用于诊断四种猪疫病病毒寡核苷酸芯片的研制
|
摘要
高发性呼吸系统、生殖系统、神经及免疫系统主要病毒性病原的混合感染和持续感染，使得集约化养猪业疫病流行状况呈现高度复杂化、多样化趋势，进而增加了临床和实验室诊断难度。目前，对猪疫病病毒性病原的诊断仍采用传统的病原分离和免疫血清学方法，其操作费时费力且灵敏度不高。分子生物学诊断方法如：PCR、标记探针杂交等技术的应用，虽然具有灵敏度高、特异性强、快速、简便的特点，但其假阳性率较高且一次仅能检测一种或同时检测两三种病毒。基于上述技术的缺点，本研究应用寡核苷酸芯片技术，结合不对称PCR和间接荧光标记技术，制备一款低密度猪疫病病原诊断基因芯片，以满足目前集约化养猪生产中流行性病疫原快速、准确、高通量的检疫要求。
本研究确定集约化养猪业常见的4种猪疫病病毒：猪流感病毒、猪伪狂犬病毒、口蹄疫病毒、猪蓝耳病毒作为检测对象。根据四种不同病毒的保守区域设计扩增短片断(100-300 bp)PCR产物的特异性引物。依据每种短片断PCR产物序列，筛选出四条高度特异、长度相同、Tm值和G+C％含量接近的检测探针，并将其固定在氨基化修饰的片基上，制备成低密度60-mer寡核苷酸诊断芯片。通过标记样本与检测探针在固液相体系中杂交，一次试验可完成对这四种猪疫病病毒的诊断。在实验过程中，对芯片上探针点样浓度的选择，每种病毒选择正义链或是反义链探针作为检测探针以及杂交条件等实验过程进行探索和优化。以上述四种猪疫病病毒为检测模型，对经过优化的猪疫病病毒诊断基因芯片集成系统的灵敏度和特异性进行评估和鉴定。
通过一系列试验，本研究取得如下的成果：(1)制作出背景低、固定效率高的(?)病毒性疫病原多重检测的基因芯片；(2)通过优化实验，确定了芯片上探针(?)浓度的最佳浓度为25μM；(3)不对称PCR体系中通过变换上下游引物作为限制性引物，杂交后结果进行比较，最终确定猪流感病毒、猪伪狂犬病毒及口蹄疫病毒以反义链探针作为检测探针，而猪蓝耳病毒则以正义链探针作为检测探针；(4)通过用间接荧光标记技术引入荧光基团，在杂交温度为60℃，杂交时间为10h的条件下，能够获得特异性强、稳定性高的荧光信号；(6)从杂交结果可以看到四种猪疫病病毒均可在在对应的检测探针位置处被检出，没有假阳性结果出现，各个病毒之间也没有出现交叉杂交的现象。芯片检测四种猪疫病病毒靶基因的最低检测限分别是：猪流感病毒7.59×10~2个／mL，猪伪狂犬病毒2.36×10~4个／mL，口蹄疫病毒6个／mL，猪繁殖与呼吸综合症病毒5.05×10~2个／mL。
The important porcine pathogens are frequently complicated with an increased incidence of viral superinfections, which endanger respiratory system, neural system, and reproductive system in the intensive swine industry. Furthermore, the difficulty of laboratory and clinical diagnosis of porcine pathogenic infections would be increased. The rapid and exact diagnosis of epidemic disease and adopting relevant measures is a precontion for loimic prevention. Current routine techniques for identification and detection of porcine pathogenic viruses are still based on virus isolation in tissue culture and serological method, but they are laborious, time-consuming and have a relatively low sensitivity. The advent of several molecular methods are established as a specific, fast and sensitive method for virus detection, such as PCR and southern blotting, but the incidence of false positivity is high and the capacity of these assays are limited. To address some of the limitations of existing methods for pathogens detection, in this paper, a low-density oligonucleotide microarray was develpoed coulped with asymmetric PCR and indirect labeled method for the rapid, accurate and high-throughput detection of epidemic viral pathogens in the swine industry.
In this study, four familiar swine virues were chosen as the investigative targets, including swine influenza virus (SIV), porcine pseudorabies virus (PRV), foot-and-mouth disease virus (FMDV) and porcine reproductive and respiratory syndrome virus (PRRSV). The specific primers were designed to amplify 100-300 bp lengh fragments based on conservative regions of each swine virus. The oligonucleotide probes immobilized on the matrix were designed based on target symmetric PCR products, at the same time ensured the same length (60-mer) as well as approximately identical GC content and Tm for each capture probe. The oligonucleotide probes immobilized on the matrix were designed using DNAstar and Oligo 6.0 software based on target symmetric PCR products, at the same time ensured the same length (60-mer) as well as approximately identical GC content and Tm for each capture probe. After the labelled samples were subjected to solid and solution phase hybridization, four swine virues could be diagnosed in one assay. In this paper we also explored optimization of the following experiment processes: spotting concentration on the microarrays, scale of of restrictive and unrestrictive primers in asymmetric PCR mixture, choice of sense stand or anti-sense probe as capture probe and hybridization condition. Finally, the sensitivity and specificity of oligonucleotides microarray were appraised using this four kinds of pathogens as the detected model.
This research attains the following achievements: (1) The diagnostic oligonucleotide microanay has the virtues of low background and high probed binding efficiency; (2) Through the optimized experiments, the optimal spotting concentration is confirmed to be 25μM; (3) The hybridization results revealed when the reverse primer was ascertained to be restrictive for PRV, SIV, FMDV, while the forward primer was ascertained to be restrictive for PRRSV, at the same time with a restrictive /unrestrictive primer ratio of 1: 100, the specificity of hybridization signals was the most strongest. (4) The hybridization temperature was at 60℃, the hybridization time was for 10h, the strong and stable fluorescent signal could be gained; (5) The hybridization results showed this technique could identify and distinguish the four swine viruses, there were no false positive results and cross hybridization. The detection limit of the oligonucleotide microarray system were 1.63×10~3 gene copy numbers for SIV, 7.08×10~4 gene copy numbers for PRV, 18 gene copy numbers for FMDV, 1.52×10~3 gene copy numbers for PRRSV.
本研究确定集约化养猪业常见的4种猪疫病病毒：猪流感病毒、猪伪狂犬病毒、口蹄疫病毒、猪蓝耳病毒作为检测对象。根据四种不同病毒的保守区域设计扩增短片断(100-300 bp)PCR产物的特异性引物。依据每种短片断PCR产物序列，筛选出四条高度特异、长度相同、Tm值和G+C％含量接近的检测探针，并将其固定在氨基化修饰的片基上，制备成低密度60-mer寡核苷酸诊断芯片。通过标记样本与检测探针在固液相体系中杂交，一次试验可完成对这四种猪疫病病毒的诊断。在实验过程中，对芯片上探针点样浓度的选择，每种病毒选择正义链或是反义链探针作为检测探针以及杂交条件等实验过程进行探索和优化。以上述四种猪疫病病毒为检测模型，对经过优化的猪疫病病毒诊断基因芯片集成系统的灵敏度和特异性进行评估和鉴定。
通过一系列试验，本研究取得如下的成果：(1)制作出背景低、固定效率高的(?)病毒性疫病原多重检测的基因芯片；(2)通过优化实验，确定了芯片上探针(?)浓度的最佳浓度为25μM；(3)不对称PCR体系中通过变换上下游引物作为限制性引物，杂交后结果进行比较，最终确定猪流感病毒、猪伪狂犬病毒及口蹄疫病毒以反义链探针作为检测探针，而猪蓝耳病毒则以正义链探针作为检测探针；(4)通过用间接荧光标记技术引入荧光基团，在杂交温度为60℃，杂交时间为10h的条件下，能够获得特异性强、稳定性高的荧光信号；(6)从杂交结果可以看到四种猪疫病病毒均可在在对应的检测探针位置处被检出，没有假阳性结果出现，各个病毒之间也没有出现交叉杂交的现象。芯片检测四种猪疫病病毒靶基因的最低检测限分别是：猪流感病毒7.59×10~2个／mL，猪伪狂犬病毒2.36×10~4个／mL，口蹄疫病毒6个／mL，猪繁殖与呼吸综合症病毒5.05×10~2个／mL。
The important porcine pathogens are frequently complicated with an increased incidence of viral superinfections, which endanger respiratory system, neural system, and reproductive system in the intensive swine industry. Furthermore, the difficulty of laboratory and clinical diagnosis of porcine pathogenic infections would be increased. The rapid and exact diagnosis of epidemic disease and adopting relevant measures is a precontion for loimic prevention. Current routine techniques for identification and detection of porcine pathogenic viruses are still based on virus isolation in tissue culture and serological method, but they are laborious, time-consuming and have a relatively low sensitivity. The advent of several molecular methods are established as a specific, fast and sensitive method for virus detection, such as PCR and southern blotting, but the incidence of false positivity is high and the capacity of these assays are limited. To address some of the limitations of existing methods for pathogens detection, in this paper, a low-density oligonucleotide microarray was develpoed coulped with asymmetric PCR and indirect labeled method for the rapid, accurate and high-throughput detection of epidemic viral pathogens in the swine industry.
In this study, four familiar swine virues were chosen as the investigative targets, including swine influenza virus (SIV), porcine pseudorabies virus (PRV), foot-and-mouth disease virus (FMDV) and porcine reproductive and respiratory syndrome virus (PRRSV). The specific primers were designed to amplify 100-300 bp lengh fragments based on conservative regions of each swine virus. The oligonucleotide probes immobilized on the matrix were designed based on target symmetric PCR products, at the same time ensured the same length (60-mer) as well as approximately identical GC content and Tm for each capture probe. The oligonucleotide probes immobilized on the matrix were designed using DNAstar and Oligo 6.0 software based on target symmetric PCR products, at the same time ensured the same length (60-mer) as well as approximately identical GC content and Tm for each capture probe. After the labelled samples were subjected to solid and solution phase hybridization, four swine virues could be diagnosed in one assay. In this paper we also explored optimization of the following experiment processes: spotting concentration on the microarrays, scale of of restrictive and unrestrictive primers in asymmetric PCR mixture, choice of sense stand or anti-sense probe as capture probe and hybridization condition. Finally, the sensitivity and specificity of oligonucleotides microarray were appraised using this four kinds of pathogens as the detected model.
This research attains the following achievements: (1) The diagnostic oligonucleotide microanay has the virtues of low background and high probed binding efficiency; (2) Through the optimized experiments, the optimal spotting concentration is confirmed to be 25μM; (3) The hybridization results revealed when the reverse primer was ascertained to be restrictive for PRV, SIV, FMDV, while the forward primer was ascertained to be restrictive for PRRSV, at the same time with a restrictive /unrestrictive primer ratio of 1: 100, the specificity of hybridization signals was the most strongest. (4) The hybridization temperature was at 60℃, the hybridization time was for 10h, the strong and stable fluorescent signal could be gained; (5) The hybridization results showed this technique could identify and distinguish the four swine viruses, there were no false positive results and cross hybridization. The detection limit of the oligonucleotide microarray system were 1.63×10~3 gene copy numbers for SIV, 7.08×10~4 gene copy numbers for PRV, 18 gene copy numbers for FMDV, 1.52×10~3 gene copy numbers for PRRSV.
引文
[1]Van Reeth K, Guan. Swine influenza: an update from Europe and Asia[M]. 2000, 6:4-7
[2]Van Reeth K, Nauwynck H, and Pensaert M. Dual infections of feeder pigs with porcine reproductive and respiratory syndrome virus followed by porcine respiratory coronavirus or swine influenza virus: A clinical and virological study[J].Vet Mic, 1996a, 48:325-335
[3]Eileen L,Thacker, Brad J, et al.Interaction between Mycoplasma hyopneumoniae and Swine Influenza Virus[J]. J Clin Microbiol, 2001, 7:2525-2530.
[4]Rohm C, Zhou N, Suss A, et al. Characterization of a novel influenza hemagglutinin, H15: criteria for determination of influenza Asubtypes[J]. Virology, 1996, 217: 508-516.
[5]Fields B N, Knipe D M, Howley P M, et al. Fields virology[M].Lippincott-Raven publishers, phildelphia, 1996: 1397-1445.
[6]斯特劳BE，阿莱尔SD，蒙加林WL等主编．赵德明，张仲秋，沈建忠主译．猪病学(第八版)[M]．北京：中国农业大学出版社，2000年9月，277-300．
[7]Ito T, Couceiro J, Kelm S, et al. Molecular basis for the generation in pigs of influenza A virus w ith pandemic potential[J]. J Virol, 1998, 72: 7367-7373.
[8]Kida H, Shortridge K F,Webster R G. Origin of the hemagglutinin gene of H3N2 influenza viruses from pigs in China[J]. Virology, 1988, 162: 160-166.
[9]Olsen C W, Carey S, Hinshaw L, et al. Virologic and serologic surveillance for human, swine and avian influenza virus infections among pigs in the north-central United States[J]. J Gen Virol, 2000, 145:1399-1419.
[10]Shope, R E..Swine influenza[J].Fitration Experiments and Etiology,1931, 54: 373-385.
[11]Straw B E, D'Allaire S,M engeling W L, et al. Disease of swine (8thed)[M]. Iown state press, Ames, Iowa, USA, 1999: 277-290.
[12]Harlbur P G. Defining the causes of PRDC[C]. Swine consultant, Pfizer Animal Health, 1996, fall: 4215.
[13]Van Reeth K, Labarque G. Efficacy of vaccination of pigs with different H1N1 swine influenza viruses using a recent challenge strain and different parameters of p rotection[J]. Vaccine, 2001,19(31): 4479-4486.
[14]Li L S, Lin Y P. Evidence for interspecies transm ission and reassorment of influenza A viruses in pigs in southern China[J].Virology,1994,202: 825.
[15]李海燕，李雁冰，于康震，等．猪流感病毒的分离与鉴定[J]．中国预防兽医学报，2002，24(1)：12-17．
[16]郭元吉，R G Webster．我国猪群中猪型(H1N1)流感病毒的发现及其来源的调查[J]．中华实验和临床病毒学杂志，1992，6(4)：347-251．
[17]Kundin W D.Hong Kong influenza virus infection among swing during a human epidemic in Taiwan[J].Nature, 1970, 47:489-491.
[18]李海燕，于康震，辛晓光，等．部分省市猪流感的血清学调查及猪流感病毒的分离与鉴定[J]．动物医学进展，2003，24(3)：67-72．
[19]Peiris J S M, Guan Y, Markwell D,et al. Coeireulation of Avian H9N2 and Contemporary" Human" H3N2 Influema A Viruses in Pigs in Southeastern Claim: Potential for Genetic Reassortment?[J]. J Vrol, 2001, 75: 9679-9686.
[20]李海燕，于康震，杨焕良，等．中国猪源H5N1和H9N2亚型流感病毒的分离鉴定[J]．中国预防兽医学报，2004，26(1)：1-6．
[21]Stephen C.H4N6 influenza virus isloated from pigs in Ontario[J].Gen.Vet.J,2000, 41:938-939.
[22]Hinshaw V S, Bean W J. Webster R G, et al.The prevalence of influenza virus in swine and the antigenic and genetic relatedness of influenza virus from man and swine[J].Virology, 1978, 84: 51.
[23]Ito T, Couceiro J N, Kelm S, et al.Molecular basis for the generation in pigs of influenza A viruses with pandemic potential[J]. J Virol, 1998, 72:7367-7373.
[24]Lin Y P M. Shaw V, Gregory K, el al.Avian to-human transmission of H9N2 subtype influenza viruses: relationship between H9N2 and H5N1 human isolates[J].Proc Natl Acad Sci USA, 2000, 97: 9654-9658.
[25]殷震、刘景华．动物病毒学．北京：科学出版社，1985，279-290，700-713．
[26]杨复华主编．病毒学．长沙：湖南科学技术出版社，1992：456-460．
[27]朱庆虎，黄俊明．近10年我国伪狂犬病研究进展[J]．中国畜禽传染病，1996，18(6)：59-61．
[28]Pensaert M, Klnge J. Virus Infection Of porcines, Elsevier SciefIce Publ[M]. Amstentam 1989, 41-43.
[29]Gidkens A L J, Peeters B P M Function of Aujeszky's disease virus proteins in virus replication and virutence[J]. Acta Veterinaria Hungarica, 1994, 42(23): 227-241.
[30]Heffner, Kovacs S F, Klupp B G, et al. Glycoiprotein gp50-negative pseudorabis virus: a never approch toward a nonspreading live herpesvirus vaccine[J]. J Virol, 1993, 67:1529-1537.
[31]殷震、刘景华．动物病毒学(第二版)[M]．北京：科学出版社，1997，998-1009．
[32]袁庆志，李卓然，南锡，等．猪伪狂犬病病毒的分离与鉴定[J]．中国畜禽传染病，1987，334(3)：10-11．
[33]江焕贤．猪伪狂犬病病毒京A株的分离与鉴定[J]．中国兽医杂志，1993，19(4)：6-7．
[34]Brown F. Vaccination against foot and mouth disease virus[J].Vaccine, 1992,10:1022-1026.
[35]谢庆阁．中国进出境动物检疫规范[M]．北京：中国农业出版社，1997．251-263．
[36]Gohara D W,Crotty S,Arnold J J, et al. Poliovirus RNA dependent RNA polymerase[J]. J Biol Chem, 2000, 275:25523-25532.
[37]Bachrach H L. Foot-and-mouth disease virus[J]. Annu Rev Microbiol,1968,22:201-244.
[38]Carroll A R, Rowlands D J,Carke B E.The complete nucleotide sequence of the RNA coding for the primary translation product of foot-and-mouth disease virus[J].Nucleic Acids Res,1984,12 (5):2461-24721.
[39]Forss S,Strebel K,Beck E,et al.Nucleotide sequence and genome organization of foot-and-mouth disease virus[J]. Nucleic Acids Res,1984,12:6587-66011.
[40]King A M, Sangar D V, Harris T J, et al.Heterogeneity of the genome-linked protein of foot-and-mouth disease virus[J].J Virol, 1980,34(3): 627-634.
[41]Anger D V, Rowland D J, Harris T J, et al. Protein covalently linked to foot-and-mouth disease virus RNA. Nature, 1977, 268(5621): 648-650.
[42]Fross S, Strebel K, Beck E, et al.Nucleotide sequence and genome organization of foot-and-mouth disease virus[J].Nucl Acids Res, 1984, 12 (16):6587-6601.
[43]张显升，刘在新，赵启祖，等．口蹄疫病毒基因组RNA结构与功能研究进展[J]．病毒学报，2001，17(4)：375-3801．
[44]Wensvoort G, Terpst ra C, Pol J M A, et al. Mystery swine disease in the Netherlands: The isolation of Lelystad virus[J]. Vet Microbiol, 1991,13: 121-130.
[45]Snijder E J, Meulenberg J M. The molecular biology of arteriviruses[J]. J Gen Virol,1998, 271: 4684-4691.
[46]Meulenberg J J M, HulstM M, De Mijer E J, et al.Lelystad virus, the causative agent of porcine epidemic abortion and respiratory syndrome (PEARS), is related to LDV and EAV[J]. Virology, 1993, 192: 62-72.
[47]Meulenberg J J M, Besten A P, De Kluyver E P, et al. Characterizat ion of proteins encoded by ORFs 2 to 7 of Lelystad virus[J]. Virology, 1995, 206: 155-163.
[48]Zhengjun Jiang, En-Min Zhou, Mehrdad Ameri-Mahabadi, et al. Identification and characterization of auto2anti2idiotypic antibodies specific for antibodies against porcine reproductive andrespiratory syndrome virus envelope glycoprotein (GPS)[J].Vet Immunol Immunopat hol, 2003,92:125-135.
[49]Pirzadeh B and Dea S. Immune response in pigs vaccinated withplasmid DNA encoding ORF5 of porcine reproductive and respiratorysyndrome virus. Journal of General Virology, 1998, 79, 989-999.
[50]Keffabern K K. Reproductive failure of unknown etiology[J].Am Asso Swine Pract Newsletter, 1989, 1(2):1-9.
[51]Wensvoort G, Kluyver E P, et al.Lelystad virus,the cause of porcine epidemic abortion and respiratory syndrome: a reviewof mystery swine disease research at lelystad[J]. Vet Microbiolog, 1992, 33:185-193.
[52]郭宝清，陈章水，刘文兴，等．从疑似PRRS流产胎儿分离PRRSV的研究[J]．中国畜禽传染病，1996，87(2)：1-4．
[53]吕晓星，丁国华．猪繁殖与呼吸综合症(PRRS)[J]．中国动物检疫，1998，15(1)：49—51．
[54]王连想，毕英佐，曹永长，等．猪流感病毒广东株的分离鉴定及其特性[J]．中国兽医科技，2003，33(2)：17-21．
[55]许传田，范伟兴，赵宏坤．A型猪流感病毒山东分离株鉴定及其HA基因序列分析[J]．中国病毒学，2004，19(1)：27-31．
[56]李海燕，于康震，杨焕良，等．中国猪源H5N1和H9N2亚型流感病毒的分离鉴定[J]．中国预防兽医学报，2004，26(1)：1-6．
[57]Duca M, Morosanu V. Efficiency of complement fixation (CF)test with internal nucleoprotein (NP) antigen and hemagglutination-inhibition (HI) test in the serodiagnosis of A and B influenza infections[J]. Arch Roum Pat hol Exp Microbiol,1979,38 (324):331-337.
[58]Jennings R, Potter C W, Mclaren C,et al. Effect of preinfection and preimmunization on the serum antibody response to subsequent immunization with heterotypic influenza vaccines[J]. J Immunol,1974,113 (6):1834-1843.
[59]Swenson S L, Vincent L L,Lute B M, et al. Acomparison of diagnostic assays for the detection of type A swine influenzavirus from nasal swabs and lungs[J]. J Vet Diagn Invest,2001,13 (1): 36-42.
[60]Voeten J T, Groen J, Van Alphen D, et al. Use of recombinant nucleoproteins in enzyme-linked immunosorbent assays for detection of virus specific immunoglobulin A or Ginfected patients[J]. J Clin Microbiol,1998,36 (12):3527-3531.
[61]亢文华，郝俊峰，张仲秋，等．PCR-ELISA快速检测高致病性禽流感病毒方法的建立[J]．中国畜牧兽医，2005，32(6)：54-56．
[62]Munch M, Nielsen L P, Handberg KJ, et al. Detection and Subtyping (H5 and H7) of avian type A influenza virus by reverse transcription PCR and PCR-ELISA[J]. Arch Virol,2001, 146:87-97.
[63]祁贤，陆承平，王贵平，等．套式RT-PCR方法检测临床样品中的猪流感病毒[J]．中国病毒学，2004，20(2)：200-202．
[64]Richt J A, Lager K M, Clouser D F, et al. Real-time reverse transcription-polymerase chain reaction assays for the detection and differentiation of North American swine influenza viruses[J]. J Vet Diagn Invest,2004,16 (5):367-373.
[65]陈焕春，方六荣，何启盖，等．猪伪狂犬病病毒鄂A株的分离鉴定[J]．畜牧兽医学报，1998，29(2)：156-161．
[66]黄伟坚，温荣辉，秦爱珍，等．猪伪狂犬病毒(PRV)桂B株的分离和鉴定[J]．广西农业大学学报，1998，17(12)：11-15．
[67]边传周，王惠杰，陈创．猪伪狂犬病毒河南株的分离与鉴定[J]．河南农业科学，2005，2：69-70
[68]Maes RK, Sussman MD, Vilnis A, et al. Recent developments in latency and recombination of Aujeszky'sdisease (pseudorabies) virus[J]. Vet Microbiol 1997,55(1-4): 13-27.
[69]杨永钦，黄德生，李乐．斑点ELISA检测口蹄疫病毒的研究[J]．中国兽医杂志，1994，20(9)：6-7．
[70]Meyer R F.Rapid and sensitive detection of foot-and-mouth disease virues in tissues by enzymatic RNA amplification of the polymerse gene[J].J Virol Method, 1991, 34(2): 161-172.
[71]Laor O. Detection of FMDV RNA amplified by PCR[J]. J Virol Method, 1992(36): 197-208.
[72]吴时友．应用RT-PCR检测口蹄疫病毒(FMDV)的研究初报[J]．动物检疫，1991，8(6)：1-2．
[73]朱彩珠．RT-PCR检测口蹄疫病毒[J]．兰州大学学报(自然科学版)，1995，31：116-122．
[74]万绍贵，何后军，尹燕博，等．猪繁殖与呼吸综合症病毒江西株的分离与鉴定[J]．江西农业大学学报(自然科学版)，2002，24(6)：87-872．
[75]程安春，汪铭书，希尼尼根，等．猪繁殖与呼吸综合症病毒四川株分离、鉴定及其病原特性研究[J]．黑龙江畜牧兽医，2004(9)：15-18．
[76]Dea S, Wilson L, Therrien D, et al. Competitive ELISAfor detection of antibodies to porcine reproductive and respiratory syndrome virus using recombinant E. coli-expressed nucleocapsid protein a antigen[J]. J Virol Methods, 2001,87(1-2): 109-122.
[77]简中友，姜平，马志勇，等．ELISA检测猪繁殖综合症病毒(PRRSV)抗体方法的建立及血清流行病学调查[J]．中国动物检疫，1997，14(5)：24-26．
[78]Suarez P, Zardoya R, Prieto C, et al. Direct Detection on the Porcine Reproductive and Respiratory Syndrome (PRRS) Virus by Reverse Polymerase Chain Reaction (RT-PCR)[J]. Archives of Virology,1994,135: 89-99.
[79]Kono Y, Shimizu M, Yamada S, et al. Nested PCRfor detection and typing of porcine reproductive and respiratory syndrome (PRRS) virus in pigs[J]. J Vet Med Sci, 1996,58(10): 941-946.
[80]Egli C, Thür B, Liu L, et al. Quantitative Taqman RT-PCR for the detection and differentiation of European and North American strain porcine reproductive and respiratory syndrome virus[J]. J Virol Methods, 2001, 98 (1): 63-75.
[81]Sur JH, Cooper VL, Galeota JA, et al. In vivo detection of porcine reproductive and respiratory syndrome virus RNA by in situ hybridization at different times postinfection[J]. Clin Microbiol, 1996, 34(9): 2280-2286.
[82]Hofman P. DNA Microarrays[J]. Nephron Physiol,2005,99 (3):85-89.
[83]Kane M D,Jatkoe T A,Stumpt C R,et al. Assessment of the sensitivity and specificity of oligonucleotide (50mer) microarrays[J]. Nucleic Acids Res, 2000, 28(22):4552-4557.
[84]Kozal M J,Shah N,et al. Extensive polymorphisms observed in HIV-1 clade B protease gene using high-density oligonucleotide arrays[J].Nature Medicine,1996,2(7):753-759.
[85]Lipshutz RJ, Morris D,Chee M, et al. Using oligonucleotide probe arrays to access genetic diversity[J].Biotechniques. 1995,19 (3):442-447.
[86]吴海，顾少华，胡志前等．检测乙丙型肝炎病毒基因芯片的制备[J]．第二军医大学学报，2001，22(6)：527-529．
[87]Sengupta S, Onodera K, Lai A, et al. Molecular detection and identification of influenza viruses by oligonucleotide microarray hybridization[J]. J Clin Microbiol, 2003, 41 (10): 4542-4550.
[88]Chambers J, Angulo A, et al.DNA microarrays of the complex human cytomegalovirus genome: profiling kinetic class with drug sensitivity of viral gene expression[J]. J V irol. 1999, 73 (7):5757-5766.
[89]陶生策，杨仁全等．SARS冠状病毒基因芯片的制作与初步临床样本验[J]．清华大学学报(自然科学版)，2003，43(5)：715-720．
[90]王艳，马文丽，毛向明，等．痘苗病毒寡核苷酸检测芯片的设计及研制[J]．第二军医大学学 报，2004，24(2)：180-183．
[91]张海燕，马文丽，李凌，等．乙型脑炎病毒寡核苷酸基因芯片研究[J]．实用医学杂志，2005，21(12)：1244-1247．
[92]Wang H, Malek R L, Kwitek A E, et al. Assessing unmodified 70-mer oligonucleotide probe performance on glass-slide microarrays[J]. Genome Biol, 2003, 4 (1): R5.
[93]Call D R, Chandler D, Brockman F. Fabrication of DNA microarrays using unmodified oligonucleotide probes[J]. Bio techniques, 2001, 30(2): 368-379.
[94]Yang S, Ghanny S, Wang W, et al. Using DNA microarray to study human cytomegalovirus gene expression[J]. Journal of Virological Methods, 2006 (131): 202-208.
[95]Cao X, Wang Y, Zhang C, et at. Visual DNA microarrays for simultaneous detection of Ureaplasma urealyticum and Chlamydia trachomatis coupled with multiplex asymmetrical PCR[J]. Biosensors and Bioelectronics, 2006, 22(3): 393-398.
[96]Gyllensten U B, Erlich H A. Generation of single-stranded DNA by the polymerase chain reaction and its application to direct sequencing of the HLA-DQA locus[J]. Proc Natl Acad Sci LISA, 1988, 85 (20): 7652-7656.
[97]Mo X, Ma W, Li L, et at. The effects of different sample labelling methods on signal intensities of a 60-met diagnostic microarray[J]. Journal of Virological Methods, 2006, 134(1-2): 36-40.
[98]Murray AE,et al. DNA/DNAhybridization to microarrays reveals gene-specific differences between closely related microbial genomes[J].Proc Natl Acad Sci USA,2001,98(17):9853-9858.
[99]万超，温国元，潘兹书，等．快速检测伪狂犬病毒荧光定量PCR技术建立及应用[J]．中国病毒学，2006，21(5)：485-489．
[100]李永东，张常印，姜平，等．口蹄疫病毒实时RT-PCR检测方法的建立[J]．中国人兽共患病学报，2006，22(3)：212-215．
[101]M．谢纳．生物芯片分析[M]．Vol影印版 北京：科学出版社，2003．
[102]Zhou X, Zhou J.Oligosaccharide microarrays fabricated on aminooxyacetyl functional glass surface for charaterization of carbohydrate-protein interaction[J].Biosensors and Bioelectronics, 2006,21:1451-1458.
[2]Van Reeth K, Nauwynck H, and Pensaert M. Dual infections of feeder pigs with porcine reproductive and respiratory syndrome virus followed by porcine respiratory coronavirus or swine influenza virus: A clinical and virological study[J].Vet Mic, 1996a, 48:325-335
[3]Eileen L,Thacker, Brad J, et al.Interaction between Mycoplasma hyopneumoniae and Swine Influenza Virus[J]. J Clin Microbiol, 2001, 7:2525-2530.
[4]Rohm C, Zhou N, Suss A, et al. Characterization of a novel influenza hemagglutinin, H15: criteria for determination of influenza Asubtypes[J]. Virology, 1996, 217: 508-516.
[5]Fields B N, Knipe D M, Howley P M, et al. Fields virology[M].Lippincott-Raven publishers, phildelphia, 1996: 1397-1445.
[6]斯特劳BE，阿莱尔SD，蒙加林WL等主编．赵德明，张仲秋，沈建忠主译．猪病学(第八版)[M]．北京：中国农业大学出版社，2000年9月，277-300．
[7]Ito T, Couceiro J, Kelm S, et al. Molecular basis for the generation in pigs of influenza A virus w ith pandemic potential[J]. J Virol, 1998, 72: 7367-7373.
[8]Kida H, Shortridge K F,Webster R G. Origin of the hemagglutinin gene of H3N2 influenza viruses from pigs in China[J]. Virology, 1988, 162: 160-166.
[9]Olsen C W, Carey S, Hinshaw L, et al. Virologic and serologic surveillance for human, swine and avian influenza virus infections among pigs in the north-central United States[J]. J Gen Virol, 2000, 145:1399-1419.
[10]Shope, R E..Swine influenza[J].Fitration Experiments and Etiology,1931, 54: 373-385.
[11]Straw B E, D'Allaire S,M engeling W L, et al. Disease of swine (8thed)[M]. Iown state press, Ames, Iowa, USA, 1999: 277-290.
[12]Harlbur P G. Defining the causes of PRDC[C]. Swine consultant, Pfizer Animal Health, 1996, fall: 4215.
[13]Van Reeth K, Labarque G. Efficacy of vaccination of pigs with different H1N1 swine influenza viruses using a recent challenge strain and different parameters of p rotection[J]. Vaccine, 2001,19(31): 4479-4486.
[14]Li L S, Lin Y P. Evidence for interspecies transm ission and reassorment of influenza A viruses in pigs in southern China[J].Virology,1994,202: 825.
[15]李海燕，李雁冰，于康震，等．猪流感病毒的分离与鉴定[J]．中国预防兽医学报，2002，24(1)：12-17．
[16]郭元吉，R G Webster．我国猪群中猪型(H1N1)流感病毒的发现及其来源的调查[J]．中华实验和临床病毒学杂志，1992，6(4)：347-251．
[17]Kundin W D.Hong Kong influenza virus infection among swing during a human epidemic in Taiwan[J].Nature, 1970, 47:489-491.
[18]李海燕，于康震，辛晓光，等．部分省市猪流感的血清学调查及猪流感病毒的分离与鉴定[J]．动物医学进展，2003，24(3)：67-72．
[19]Peiris J S M, Guan Y, Markwell D,et al. Coeireulation of Avian H9N2 and Contemporary" Human" H3N2 Influema A Viruses in Pigs in Southeastern Claim: Potential for Genetic Reassortment?[J]. J Vrol, 2001, 75: 9679-9686.
[20]李海燕，于康震，杨焕良，等．中国猪源H5N1和H9N2亚型流感病毒的分离鉴定[J]．中国预防兽医学报，2004，26(1)：1-6．
[21]Stephen C.H4N6 influenza virus isloated from pigs in Ontario[J].Gen.Vet.J,2000, 41:938-939.
[22]Hinshaw V S, Bean W J. Webster R G, et al.The prevalence of influenza virus in swine and the antigenic and genetic relatedness of influenza virus from man and swine[J].Virology, 1978, 84: 51.
[23]Ito T, Couceiro J N, Kelm S, et al.Molecular basis for the generation in pigs of influenza A viruses with pandemic potential[J]. J Virol, 1998, 72:7367-7373.
[24]Lin Y P M. Shaw V, Gregory K, el al.Avian to-human transmission of H9N2 subtype influenza viruses: relationship between H9N2 and H5N1 human isolates[J].Proc Natl Acad Sci USA, 2000, 97: 9654-9658.
[25]殷震、刘景华．动物病毒学．北京：科学出版社，1985，279-290，700-713．
[26]杨复华主编．病毒学．长沙：湖南科学技术出版社，1992：456-460．
[27]朱庆虎，黄俊明．近10年我国伪狂犬病研究进展[J]．中国畜禽传染病，1996，18(6)：59-61．
[28]Pensaert M, Klnge J. Virus Infection Of porcines, Elsevier SciefIce Publ[M]. Amstentam 1989, 41-43.
[29]Gidkens A L J, Peeters B P M Function of Aujeszky's disease virus proteins in virus replication and virutence[J]. Acta Veterinaria Hungarica, 1994, 42(23): 227-241.
[30]Heffner, Kovacs S F, Klupp B G, et al. Glycoiprotein gp50-negative pseudorabis virus: a never approch toward a nonspreading live herpesvirus vaccine[J]. J Virol, 1993, 67:1529-1537.
[31]殷震、刘景华．动物病毒学(第二版)[M]．北京：科学出版社，1997，998-1009．
[32]袁庆志，李卓然，南锡，等．猪伪狂犬病病毒的分离与鉴定[J]．中国畜禽传染病，1987，334(3)：10-11．
[33]江焕贤．猪伪狂犬病病毒京A株的分离与鉴定[J]．中国兽医杂志，1993，19(4)：6-7．
[34]Brown F. Vaccination against foot and mouth disease virus[J].Vaccine, 1992,10:1022-1026.
[35]谢庆阁．中国进出境动物检疫规范[M]．北京：中国农业出版社，1997．251-263．
[36]Gohara D W,Crotty S,Arnold J J, et al. Poliovirus RNA dependent RNA polymerase[J]. J Biol Chem, 2000, 275:25523-25532.
[37]Bachrach H L. Foot-and-mouth disease virus[J]. Annu Rev Microbiol,1968,22:201-244.
[38]Carroll A R, Rowlands D J,Carke B E.The complete nucleotide sequence of the RNA coding for the primary translation product of foot-and-mouth disease virus[J].Nucleic Acids Res,1984,12 (5):2461-24721.
[39]Forss S,Strebel K,Beck E,et al.Nucleotide sequence and genome organization of foot-and-mouth disease virus[J]. Nucleic Acids Res,1984,12:6587-66011.
[40]King A M, Sangar D V, Harris T J, et al.Heterogeneity of the genome-linked protein of foot-and-mouth disease virus[J].J Virol, 1980,34(3): 627-634.
[41]Anger D V, Rowland D J, Harris T J, et al. Protein covalently linked to foot-and-mouth disease virus RNA. Nature, 1977, 268(5621): 648-650.
[42]Fross S, Strebel K, Beck E, et al.Nucleotide sequence and genome organization of foot-and-mouth disease virus[J].Nucl Acids Res, 1984, 12 (16):6587-6601.
[43]张显升，刘在新，赵启祖，等．口蹄疫病毒基因组RNA结构与功能研究进展[J]．病毒学报，2001，17(4)：375-3801．
[44]Wensvoort G, Terpst ra C, Pol J M A, et al. Mystery swine disease in the Netherlands: The isolation of Lelystad virus[J]. Vet Microbiol, 1991,13: 121-130.
[45]Snijder E J, Meulenberg J M. The molecular biology of arteriviruses[J]. J Gen Virol,1998, 271: 4684-4691.
[46]Meulenberg J J M, HulstM M, De Mijer E J, et al.Lelystad virus, the causative agent of porcine epidemic abortion and respiratory syndrome (PEARS), is related to LDV and EAV[J]. Virology, 1993, 192: 62-72.
[47]Meulenberg J J M, Besten A P, De Kluyver E P, et al. Characterizat ion of proteins encoded by ORFs 2 to 7 of Lelystad virus[J]. Virology, 1995, 206: 155-163.
[48]Zhengjun Jiang, En-Min Zhou, Mehrdad Ameri-Mahabadi, et al. Identification and characterization of auto2anti2idiotypic antibodies specific for antibodies against porcine reproductive andrespiratory syndrome virus envelope glycoprotein (GPS)[J].Vet Immunol Immunopat hol, 2003,92:125-135.
[49]Pirzadeh B and Dea S. Immune response in pigs vaccinated withplasmid DNA encoding ORF5 of porcine reproductive and respiratorysyndrome virus. Journal of General Virology, 1998, 79, 989-999.
[50]Keffabern K K. Reproductive failure of unknown etiology[J].Am Asso Swine Pract Newsletter, 1989, 1(2):1-9.
[51]Wensvoort G, Kluyver E P, et al.Lelystad virus,the cause of porcine epidemic abortion and respiratory syndrome: a reviewof mystery swine disease research at lelystad[J]. Vet Microbiolog, 1992, 33:185-193.
[52]郭宝清，陈章水，刘文兴，等．从疑似PRRS流产胎儿分离PRRSV的研究[J]．中国畜禽传染病，1996，87(2)：1-4．
[53]吕晓星，丁国华．猪繁殖与呼吸综合症(PRRS)[J]．中国动物检疫，1998，15(1)：49—51．
[54]王连想，毕英佐，曹永长，等．猪流感病毒广东株的分离鉴定及其特性[J]．中国兽医科技，2003，33(2)：17-21．
[55]许传田，范伟兴，赵宏坤．A型猪流感病毒山东分离株鉴定及其HA基因序列分析[J]．中国病毒学，2004，19(1)：27-31．
[56]李海燕，于康震，杨焕良，等．中国猪源H5N1和H9N2亚型流感病毒的分离鉴定[J]．中国预防兽医学报，2004，26(1)：1-6．
[57]Duca M, Morosanu V. Efficiency of complement fixation (CF)test with internal nucleoprotein (NP) antigen and hemagglutination-inhibition (HI) test in the serodiagnosis of A and B influenza infections[J]. Arch Roum Pat hol Exp Microbiol,1979,38 (324):331-337.
[58]Jennings R, Potter C W, Mclaren C,et al. Effect of preinfection and preimmunization on the serum antibody response to subsequent immunization with heterotypic influenza vaccines[J]. J Immunol,1974,113 (6):1834-1843.
[59]Swenson S L, Vincent L L,Lute B M, et al. Acomparison of diagnostic assays for the detection of type A swine influenzavirus from nasal swabs and lungs[J]. J Vet Diagn Invest,2001,13 (1): 36-42.
[60]Voeten J T, Groen J, Van Alphen D, et al. Use of recombinant nucleoproteins in enzyme-linked immunosorbent assays for detection of virus specific immunoglobulin A or Ginfected patients[J]. J Clin Microbiol,1998,36 (12):3527-3531.
[61]亢文华，郝俊峰，张仲秋，等．PCR-ELISA快速检测高致病性禽流感病毒方法的建立[J]．中国畜牧兽医，2005，32(6)：54-56．
[62]Munch M, Nielsen L P, Handberg KJ, et al. Detection and Subtyping (H5 and H7) of avian type A influenza virus by reverse transcription PCR and PCR-ELISA[J]. Arch Virol,2001, 146:87-97.
[63]祁贤，陆承平，王贵平，等．套式RT-PCR方法检测临床样品中的猪流感病毒[J]．中国病毒学，2004，20(2)：200-202．
[64]Richt J A, Lager K M, Clouser D F, et al. Real-time reverse transcription-polymerase chain reaction assays for the detection and differentiation of North American swine influenza viruses[J]. J Vet Diagn Invest,2004,16 (5):367-373.
[65]陈焕春，方六荣，何启盖，等．猪伪狂犬病病毒鄂A株的分离鉴定[J]．畜牧兽医学报，1998，29(2)：156-161．
[66]黄伟坚，温荣辉，秦爱珍，等．猪伪狂犬病毒(PRV)桂B株的分离和鉴定[J]．广西农业大学学报，1998，17(12)：11-15．
[67]边传周，王惠杰，陈创．猪伪狂犬病毒河南株的分离与鉴定[J]．河南农业科学，2005，2：69-70
[68]Maes RK, Sussman MD, Vilnis A, et al. Recent developments in latency and recombination of Aujeszky'sdisease (pseudorabies) virus[J]. Vet Microbiol 1997,55(1-4): 13-27.
[69]杨永钦，黄德生，李乐．斑点ELISA检测口蹄疫病毒的研究[J]．中国兽医杂志，1994，20(9)：6-7．
[70]Meyer R F.Rapid and sensitive detection of foot-and-mouth disease virues in tissues by enzymatic RNA amplification of the polymerse gene[J].J Virol Method, 1991, 34(2): 161-172.
[71]Laor O. Detection of FMDV RNA amplified by PCR[J]. J Virol Method, 1992(36): 197-208.
[72]吴时友．应用RT-PCR检测口蹄疫病毒(FMDV)的研究初报[J]．动物检疫，1991，8(6)：1-2．
[73]朱彩珠．RT-PCR检测口蹄疫病毒[J]．兰州大学学报(自然科学版)，1995，31：116-122．
[74]万绍贵，何后军，尹燕博，等．猪繁殖与呼吸综合症病毒江西株的分离与鉴定[J]．江西农业大学学报(自然科学版)，2002，24(6)：87-872．
[75]程安春，汪铭书，希尼尼根，等．猪繁殖与呼吸综合症病毒四川株分离、鉴定及其病原特性研究[J]．黑龙江畜牧兽医，2004(9)：15-18．
[76]Dea S, Wilson L, Therrien D, et al. Competitive ELISAfor detection of antibodies to porcine reproductive and respiratory syndrome virus using recombinant E. coli-expressed nucleocapsid protein a antigen[J]. J Virol Methods, 2001,87(1-2): 109-122.
[77]简中友，姜平，马志勇，等．ELISA检测猪繁殖综合症病毒(PRRSV)抗体方法的建立及血清流行病学调查[J]．中国动物检疫，1997，14(5)：24-26．
[78]Suarez P, Zardoya R, Prieto C, et al. Direct Detection on the Porcine Reproductive and Respiratory Syndrome (PRRS) Virus by Reverse Polymerase Chain Reaction (RT-PCR)[J]. Archives of Virology,1994,135: 89-99.
[79]Kono Y, Shimizu M, Yamada S, et al. Nested PCRfor detection and typing of porcine reproductive and respiratory syndrome (PRRS) virus in pigs[J]. J Vet Med Sci, 1996,58(10): 941-946.
[80]Egli C, Thür B, Liu L, et al. Quantitative Taqman RT-PCR for the detection and differentiation of European and North American strain porcine reproductive and respiratory syndrome virus[J]. J Virol Methods, 2001, 98 (1): 63-75.
[81]Sur JH, Cooper VL, Galeota JA, et al. In vivo detection of porcine reproductive and respiratory syndrome virus RNA by in situ hybridization at different times postinfection[J]. Clin Microbiol, 1996, 34(9): 2280-2286.
[82]Hofman P. DNA Microarrays[J]. Nephron Physiol,2005,99 (3):85-89.
[83]Kane M D,Jatkoe T A,Stumpt C R,et al. Assessment of the sensitivity and specificity of oligonucleotide (50mer) microarrays[J]. Nucleic Acids Res, 2000, 28(22):4552-4557.
[84]Kozal M J,Shah N,et al. Extensive polymorphisms observed in HIV-1 clade B protease gene using high-density oligonucleotide arrays[J].Nature Medicine,1996,2(7):753-759.
[85]Lipshutz RJ, Morris D,Chee M, et al. Using oligonucleotide probe arrays to access genetic diversity[J].Biotechniques. 1995,19 (3):442-447.
[86]吴海，顾少华，胡志前等．检测乙丙型肝炎病毒基因芯片的制备[J]．第二军医大学学报，2001，22(6)：527-529．
[87]Sengupta S, Onodera K, Lai A, et al. Molecular detection and identification of influenza viruses by oligonucleotide microarray hybridization[J]. J Clin Microbiol, 2003, 41 (10): 4542-4550.
[88]Chambers J, Angulo A, et al.DNA microarrays of the complex human cytomegalovirus genome: profiling kinetic class with drug sensitivity of viral gene expression[J]. J V irol. 1999, 73 (7):5757-5766.
[89]陶生策，杨仁全等．SARS冠状病毒基因芯片的制作与初步临床样本验[J]．清华大学学报(自然科学版)，2003，43(5)：715-720．
[90]王艳，马文丽，毛向明，等．痘苗病毒寡核苷酸检测芯片的设计及研制[J]．第二军医大学学 报，2004，24(2)：180-183．
[91]张海燕，马文丽，李凌，等．乙型脑炎病毒寡核苷酸基因芯片研究[J]．实用医学杂志，2005，21(12)：1244-1247．
[92]Wang H, Malek R L, Kwitek A E, et al. Assessing unmodified 70-mer oligonucleotide probe performance on glass-slide microarrays[J]. Genome Biol, 2003, 4 (1): R5.
[93]Call D R, Chandler D, Brockman F. Fabrication of DNA microarrays using unmodified oligonucleotide probes[J]. Bio techniques, 2001, 30(2): 368-379.
[94]Yang S, Ghanny S, Wang W, et al. Using DNA microarray to study human cytomegalovirus gene expression[J]. Journal of Virological Methods, 2006 (131): 202-208.
[95]Cao X, Wang Y, Zhang C, et at. Visual DNA microarrays for simultaneous detection of Ureaplasma urealyticum and Chlamydia trachomatis coupled with multiplex asymmetrical PCR[J]. Biosensors and Bioelectronics, 2006, 22(3): 393-398.
[96]Gyllensten U B, Erlich H A. Generation of single-stranded DNA by the polymerase chain reaction and its application to direct sequencing of the HLA-DQA locus[J]. Proc Natl Acad Sci LISA, 1988, 85 (20): 7652-7656.
[97]Mo X, Ma W, Li L, et at. The effects of different sample labelling methods on signal intensities of a 60-met diagnostic microarray[J]. Journal of Virological Methods, 2006, 134(1-2): 36-40.
[98]Murray AE,et al. DNA/DNAhybridization to microarrays reveals gene-specific differences between closely related microbial genomes[J].Proc Natl Acad Sci USA,2001,98(17):9853-9858.
[99]万超，温国元，潘兹书，等．快速检测伪狂犬病毒荧光定量PCR技术建立及应用[J]．中国病毒学，2006，21(5)：485-489．
[100]李永东，张常印，姜平，等．口蹄疫病毒实时RT-PCR检测方法的建立[J]．中国人兽共患病学报，2006，22(3)：212-215．
[101]M．谢纳．生物芯片分析[M]．Vol影印版 北京：科学出版社，2003．
[102]Zhou X, Zhou J.Oligosaccharide microarrays fabricated on aminooxyacetyl functional glass surface for charaterization of carbohydrate-protein interaction[J].Biosensors and Bioelectronics, 2006,21:1451-1458.