SARS相关冠状病毒PPA-ELISA和nest-PCR检测方法的建立及联合应用
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摘要
重症急性呼吸综合症(Severe Acute Respiratory Syndrome，SARS)的病原是SARS冠状病毒(SARS.CoV)，为了调查野生动物和家畜家禽体内携带的冠状病毒的情况以及其与SARS.CoV的基因序列同源性，本研究根据SPA能与多种动物血清IgG结合的特性，建立了间接PPA-ELISA检测多种动物血清中SARS病毒相关抗体的方法；根据SARS冠状病毒聚合酶基因序列设计引物，建立套式PCR(nest-PCR)检测方法。对11种动物146个样品进行RT-PCR检测，并用PCR产物测序。与Genbank中的SARS相关冠状病毒的聚合酶基因进行序列比较，构建系统发育树，分析不同宿主来源动物冠状病毒与SARS冠状病毒的同源性。两种检测方法的实验结果表明：
SPA与多种哺乳动物血清中的IgG有较好的结合能力，本实验确定了PPA-ELISA的抗原-抗体最佳反应时间，HRP-SPA与抗体反应最佳时间及底物溶液的显色时间，在此基础上，建立了间接PPA-ELISA检测多种动物血清中SARS病毒相关抗体的检测程序。
通过对冠状病毒聚合酶基因的扩增和序列分析，结果表明被检测的动物样品中的冠状病毒聚合酶的保守基因序列与SARS冠状病毒聚合酶的同一位置的基因序列同源性为100％～95.8％。
通过对同一动物的PPA-ELISA和nest-PCR检测表明，两种检测的约为符合率60％～70％，两种方法的同时使用对动物冠状病毒的检测有着重要的参考价值。
The pathogen of Severe Acute Respiratory Syndrome(SARS) is Corona Virus, For investigating the truth about Corona virus in animals, and find the homologous with SARS. CoV, a essay have been establish that based on the characters that SPA can combine the IgG in the serum of many kinds of animals, indirect PPA-ELISA. To detect antibody of SARS virus in the serum of many animals was established. Two pairs of primer was designed according to the partly polymerase gene of SARS associated corona virus sequences published in Genbank. 146 samples from 11 kinds of animals were detected by nest-PCR., Then PCR product was sequenced. Phylogenetic analysis was done on the amplified region .And the relationship between Corona virus for the animals and SARS. CoV was analyzed.The results indicated that SPA can combine with many mammals IgG in the serum well, and determined the best time of the antigen combined antibody reaction HRP-SPA commeded antibody, At the same time, we made the method of PPA-LEISA which detected the SARS antibody dedicational.The result of amplifying of polymerase gene of SARS associated corona virus and sequence analyze showed that there were high homology between the samples from human and animals, around 100%~95. 8% in nucleotide acid.By detect the sample of the same animals using PPA-ELISA and nest-PCR separated, the accordance rate of the two methods is around 60%~70%, it indicated that it' s valuable for the detection of Corona virus in animals by using the two assay.
SPA与多种哺乳动物血清中的IgG有较好的结合能力，本实验确定了PPA-ELISA的抗原-抗体最佳反应时间，HRP-SPA与抗体反应最佳时间及底物溶液的显色时间，在此基础上，建立了间接PPA-ELISA检测多种动物血清中SARS病毒相关抗体的检测程序。
通过对冠状病毒聚合酶基因的扩增和序列分析，结果表明被检测的动物样品中的冠状病毒聚合酶的保守基因序列与SARS冠状病毒聚合酶的同一位置的基因序列同源性为100％～95.8％。
通过对同一动物的PPA-ELISA和nest-PCR检测表明，两种检测的约为符合率60％～70％，两种方法的同时使用对动物冠状病毒的检测有着重要的参考价值。
The pathogen of Severe Acute Respiratory Syndrome(SARS) is Corona Virus, For investigating the truth about Corona virus in animals, and find the homologous with SARS. CoV, a essay have been establish that based on the characters that SPA can combine the IgG in the serum of many kinds of animals, indirect PPA-ELISA. To detect antibody of SARS virus in the serum of many animals was established. Two pairs of primer was designed according to the partly polymerase gene of SARS associated corona virus sequences published in Genbank. 146 samples from 11 kinds of animals were detected by nest-PCR., Then PCR product was sequenced. Phylogenetic analysis was done on the amplified region .And the relationship between Corona virus for the animals and SARS. CoV was analyzed.The results indicated that SPA can combine with many mammals IgG in the serum well, and determined the best time of the antigen combined antibody reaction HRP-SPA commeded antibody, At the same time, we made the method of PPA-LEISA which detected the SARS antibody dedicational.The result of amplifying of polymerase gene of SARS associated corona virus and sequence analyze showed that there were high homology between the samples from human and animals, around 100%~95. 8% in nucleotide acid.By detect the sample of the same animals using PPA-ELISA and nest-PCR separated, the accordance rate of the two methods is around 60%~70%, it indicated that it' s valuable for the detection of Corona virus in animals by using the two assay.
引文
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[2]Paul A, Rota M, Steven O, et al Characterization of a novel corona virus Associated with Severe Acuter Respiratory Syndrome. Science, 2003,11.26
[3]Lavi E , Weiss SR , Hingley ST , The Nido viruses (Coronaviruses and Arteriviruses) . Dordrechd, Netherlands : KluwerAcademic/Plenum Publicans, 2001. 12742
[4]Peiris JSM. Chu CM. Cheng VC. et al. Clinical progression and viral load in a community outbreak of coronaviras-associated SARS pneumonia:A prospective study[J].lancet, 2003.361:17766～1772
[5]钟南山主编．传染性非典型肺炎临床诊断与治疗．广东教育出版社，2003，21-26
[6]Ruan Y, Wei CL, Et Ah, et al. Comparative full-length genone sequence analysis of 14 SARS coronavirus isolates and common mutations associated with putative origins of infaction, lancet, 2003.361:1779～1785
[7]Holmes KV. SARS2associated coronavirus [J]. N Engl J Med, 2003, 348 (20) :1948-1951.
[8]陈宝峰 任金政 从SARS危机看畜牧中小企业发展 中国农业大学经济管理学院，2003
[9]杨利国，胡少昶等．酶免疫测定技术．南京大学出版社1998(7)，1
[10]王皓，曹之舫．SPA在ELISA中应用条件探讨．上海免疫学杂志．1994．(14)．(2)
[11]何昭阳，胡桂学，王春风主编．动物免疫学实验技术．吉林科学技术出版社．2002 10(1)
[12]王淑敏，冼琼珍，温广杰．PPA—ELISA检测猪传染性胸膜肺炎抗体方法的建立．黑龙江畜牧兽医2003，(1)
[13]姜迪来，郭福生等．PPA-ELISA监测猪传染性胃肠炎病毒血清抗体水平的研究．中国动物检疫．2000(20)(8)
[14]蒋正军，马世东，蔡丽娟等．非洲猪瘟间接ELISA诊断试剂盒的研究．中国预防兽医学报2002(9)，220
[15]丁宗武，临床免疫与实验免疫，1(4)：13，1980
[16]张咏，临床免疫与实验免疫，1(4)：16，1980
[17]Ksiazek TG Erdman D, Goldsmith CS, et al. A Novel Corona virus Associated with Severe Acute Respiratory Syndrome [J]N Engl J Med, 2003,348(20):1947-1958
[18]Drazen JM ,Campion Ew. Severe Acute Respiratory Syndrome. N Engl J MED ,2003 ,348 :1 589.
[19]Haan CA ,Masters PS ,Shen X, et al. The group2specific murine coron2 avirus genes are not essential ,but their deletion,by reverse genetics ,is attenuating in the natural host Virology ,2002 ,296 (1) :1772189.
[20]WHO. SARS : Availability and use of laboratory testing. 24 April 2003. []KathrynV. Holmes:SARS- Associated coronavirus[J]. The New England Journal of Medicine,2003,348:201
[21]Biosinol SARS病毒的背景和初步分析[EB／OL ]1中国生物信息网，2003-04-211
[22]Ortego J, Escors D, Laude H, et al. Generation of a replication2 competent, propagation2deficient virus vector based on the transmissi2 ble gastroenteritis coronavirus genome[J]. J Virol, 2002 , 76 (22) : 11518-11529.
[23]Izata A , Smerdou C ,Alonso S , et al. Replication of transmissible gastroenteritis coronavirus - derived synthetic minigenomes [J ]. J Virol ,1999 ,73:1535-1545.
[24]范宝昌，姜涛，于曼等，4株SARS冠状病毒中国分离株3′非编码区的序列测定及分析，中国生物化学与分子生物学报，2003，19(2)273—277
[25]Yu Man, HuZhijun, Zhao Wei, ChenShuiping, Wang Pengcheng, FanBaochang, Yang Peiying, Qin E de. Analysis of complete genomicsequence of the dengue type 3 virus 80-2 strain isolated from Ouangxi inChina . Chin J Biochem Al Biol) ,2001 ,17(5) :621—625
WHO :WHO Multicentre Collaborative Network for Severe AcuteRespiratory Syndrome (SARS) diagnosis. Weekly Epidemioogical Record ,2003 ,78:121～122
[26]J．萨母布鲁克，E．F．弗里奇，T．曼尼阿蒂斯。分子克隆实验指南(第二版)。科学出版社，1999。
[27]F．奥斯白，R．布伦特，R．E．金斯顿，D．D．穆尔，J．G．塞德曼，J．A．史密斯，K．斯特拉尔。精编分子生物学实验指南。科学技术出版社，1998。
[28]林万明PCR技术操作和应用指南人民军医出版社1993
[29]蔡宝祥主编 家畜传染病学(第四版)北京：中国农业出版社，1999。
[30]甘肃农业大学主编 兽医微生物学(第二版) 北京：中国农业出版社，1999。
[31]Drosten C, Gunther S, Preiser W,et al, Identification of a novel coronavirus in Patients with Severe Acute Respiratory Syndrome. N Engl J Med ,2003,20 (348) :1 967～1 976.
[32]Yang, Weili Severe acute respiratory syndrome (SARS): infection control The Lancet 3 Apr.2003:1386-1387,9366
[33]Williams G D, Chang R Y, Brian D A. A phylogenetically conserved hairpin7type 3' untranslated region pseudoknot functions in coronavirus RNA replication. J Uirol, 1999, 73:8349—8355
[34]殷震，刘景华动物病毒学(第二版)北京：科学出版社1997。
[35]Rest J S, M indell D R SARS associated co ronavims has a recombinant polymerase and coronavirus has a history of host shifting[J ]. Infect Genel Evo 1, 2003 (3) : 192225.
[36]Van Made G,Dobbe J C ,Gultyaev A P ,et al. Arterivirus discontinu2 ous mRNA transcription is guided by base pairing between sense and antisense transcription - regulating sequences [J ]. Proc Natl Acad Sci ,USA ,1999,96:12056 - 12061.
[37]Poutanen SM , Low DE , Henry B. et al , Identification of severe acute respiratory syndrome in Canada. N gngl J Med , 2003 , 20 (348) :1 995～2 005.
[38]丁清泉RNA病毒RNA聚合酶的研究进展 中国病毒学，1998，6：107～113
[39]吴乃虎基因工程原理(第二版)北京：科学出版社，1999。
[40]祝庆余，秦鄂德，王翠娥，等。非典型肺炎病例标本中新型冠状病毒的分离与鉴定。中国生物工程杂志，2003，23：106—108
[41]王艳 马文丽 宋艳斌 反转录巢式PCR方法检测SARS冠状病毒 中国第一军医学报 2003，23(5)：421-423
[2]Paul A, Rota M, Steven O, et al Characterization of a novel corona virus Associated with Severe Acuter Respiratory Syndrome. Science, 2003,11.26
[3]Lavi E , Weiss SR , Hingley ST , The Nido viruses (Coronaviruses and Arteriviruses) . Dordrechd, Netherlands : KluwerAcademic/Plenum Publicans, 2001. 12742
[4]Peiris JSM. Chu CM. Cheng VC. et al. Clinical progression and viral load in a community outbreak of coronaviras-associated SARS pneumonia:A prospective study[J].lancet, 2003.361:17766～1772
[5]钟南山主编．传染性非典型肺炎临床诊断与治疗．广东教育出版社，2003，21-26
[6]Ruan Y, Wei CL, Et Ah, et al. Comparative full-length genone sequence analysis of 14 SARS coronavirus isolates and common mutations associated with putative origins of infaction, lancet, 2003.361:1779～1785
[7]Holmes KV. SARS2associated coronavirus [J]. N Engl J Med, 2003, 348 (20) :1948-1951.
[8]陈宝峰 任金政 从SARS危机看畜牧中小企业发展 中国农业大学经济管理学院，2003
[9]杨利国，胡少昶等．酶免疫测定技术．南京大学出版社1998(7)，1
[10]王皓，曹之舫．SPA在ELISA中应用条件探讨．上海免疫学杂志．1994．(14)．(2)
[11]何昭阳，胡桂学，王春风主编．动物免疫学实验技术．吉林科学技术出版社．2002 10(1)
[12]王淑敏，冼琼珍，温广杰．PPA—ELISA检测猪传染性胸膜肺炎抗体方法的建立．黑龙江畜牧兽医2003，(1)
[13]姜迪来，郭福生等．PPA-ELISA监测猪传染性胃肠炎病毒血清抗体水平的研究．中国动物检疫．2000(20)(8)
[14]蒋正军，马世东，蔡丽娟等．非洲猪瘟间接ELISA诊断试剂盒的研究．中国预防兽医学报2002(9)，220
[15]丁宗武，临床免疫与实验免疫，1(4)：13，1980
[16]张咏，临床免疫与实验免疫，1(4)：16，1980
[17]Ksiazek TG Erdman D, Goldsmith CS, et al. A Novel Corona virus Associated with Severe Acute Respiratory Syndrome [J]N Engl J Med, 2003,348(20):1947-1958
[18]Drazen JM ,Campion Ew. Severe Acute Respiratory Syndrome. N Engl J MED ,2003 ,348 :1 589.
[19]Haan CA ,Masters PS ,Shen X, et al. The group2specific murine coron2 avirus genes are not essential ,but their deletion,by reverse genetics ,is attenuating in the natural host Virology ,2002 ,296 (1) :1772189.
[20]WHO. SARS : Availability and use of laboratory testing. 24 April 2003. []KathrynV. Holmes:SARS- Associated coronavirus[J]. The New England Journal of Medicine,2003,348:201
[21]Biosinol SARS病毒的背景和初步分析[EB／OL ]1中国生物信息网，2003-04-211
[22]Ortego J, Escors D, Laude H, et al. Generation of a replication2 competent, propagation2deficient virus vector based on the transmissi2 ble gastroenteritis coronavirus genome[J]. J Virol, 2002 , 76 (22) : 11518-11529.
[23]Izata A , Smerdou C ,Alonso S , et al. Replication of transmissible gastroenteritis coronavirus - derived synthetic minigenomes [J ]. J Virol ,1999 ,73:1535-1545.
[24]范宝昌，姜涛，于曼等，4株SARS冠状病毒中国分离株3′非编码区的序列测定及分析，中国生物化学与分子生物学报，2003，19(2)273—277
[25]Yu Man, HuZhijun, Zhao Wei, ChenShuiping, Wang Pengcheng, FanBaochang, Yang Peiying, Qin E de. Analysis of complete genomicsequence of the dengue type 3 virus 80-2 strain isolated from Ouangxi inChina . Chin J Biochem Al Biol) ,2001 ,17(5) :621—625
WHO :WHO Multicentre Collaborative Network for Severe AcuteRespiratory Syndrome (SARS) diagnosis. Weekly Epidemioogical Record ,2003 ,78:121～122
[26]J．萨母布鲁克，E．F．弗里奇，T．曼尼阿蒂斯。分子克隆实验指南(第二版)。科学出版社，1999。
[27]F．奥斯白，R．布伦特，R．E．金斯顿，D．D．穆尔，J．G．塞德曼，J．A．史密斯，K．斯特拉尔。精编分子生物学实验指南。科学技术出版社，1998。
[28]林万明PCR技术操作和应用指南人民军医出版社1993
[29]蔡宝祥主编 家畜传染病学(第四版)北京：中国农业出版社，1999。
[30]甘肃农业大学主编 兽医微生物学(第二版) 北京：中国农业出版社，1999。
[31]Drosten C, Gunther S, Preiser W,et al, Identification of a novel coronavirus in Patients with Severe Acute Respiratory Syndrome. N Engl J Med ,2003,20 (348) :1 967～1 976.
[32]Yang, Weili Severe acute respiratory syndrome (SARS): infection control The Lancet 3 Apr.2003:1386-1387,9366
[33]Williams G D, Chang R Y, Brian D A. A phylogenetically conserved hairpin7type 3' untranslated region pseudoknot functions in coronavirus RNA replication. J Uirol, 1999, 73:8349—8355
[34]殷震，刘景华动物病毒学(第二版)北京：科学出版社1997。
[35]Rest J S, M indell D R SARS associated co ronavims has a recombinant polymerase and coronavirus has a history of host shifting[J ]. Infect Genel Evo 1, 2003 (3) : 192225.
[36]Van Made G,Dobbe J C ,Gultyaev A P ,et al. Arterivirus discontinu2 ous mRNA transcription is guided by base pairing between sense and antisense transcription - regulating sequences [J ]. Proc Natl Acad Sci ,USA ,1999,96:12056 - 12061.
[37]Poutanen SM , Low DE , Henry B. et al , Identification of severe acute respiratory syndrome in Canada. N gngl J Med , 2003 , 20 (348) :1 995～2 005.
[38]丁清泉RNA病毒RNA聚合酶的研究进展 中国病毒学，1998，6：107～113
[39]吴乃虎基因工程原理(第二版)北京：科学出版社，1999。
[40]祝庆余，秦鄂德，王翠娥，等。非典型肺炎病例标本中新型冠状病毒的分离与鉴定。中国生物工程杂志，2003，23：106—108
[41]王艳 马文丽 宋艳斌 反转录巢式PCR方法检测SARS冠状病毒 中国第一军医学报 2003，23(5)：421-423