春，猪环状病毒2型的PCR检测方法的建立及应用．华中农业大学学报．2001，20(1)：53-56
[35] 李媛，崔尚金，全艳平，等：检测与诊断猪圆环病毒PCR技术的建立及病毒的分离鉴定，中国预防兽医学报，2002，24(增)：164-168．
[36] 吕艳丽，杨汉春，郭鑫，猪圆环病毒2型分离毒株基因组序列测定与分析[J]中国预防兽医学报(2002)增刊：63-66．
[37] Tischer, I.; Gelderblom, H.; Vettermann, W., etc. A very small porcine virus with a circular single-stranded DNA. Nature. 1982; 295:64-66.
[38] Tischer I, Peters D, Rasch R, Replication of Porcine Circovirus: Induction by Glycosamine and Cell Cycle Depensence. Arch Virol. 1987, 96:39-57
[39] Allan,G.M,Phenix.K.V, Todd.D.Some biological and physicochemical properties of porcine circovirus [J].J Vet Med. 1994,1341:17-26
[40] Liu, Q Wang L.; Willson, P., and Babiuk, L. Quantitative, competitive PCR analysis of porcine circovirus DNA in serum from pigs with postweaning multisystemic wasting syndrome. Journal of Clinical Microbiology. 2000; 38(9):3474-3477.
[41] Clark, E. Pathology of the post-weaning multisystemic wasting syndrome of pigs. Proceedings of the West Canadian Association of Swine Practitioners. 1996; 22-25.
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[53] Choi Changsun, Chae Chanhee, Clark EG. Porcine postweaning multisystemic wasting syndrome in Korean pig: detection of porcine circovirus 2 infection by immunohistochemistry and polymerase chain reaction. Journal of Veterinary Diagnostic Investigation (2000)12(2): 151-153.
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[63] 张强．图象分析仪在定量RTPCR方法中的应用．国外医学遗传学分册．1998，2(3)：152-154．
[2] Homer, G. Pig circovirus antibodies present in New Zealand pigs. Surveillance. 1991. 18: 5,23.
[3] Helie, P.; Drolet, R.; Germain, M. C., etc. Systemic necrotizing vasculitis and glomerulonephritis in grower pigs in southwestern Quebec. Canadian Veterinary Journal. 1995.36: 3, 150-154.
[4] Tischer, I.; Bode, L.; Peters, D.; etc. B Distribution of antibodies to porcine circovirus in swine populations of different breeding farms. Archives of Virology. 1995. 140: 4, 737-743.
[5] Clark, E. Pathology of the post-weaning multisystemic wasting syndrome of pigs. Proceedings of the West Canadian Association of Swine Practitioners. 1996; 22-25.
[6] Harding, J. Post-weaning muhisystemic wasting syndrome (PMWS), preliminary epidemiology and clinical presentation. Proceedings of the American Association of Swine Practitioners. 1997; 503.
[7] Choi Changsun, Chae Chanhee, Clark EG. Porcine postweaning multisystemic wasting syndrome in Korean pig: detection of porcine circovirus 2 infection by immunohistochemistry and polymerase chain reaction. Journal of Veterinary Diagnostic Investigation (2000) 12(2): 151-153.
[8] 曹胜波，陈涣春，猪环状病毒2型的PCR检测方法的建立及应用．华中农业大学学报．2001(20)1：53-56
[9] Allan,G. M.; McNeilly, E.; Kennedy, S.; etc. Isolation of porcine circovirus-like viruses from pigs with a wasting disease in the USA and Europe. Journal of Veterinary Diagnostic Investigation. 1998; 103-110.
[10] Spillane, P ; Kennedy, S; Meehan, B, etc. Porcine circovirus infection in the Republic of Ireland. Veterinary Record. 1998; 143:511-512.
[11] Onuki, A.; Abe, K.; Togashi, K.; etc. Detection of porcine circovirus from lesions of a pig with wasting disease in Japan. Journal of Veterinary Medical Science. 1999; 61(10): 1119-1123.
[12] Allan, G. M.; McNeilly, E.; Kennedy, S.; etc. PCV-2-associated PDNS in Northern Ireland in 1990. Veterinary Record. 2000b; 146(24):711-712.
[13] Ramos-Vara, J.;. Duran, O.; Render, J. etc. Porcine dermatitis and nephropathy syndrome in the USA. Veterinary Record. 1997. 141: 18, 479-480.
[14] Segales, J.; Piella, J.; Marco, E.; Mateu-de-Antonio, E.; Espuna, E., and Domingo, M. Porcine dermatitis and nephropathy syndrome in Spain. Veterinary Record. 1998. 142: 18,
483-486.
[15] Bogdan, J.; West, K.; Clark, E.; etc. Association of porcine circovirus 2 with reproductive failure in pigs: a retrospective study, 1995-1998. Canadian Veterinary Journal. 2001; 42(7):548-550.
[16] Hines, R and Lukert, P. Porcine circovirus as a cause of congential tremors in newborn piglets. Proceedings of the American Association of Swine Practitioners. 1994; 344-345.
[17] Tischer I, Rasch R, Tochtermann G. Characterization of papovavirus-and picornavirus-like particles in permanent pig kidney cell lines. Zentralblatt fur Baktedologie Mikrobiologie und Hygiene seres A. 1974, 226: 153-67.
[18] Dulac GC, Afshar A: Porcine circovirus antigens in PK-15 cell line (ATCC CCL-33) and evidence of antibodies in Canadian pigs. Can J Vet Res 53:431-433, 1989
[19] Allan, G.M.; McNeilly, E.; Kennedy, S.; etc. Isolation of porcine circovirus-like viruses from pigs with a wasting disease in the USA and Europe. Journal of Veterinary Diagnostic Investigation. 1998; 103-110.
[20] Ellis, J.; Hassard, L.; Clark, E.; etc. Isolation of circovirus from lesions of pigs with postweaning multisystemic wasting syndrome. Canadian Veterinary Journal. 1998; 39(1):44-51.
[21] Allan, G. M.; Kennedy, S.; McNeilly, F; etc. Experimental reproduction of severe wasting disease by co-infection of pigs with porcine circovirus and porcine parvovirus. Journal of Comparative Pathology. 1999; 121(1):1-11.
[22] Balasch, M.; Segales, J.; Rosell, C.; etc. Experimental inoculation of conventional pigs with tissue homogenates from pigs with post-weaning multisystemic wasting syndrome. Journal of Comparative Pathology. 1999; 121(2):139-148.
[23] Ellis, J.; Krakowka, S.; Allan, G; etc. "The clinical scope of Podrcine Reproductive and Respiratory Syndrome Virus infection has expanded since 1997": An alternative hypothesis. Veterinary Pathology. 1999b; 36(1): 262-265.
[24] West, K. H. ; Bystrom, J. M.; Wojnarowicz, C.; etc. Myocarditis and abortion associated with intrauterine infection of sows with porcine circovirus 2. Journal of Veterinary Diagnostic Investigation. 1999; 11(6):530-532.
[25] Tischer I, Mields W, Wolfe D.Studies on the pathogenicity of porcine circovirus [J] Vet Diagn Invest. 1999,143:1723-1744.
[26] Allan, G. M. and Ellis, J. A. Porcine circoviruses: a review. Journal of Veterinary Diagnostic Investigation. 2000; 12(1): 3-14.
[27] Ellis, J., S. Krakowka, M. Lairmore, D. etc. Reproduction of lesions of postweaning multisystemic wasting syndrome in gnotobiotic piglets. J. Vet. Diagn. Investig. 1999,11:3-14.
[28] Ellis, J. A., A. Bratanich, E. G. Clark, G.ect. Coinfection by porcine circoviruses and porcine parvovirus in pigs with naturally acquired postweaning multisystemic wasting syndrome. J. Vet. Diagn. Investig. 2000, 12:21-27.
[29] Larochelle, R.; Bielanski, A., Muller, P., and Magar, R. PCR detection and evidence of shedding of porcine circovirus type-2 in boar semen. Journal of Clinical Microbiology. 2000; 38:4629-4632.
[30] Stevenson. GW, Kiupel, M,Mittal S.K. Ultrastructure of porcine circovirus in persistently infected PK-15 cell. Vet Path.(1999)36(5):368-378
[31] Tischer, I.; Mields, W.; Wolff, D.; etc. Studies on epidemiology and pathogenicity of porcine circovirus. Archives of Virology. 1986. 91: 3/4, 271-276.
[32] Magar, R.; Larochelle, R.; Thibault, S., etc. Experimental transmission of porcine circovirus type 2 (PCV-2) in weaned pigs: a sequential study. Journal of Comparative Pathology. 2000; 123(4):258-69.
[33] 郎洪武，王力，张广川等．猪圆环病毒分离鉴定及猪断奶多系统衰弱综合征的诊断．中国兽医科技．2001，31(3)：3-5．
[34] 曹胜波，陈涣春，猪环状病毒2型的PCR检测方法的建立及应用．华中农业大学学报．2001，20(1)：53-56
[35] 李媛，崔尚金，全艳平，等：检测与诊断猪圆环病毒PCR技术的建立及病毒的分离鉴定，中国预防兽医学报，2002，24(增)：164-168．
[36] 吕艳丽，杨汉春，郭鑫，猪圆环病毒2型分离毒株基因组序列测定与分析[J]中国预防兽医学报(2002)增刊：63-66．
[37] Tischer, I.; Gelderblom, H.; Vettermann, W., etc. A very small porcine virus with a circular single-stranded DNA. Nature. 1982; 295:64-66.
[38] Tischer I, Peters D, Rasch R, Replication of Porcine Circovirus: Induction by Glycosamine and Cell Cycle Depensence. Arch Virol. 1987, 96:39-57
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