摘要
随着分子生物学技术发展,许多分子生物学方法在动物疾病的诊断中得到了应用。基因芯片技术作为一项高新技术,在面积不大的载体上如玻片上可实现对多种目的基因的同时检测。它是基于PCR技术和核酸杂交的原理,事先对载体表面进行特定处理,将寡核苷酸片段或cDNA片段有序地、高密度地排列于固相载体上,形成DNA微阵列,用高精密度的激光共聚焦荧光检测系统进行杂交信号的检测。其优点为高通量、并行性、微型化、自动化、准确快速、多功能及防止污染从而有效排除外界干扰。目前国内外有许多学者都在从事基因芯片的研究工作,基因芯片技术无疑为动物疾病诊断和微生物检测提供了一项有效快速的检测技术。本试验尝试性地制备了用于检测猪伪狂犬病病毒和三种主要致病菌的基因芯片。
伪狂犬病病毒(PRV)是引起猪繁殖障碍的主要病原体之一,这种疾病给养猪业造成了巨大的经济损失,常规的病因学实验室诊断方法大多耗时长、敏感性低和需要较复杂的操作技术。本研究在分析猪伪狂犬病毒基因组结构特征的基础上,研究了PRV检测基因芯片靶基因和用于检测PRV的探针、芯片的杂交温度和时间等;将gE基因作为目的基因,并根据其基因设计引物与探针,采用不对称PCR技术扩增目的基因,将探针固定在表面经过醛基化处理过的芯片上来检测猪伪狂犬病病毒,构建了PRV病原检测基因芯片,达到了对猪伪狂犬病病毒检测的目的。
临床上常用的病原微生物诊断方法有培养技术、免疫学技术和PCR技术等。这些技术在临床诊断中发挥了巨大的作用,但仍存在一些缺点。如培养技术繁琐而费时,免疫技术要有特异的血清等;快速检测致病菌能够及时有效地预防、治疗和控制致病菌的传染。为了探索一种准确快速的检测方法,本试验尝试将基因芯片技术应用到致病菌检测领域,利用原核生物16S rRNA基因保守性兼变异性的特点,设计筛选了1对通用引物,在其保守区设计通用引物,在其变异区设计探针,实现多病原菌的检测,真正的实现高通量检测,在相同的条件下对沙门氏菌、金黄色葡萄球菌和大肠杆菌进行检测,达到了对细菌进行检测鉴定的目的,制备的基因芯片能够同时检测沙门氏菌、金黄色葡萄球菌和大肠杆菌。
With the development of molecular biology techniques, many molecular biology methods in the diagnosis of animal diseases has been applied. Microarray technology as a high-tech,in the area of small carriers such as slides can be realized simultaneously detect multiple target genes. It is based on PCR, and nucleic acid hybridization principles, prior to the carrier surface of a specific treatment, the oligonucleotide or cDNA fragment in an orderly, high-density land arranged in solid phase carrier to form a DNA microarray, with high precision degree of laser confocal fluorescence detection system for detection of hybridization signals. The advantage of high throughput, parallelism, miniaturization, automation, fast and accurate, multi-function and prevent pollution in order to effectively rule out outside interference. Many scholars at home and abroad are engaged in research on gene chip, gene chip technology has undoubtedly animal disease diagnosis and microbiological test provides a rapid and effective detection. This study tested the preparation for detection of porcine pseudorabies virus and several pathogenic gene chips.
Pseudorabies virus (PRV) is a disorder caused by porcine reproductive one of the major pathogenic bacteria,the disease to the swine industry has caused tremendous economic losses, the conventional methods of laboratory diagnosis of the etiology most time-consuming, low sensitivity and require high the operating techniques. In this study, the analysis of porcine pseudorabies virus genome structure of the basis of the PRV and the target gene microarray probes for detection of PRV, chip hybridization temperature and time; The gE gene as a target gene, and primers were designed according to the gene probe, amplified by asymmetric PCR gene, the probes immobilized on aldehyde-treated after chip up detection of pseudorabies virus, was constructed PRV pathogen microarray and reached the detection of porcine pseudorabies virus purposes.
Clinical diagnosis of pathogenic microorganisms commonly used methods of cultivation techniques, immunoassay, PCR technology, these techniques in clinical diagnosis played a huge role, but let some shortcomings. Cumbersome and time-consuming culture techniques; immune serum specific technical need, rapid detection and identification of intestinal bacteria to timely and effective prevention, treatment and control of intestinal infectious diseases. In order to explore an accurate, rapid detection method, this study attempts to gene chip technology to the field of pathogen detection, use of prokaryotic 16S rRNA gene conservation and variability of the characteristics of the design selection of the one pair of universal primers, in its conservative area design universal primers, probes in the design of its variable region, multi pathogen detection, the true high throughput detection, under the same conditions as Salmonella, Staphylococcus aureus and Escherichia coli, to detection and identification of bacteria The purpose of preparation of the gene chip can detect Salmonella, Staphylococcus aureus and Escherichia coli.
引文
[1]Schena M, Davis R W, Brown P O, et al. Quantiative monitoring of gene expression patterns with a complementary DNA microarray[J].Science, 1995, 270 (5235):467-470.
[2]Spellman P T, Zhang M Q, Iyer V R, et al. Comprehensive identification of cell cycle-regulated genes of the yeast Saccharomyces cerevisiaeby microarray hybridization[J].Mol Biol Cell, 1998, 7 (9):3273-3297.
[3]Raymond J, Steinmetz L, Conway A, et al. A genome-wide transcriptional analysis of the mitotic [J].Molecular Cell, 1998, 1 (2):65-73.
[4]Chu S, Eisen M, Mulholland J,et al. The transcriptional progrom of sporulation in budding yeast [J].Science 1998, 23 (282):699-705.
[5]DeRisi J L, Lyer V R, Brown P O. Exploring the metabolic and genetic control of gene expression on a genomic scale[J].Science, 1997, 24 (278):680-686.
[6]Tao H, Bausch C, Richmond C, et al. Functional genomics: expression analysis of Escherichia colingrowing on minimal and rich media [J].J Bacteriol, 1999, 20(181):6420-6440.
[7]Richmond C S, Mau R, Jin H, et al. Genome-wide expression profiling in Escherichia coliK-12[J].Nucleic Acids Res, 1999, 27 (19):3821-3835.
[8]Khan J, Simon R, Bittner M, et al. Gene expression profiling of alveolar rhabdomyosarcoma with cDNA microarrays.Cancer Res, 1998, 15 (58):5009-5013.
[9]Perou C M, Jeffrey S S, Rees C A, et al. Distinctive gene expression patterns in human mammary epithelial cells and breast cancers[J].Proc Natl Acad Sci U S A, 1999, 16 (96):9212-9217.
[10]Alizadeh A A, Davis R E, Ma C,et al. Distinct types of diffuse large B-cell lymphoma identified by gene expression profiling[J].Nature, 2000, 12 (403):503-511.
[11]Golub T R, Tamayo P, Huard C,et al. Molecular classification of cancer:class discovery and class prediction by gene expression monitoring[J].Science, 1999, 286 (5439):531-537.
[12]Alon U, Notterman D A, Gish K,et al. Broad patterns of gene expression revealed by clustering analysis of tumor and normal colon tissues probed by olionucleotide arrays[J].Proc Natl Acad Sci U S A, 1999, 96(9):6745-6750.
[13]Wang K, Jeffery E, Gayle M,et al. Monitoring gene expression profile changes in ovarian carcinomas using cDNA microarray[J].Gene, 1999, 299 (18):101-108.
[14]Chen C C, Shieh B, Jin Y T, et al. Microarray profiling of gene expression patterns in bladder tumor cells treated with genistein[J].J Biomed Sci, 2001,8 (2):214-222.
[15]Kobayashi T, Yamaguchi M, Kim S, et al. Microarray Reveals Differences in Both Tumors and Vascular Specific Gene Expression in de Novo CD5(+)andCD5(-)Diffuse Large B-CellLymphomas[J].Cancer Research, 2003, 63 (1):60-66.
[16]Marton M J, Bennett H A, Iyer V R, et al. Drug target validation and identification of secondary drug target effects using DNA microarrays[J].Nature Medicine,1998, 4(11):1293-1301.
[17]Scherf U, Waltham M, Smith L H, et al. A gene expression database for the molecular pharmacology of cance [J].Nat Genet, 2006, 16(24):236-244.
[18]Clake P A, Poele R T, Wooster R P. Workman.Gene expression microarray analysis in cancer biology,pharmacology,and drug development:progress and potential[J].Biochem Pharmacol, 2007, 64(15):1311-1336.
[19]Marcotte E R, Srivastava L K, Quirion R. DNA microarrays in neuropsy chophar macology[J].Trends Pharmacol Sci, 2001, 22(8):426-436.
[20]Wallqvist A, Rabow A A, Shoemaker R H, et al. Establishing connections between microarray expression data and chemotherapeutic cancer pharmacology[J].Mol Cancer Ther, 2005, 1(3):311-320.
[21]Debouck C, Goodfellow P N. DNA microarrays in drug discovery and development [J]. Nat Gent, 1999, 21(1):48-50.
[22]Ang S, Lee C Z, Peck K, et al. Acid-induced gene expression in Helicobacter pylori: study in genomic scale by microarray [J].Infect Immun, 2001, 69(3):1679-1686.
[23]Fitzgerald J R, Musser J M. Evolutionary genomics of pathogenic bacteria [J].Trends Microbiol, 2001, 11(9):547-553.
[24]Janssen P J, Audit B, Ouzounis C A. Strain-specific genes of Hellicobacter pylori: distribution, function and dynamics [J].Nuceleic Acids Res, 2005, 29(21):4395-4404.
[25]Khaldi S F, Martin S A, Evans J D, et al. DNA microarray technology used for studying foodboren pathogens and microbial habitats:minireview[J].J AOAC int, 2005, 85(4):906-910.
[26]Arendt C W, Littman D R. HIV: master of the host cell [J].Genome Biol, 2001, 11(2): 1031-1034.
[27]Campbell J W, Morgan-Kiss R M. A new Escherichia coli metabolic competency: growth on fatty acids by o novel anaerobic beta-oxidation pathway [J].Mol Microbiol, 2003, 186(1):793-805.
[28]Striebel H M, Egerer R, Papp Z F. Virus diagnostics on microarray[J].Current Pharmaceutical Biotechnology, 2003, 4(6):401-415.
[29]Strachan T, Abitbol M, Davidson D, et al. A new dimension for the human genic project: towards comprehensive expression maps [J]. Nature Cenetic, 1999, 16(10):126-131.
[30]Lipshuze R J, Fodor S P.High density synthetic oligonucleotide arrays [J]. Nature Cenetics,1999, 1(21):20-24.
[31]陈亚利,陆祖宏.微芯片—生命科学领域的新工具[J].生物化学与生物物理进展,1998, 25(6):517-522.
[32]Cheung V G. Making and reading microarrays [J]. Nature Cenetics, 1999, 21(1):15-19.
[33]Lemieux B, Aharoni A, shena M. Overview of DNA chip technology [J].Molecular Breeding, 1998, 4(4):277-289.
[34]Stephen P A. Light-Directed spatially dressable parallel chemical synthesis [J].Science, 1991, 251(4995):767-773.
[35]Hone P A, Kort F, van Ommen G J,et al. Fluorescent labeling of cRNA for microarray application[J].Nucleic Acids Res, 2007, 31(5):257-272.
[36]石嵘,马文丽,宋艳斌,等.两种限制性标记方法提高基因芯片杂交结果的噪音比[J].第一军医大学学报, 2003, 23(2):124-126.
[37]徐秋林,马文丽,李麦,等.一种新的基因芯片检测荧光标记技术:通用引物U2联合标记法[J].第一军医大学学报, 2005,25(3):289-292.
[38]文思远,陈苏红,张敏丽,等. 3种基于PCR的荧光标记方法的比较[J].军事科学院院刊, 2004, 28(4):364-368.
[39]Song Y J, Stingski M F. Effect of the human cytomegalovirus IE86 protein on expression of E2F-responsive genes:a DNA microarray analysis[J].Proc Natl Acad Sci U S A, 2006, 99(5):2836-2841.
[40]Anthony R M ,French G L. Rapid diognosis of bacteria by universal amplification of 23S ribosomal DNA followed by hybridization to an oligonucleotide array[J].Clin Microbiol, 2000, 38(20):781-788.
[41]Aizaki H, Harada T, Otsuka M,et al. Expression profiling of liver cell lines expressing entire or parts of hepatitis C virus open reading frame[J].Hepatology, 2002, 36(6):1431-1438.
[42]孙慧,董陆佳,田方,等.应用寡核苷酸芯片检测白血病与其同胞供者的白血病基因表达差异[J].中国实验血液学杂志, 2004, 12(4):450-454.
[43]靳连群,王升启.基因芯片技术检查环境中常见致病菌的初步研究[J].中华微生物和免疫学杂志, 2003,(23)1:74-78.
[44]刘爱英,孙建方,尹跃平.基因芯片技术检测生殖器溃疡性病病原体[J].中华皮肤科杂志,2004,37(5):265-267.
[45]杨毅.基因芯片在小儿败血症病原诊断中的初步应用研究[D].Master,上海:复旦大学,2003.
[46]侯亭.菌血症常见细菌的基因芯片检验研究[D].Master,天津:南开大学,2003.
[47]马晓冬,马文丽,孙朝辉,等.制备炭疽芽孢杆菌检测基因芯片的初步研究[J].微生物学报, 2004,44(3):299-303.
[48]Chizhikov V A. Rasooly.Microarray analysis of microbial virulence factor[J].Appl Environ Microbiol, 2007, 67(7):3258-3263.
[49]Linton D, Lawson A J, Owen R J,et al. Linton PCR detection identification to species level and fingerprinting of Carmpylobacter jejuni and campylobacter coli direct from diaaaheic samples[J].Clin microbial, 1997, 35(10):2568-2572.
[50]Bavykin S G, Akowski J P, Zakhariev V M,et al. Portable system for microbial sample preparation and oligonucleotide microarray analysis[J].Appl Environ Microbiol, 2006, 67(2):922-928.
[51]Bordoni R, Consolandi C, Castiglioni B,et al. Investigation of multiple anchors approach in oligonnucleotide microarray preparation using linear and stem-loop structured probes[J].Nucleic Acids Res, 2002, 30(8):E34.
[52]Busti E, Bordoni R, Castiglionni B, et al. Bacterial discrimmation by means of a universal array approach mediated by LDR (ligase detection reaction) [J].BMC Microbiol, 2007, 2(1):27.
[53]Peplies J, Glockner F O, Amann R. Optimization Strategies for DNA Microarray-Based Detection of Bacteria with 16S Rrna-targeting Oligonucleotide Probes[J].Appl Environ Microbiol, 2003, 69(3):1397-1407.
[54]Masao H H. Detection and identification of Mycobacterium species isolates by DNA microarray [J].J Clin Microbiol, 2006, 41(6):2605-2615.
[55]Kato-Maeda M, Rhee J T, Gingeras T R, et al. Comparing genomes within the species Mycobacterium tuberculosis[J].Genome Res, 2001, 11(4):547-554.
[56]Heller R A, Schena M, Chai,et al. Discovery and analysis of inflammatory disease-related genes using cDNA microarrays[J].Proc Natl Acad Sci U S A, 1997, 94(6):2150-2155.
[57]Head S R, Parikh K, Rogers Y H. Solid-phase sequence scanning for drug resistance detection in tuberculosis [J].Mol Cell Probes, 1999, 13(2):81-87.
[58]郑文岭,毛向明,张宝,等.应用PCR快速制备乙型、丁型肝炎病毒诊断基因芯片探针[J].第一军医大学学报, 2003, 23(7):677-679.
[59]孙朝辉,马文丽,郑文玲.基因芯片诊断病毒性乙型、丙型肝炎[J].解放军医学杂志, 2003, 28(4):375-376.
[60]张香玲,周亚凤,王建春,等.采用基因芯片检出一例丙型肝炎病毒3b亚型的报告[J].病毒学报, 2003, 19(3):274-277.
[61]宋世会,齐俊英,杨道锋,等.乙型肝炎病毒基因型与YMDD变异的关系[J].中西医结合肝病杂志, 2005, 15(4):199-201.
[62]赵伟,刘全俊,等.肝炎基因诊断芯片对丙型肝炎病毒基因分型检测的初步研究[J].中华医学杂志, 2002, 82(18):1249-1253.
[63]赵伟,刘伟,等.肝炎基因诊断芯片检测肝炎患者血清及肝组织中HBVDNA、HCVRNA的临床研究[J].中华临床医药杂志(北京),2003, 4(1):50-54,49.
[64]李刚,舒欣,等.基因芯片技术检测HBV HCV及HBV YMDD变异株[J].世界华人消化杂志, 2003, 11(2):178-181.
[65]肖琳,张跃新,阿曼古丽,等. 3种方法检测HBVYMDD变异的比较[J].中华临床医药杂志(北京), 2004, 5(12):11-14.
[66]Chiou L Y, Huang T S. Extrenely high prevalence of nasopharyngeal carriage of penicillin resistant streptococcus pneumonia armong children in kayo hsiung [J].J Clin microbial, 1998, 36(7):1933-1937.
[67]赵伟.基因芯片及技术诊断乙型病毒性肝炎的初步研究[J].江苏医药,2001,27(6):425-426.
[68]赵伟.病毒性肝炎基因诊断芯片测定HBVDNA、HCVRNA的价值[J].世界感染杂志,2001,1(4):298-302.
[69]赵伟.慢性乙型肝炎患者血清及肝组织HBVDNA表达及基因芯片检测研究[J].山东医药,2002,42(1):10-12.
[70]赵伟. 80例乙型、丙型病毒性肝炎血清,组织基因芯片检测分析[J].临床肝胆病杂志,2002,18(1):9-11.
[71]赵伟,万建民,刘伟.基因芯片法对丙型肝炎病毒RNA的基因型分析[J].第二军医大学学报,2003,24(5):491-495.
[72]曹三杰.鸡新城疫、鸡传染性支气管炎基因芯片的构建及其检测技术研究[D].2004,四川农业大学:雅安.
[73]秦智峰,钟安清,杨宝华,等.用基因芯片鉴别诊断四种水泡性疾病[J].中国病毒学, 2003,18(2):174-177.
[74]肖驰.猪繁殖与呼吸综合征、猪瘟和猪圆环病毒病诊断DNA微阵列的构建及其检测技术研究[D].2005,四川农业大学:雅安.
[75]杨素,花群义,徐自忠,等.口蹄疫等5种动物病毒基因芯片检测技术的研究[J].微生物学报, 2004, 44(4):479-482.
[76]耿捷,罗卫,林苗,等.基因芯片技术检测新城疫病毒[J].黑龙江畜牧兽医医, 2004, (12):72-73.
[77]王秀荣.禽流感病毒基因芯片检测技术探索及相关基础工作研究[D].2002,中国农业大学:北京.
[77]Chan V, Mekehzie S E. The biophysics of DNA hybridization with immobilizedoligonucleotide probes [J].Biophysical J, 1999, 69(6):2243-2255.
[78]Southern E, Shchepinov M. Molecular interactions on microarrays [J].Nat Genet, 1999, 21(5):5-9.
[79]殷震,刘景华.动物病毒学[M].北京:科学出版社,1997.998-1009.
[80]殷震,刘景华.动物病毒学[M].第二版.北京:科学出版社.2002.
[81]Kritas S K, Pensaert M B, Mettenleiter T C. Role of envelope glycoprotein gI, gp63 and gIlI in the invasion and spread of Aujeszly's disease virus in the olfactory nervous pathway of the pig[J].Gen.Virol, 1994, 75(10):2319-2327.
[82]Pursel V, Linto P J, Sandgren E,et al. Expression of mouse IgA by transgenic mice pigs and sheep. Eur[J]. Journal of Virology, 1991, 21(3):1001-1006.
[83]吴清民.兽医传染病学[M].北京:中国农业大学出版所,2002.
[84]Peeters B , De Wind N , Minetta H,et al. Prv gp50 and gII are essential for virus penetration[J]. Journal of Virology, 1991, 65(10):5336-5348.
[85]Geraghty R J , Krummenacher C , Cohen G H et al. Entry of alphaherpesvirus mediated by poliovirus receptor-related Protein 1 and poliovirus receptor [J]. Science, 1998, 280(5369):1618-1620.
[86]Favoreel H W , Nauwynck H J, Enquist L W,et al. A Tyrosine-Based Motif in the Cytoplasmic tail of Pseudorabies Virus Glycoprotein B is important for both Antibody-induced intermalization of Viral Glycoproteins and Efficient cell-to-cell Spread[J]Journal of Virology, 76(13):6845-6851.
[87]Rauh I, Mettenleitier T C.Pseudorabies virus glycoproteins gII and gp50 are essential for virus penetration [J] Virol, 1991, 65(10):5336-5348.
[88]Klupp B C, Fuchs W, Weiland E,et al. Pseudorabies virus glycoprotein L is necessary for virus infectivity but dispensable for viron localization of glycoprotein H [J].virol, 1997, 71(10):7687-7695.
[89]Klupp B C, Baumeisfer J, Dietz P. Pseudorabies Virus gK is a Virion Structural Component Involved in Virus Release but is Not Required for Entry [J]. Virol, 1998, 72(3):1949-1958.
[90]Harriey C A, Drummer H E, Studdert M J. The nucleotidese 2 quence of the glycoprotein gG homologue of equeneherpoes virus3 (EHV3) indicates EHV3 isadistincte quidalpherpes virus [J].Arch Virol, 1999, 144(6):2023-2033.
[91]Tran L C, Kissner J M, Westerman L E,et al. A herpes sim-plex virus I recombinant lacking the glycoprotein gG coding sequences is defective in entry throμgh apical surfaces of polarized epithelial cells in cμLture and in vivo[J].PNAS, 2000, 97(12):1818-1822
[92]Nakamichi K, Ohara K, Kuroki D, et al. Bovine herpesvirus 1 glycoprotein G is requiredfor viral growth by cell-to-cell infection[J].Virus Res, 2000, 68(2): 175-181.
[93]Hartiep C A, Drummer H E, Studdert M J. The nucleotide Sequence of the glycoprotein gG homologue of equine herpesvirus 3 (EHV3) indicates EHV3 is a distinct equid alphaherpesvirus [J].Arch Virol, 1999, 144(10):2023-2033.
[94]Klupp B G, Baumeister J, Mettenleiter T C, et al. PRV glycoprotein gK is a virion structural compinent involved in virus release but is not required for entry.[J].Virol, 1999, 72(3):1949-1958.
[95]Dijkstra J, Mettenleiter T C, K1upp B G. Deletion of glycoprotein gM 76 of pseudorabies virus rests in attenuation for the natural host [J]. Gen Virol, 1997, 78(5):2147-2151.
[96]赵耘,李健强.猪伪狂犬病血清学诊断概况[J].国外兽医学:畜禽疾病,1992,13(4):7-10.
[97]Stewart W C.A comparison of three serologic techniques for the detection of pseudorabies antibodies [J].Proceeding of the American Association of Veerinary Laboratory Diagnosticians, 1998, (21):43-51.
[98]Allan G M. Rapid diagnosis of Aujeszky,s disease in pigs by immunofluorescence[J].Research in Veterinary Science, 1994, 36(2):235-239.
[99]Ducatelle R, Coussement W, Hoorens J. Immunoperoxidase study of Aujeszky,s disease in pigs[J].Research in Veterinary Science, 1992, 32(3):294-302.
[100]Afshar A. Immunoperoxidase plaque staining for the detection of pseudorabies virus[J].Can J Vet Res, 1999, 50(1):118-119.
[101]Neill J D. Comparison of a direct radioimmunoassay with virus isolation in cell culture for detection of pseudorabies virus in tissues of infected swine [J].Am J Vet Res, 1998, 43(4):708-710.
[102]娄高明,陈志荣,郭万柱.双抗体夹心ELISA检测伪狂犬病病毒的研究[J].中国畜禽传染病,1998, 20(4):236-240.
[103]Kimman T G. An indirect double-antibody sandwich enzyme-linked immunosorbent assay (ELISA) using baculovirus-expressed antigen for the detection of antibodies to glycoprotein E of pseudorabies virus and comparison of the method with blocking ELISA[J].Clin Diagn Lab Immunol, 1996, 3(2):167-174.
[104]苗得园,杨兵,样富康.单抗夹心LAB-ELISA检测猪伪狂犬病病毒的研究[J].华北农学报,2001,16(1):127-131.
[105]Jestin A F,Oulon T, Pertuiset B, et al. Rapid detection of pseudorabies virus genomic sequences inbiological smaples from in fected pig susing polymerise chain reaction DNA amLification [J]. Microbiol, 1996, 23(5): 317-328.
[106]Maes R K, Beisel C E, Spatz S J, et al. Polymerase chain reaction amplification of pseudorabies virus DNA from acutely and latently infected cells[J]. Vet Microbiol, 1996, 24(4):281-295.
[107]Wheeler J G, Osorio F A. Investigation of sites of pseudorables virus latency using polymerise chainreaction [J].Am J Vet Res, 1997, 52(11): 1799-1803.
[108]Leman A D, Straw B, Glock R D,et al.刘文军,高福译.猪病学[M].第6版.北京:北京农业大学出版社,1990, 255-269.
[109]Bitsch V. Application of a highly sensitive virus-serum neutralization test in screening examinations of large numbers of swine sera for antibody to Aujeszky,s disease virus[J].Proceeding of the International Pig Veterinary Society Congress, 1993, 101(4):191-206.
[110]万六荣,陈焕春,何启盖.应用微量中和试验进行伪狂犬病血清学调查[J].中国畜禽传染病,1998,20(3):151-153.
[111]柴田勋.应用乳胶凝集试验检测伪狂犬病病毒抗体[J].国外兽医学-畜禽传染病,1991,11(2):40-41.
[112]国际兽医局编著.诊断试剂盒疫苗标准手册[M].青岛:青岛新闻出版局,1996.
[113]Belak S. Rapid diagnosis of Aujeszky,s disease (pseudorabies)by various DNA hybridization methods [J].Paper presented at:European Society for Veterinary Virology,First Congress, 1999, 108(4):279-286.
[114]McFarlane R G, Thawley D G. DNA hybridization procedure to detect pseudorabies virus DNA in swine tissue[J].American Journal of Veterinary Research, 2003, 46(5):1133-1136.
[115]郭万柱,冯炳芳.应用32P标记DNA探针检测伪狂犬病病毒的研究[J].四川农业大学学报,1991,9(1):52-56.
[116]Gutekunst D E. Latent pseudorabies virus infection in swine detected by RNA-DNA hydridization [J].American Journal of Veterinary Research, 1979, 40(11):1568-1572.
[117]Mase R S, Spatz S J, Thacker B. Nonradioactive probes to detect acute and latent pseudorabies virus (Aujeszky,s disease)infections[J].Proceedings International Pig Veterinary 10th Congress, 1998, 169(12):281-295.
[118]石建平,宜华.伪狂犬病病毒PCR检测方法的建立及初步应用[J].中国畜禽传染病,1996,(5):16-20.
[119]吴斌,陈焕春,万六荣,等.聚合酶链反应在猪伪狂犬病临床诊断中的应用[J].中国兽医科技,1988,28(1):3-4.
[120]Maes R K, Sussman M D, Vilnis A, et al.Recent development in latency and recombination of Aujeszky,s disease(pseudorabies)virus[J].Vet Microbiol, 1997, 55(1-4):13-27.
[121]Belak S, Ballagi-Pordany A, Flensburg J ,et al. Detection of pseudorabies virus DNA sequences by the polymerase chain reaction[J].Arch Virol, 1999, 108(3-4):279-286.
[122]Wheeler J Q, Osorio F A. Investigation of sites of pseudorabies virus latency using polymerase chain reaction[J].Am J Vet Res, 1991, 52(11):1799-1803.
[123]Scherba G, Gin L. Differential polymerase chain reaction for detection of wild-type and a vaccine strain of Aujeszkys disease (pseudorabies) virus [J].J Virol Methods, 1992, 38(1):131-143.
[124]Katz J B, Pedersen J C. Molecular analysis of pseudorabies viral vaccines and rapid differentiation from wild-type isolates using DNA-amplified glycoprotein I and thymidine kinase gene segment polymorphisms[J].Biologicals, 1992, 20(3):187-195.
[125]冉智光,童光志,孔令达,等.利用复合多聚酶链反应方法快速鉴别伪狂犬弱毒苗和野毒[J].中国兽医杂志,1999,25(5):3-4.
[126]Hasebe H, Wheeler J G, Osorio F A. Gene specific assay to differentiate strains of pseudorabies virus [J].Veterinary Microbiology, 2003, 34(3):221-231.
[127]Jacobs L, Mulder W. Detection of wild-type Aujeszky,s disease virus by polymerase chain reaction in sheep vaccinated with a modified live vaccine atrain[J].Res Vet Sci, 1997, 62(3):271-274.
[128]Kenji H, Ken-Ichiro I, Hiroshi N,et al. Selective Amplification of tyv(rfbE),prt(rfbs),viaB,flic Genes by Multiplex PCR for identification of Salmonella enterica Serovars Typhi and Paratyphi A[J].Journal of Clinical Microbiology, 2002, 40(2):633-636.
[129]Campbell G R, Prosser J, Glover K, et al. Detection of Escherichia coli O157:H7 in soil and water using multiplex PCR[J].Journal of Applied Microbiology, 2001, (91):1004-1010.
[130]Wim J B, Michiel R, Henny M E, et al. Detection of Pathogenic Yersinia enterocolitica by a Rapid and Sensitive Duplex PCR Assay[J].Journal of Clinical Microbiology, 2007, 39(12):4483-4486.
[131]Hege K N, Knut R , Kristine N, et al. Application of 5’-Nuclease PCR for Quantitative Detection of listeria monocytogenes in Pure Cultures[J].Unpasteurized Whole Milk, 2000, 66(10):4266-4271.
[132]Dawn M N, Carl A. Detection of Viable listeria monocytogenes with a 5, Nuclease PCR Assay [J].Applied and Environment Microbiology, 1999, 65(5):2122-2127.
[133]Elfaki M G, Taher U Z, Abdullah A, et al. Detection of Brucella DNA in sera from patients with brucellosis by polymerase by polymerase chain reaction[J].Diagnostic Microbiology and infectious Disease, 2006, 53(7):1-7.
[134]AOAC.Official Methods of Analysis[M]. Arrlington VA, 1990, 194-200.
[135]Rudolf A, Gloeckner F, Neef A. Modern Methods in Subsurface Microbiology In situ identification of Microorganisms with Nucleic Acid Probes[J].FEMS Microbiology Reviews, 1997, 20(3-4):191-200.
[136]Wang Y R, Bedzyk T, Croker,K. Applications of DNA microarrays in microbial systems[J].Microbiol.Methods, 2007, 47(3):257-272.
[137]Schena M. Microarray Biochip Technology Eaton Publishing [M].Natick, MA.2006
[138]Giovanna F, Stefania T,Christina W N, et al. Paolo Aureli.Characterization of listeria monocytogenes Strains Involved in Invasive and Noninvasive Listeriosis Outbreaks by PCR-Based Fringerprinting Techniques[J].Applied and Environmental Microbiology, 2001, 67(4):1793-1799.
[139]Gonza,lez S F, Krug M E, Niselsen Y, et al. Simultaneous detection of marine fish pathogens by using multiplex PCR and a DNA microarray[J].J Clin Microbiol, 2004, 42(4):1414-1419.
[140]Fratamico P M, Bhunia L, Smith J. Food pathogens: Microbiology and Molecular Biology[M].Norwich Caister Academic Press, 2005, 395-410.
[141]Volker G, Vilma A. New approaches to typing and identification of bacteria using the 16S-23S rDNA spacer region [J].Microbiology, 1996, 142(1):3-16.
[142]Gray M W, David S, Robert J C. On the evolutionary of organisims and organdies:a global phylogeny based on a highly conserved structural core in small subunit ribosomal RNA[J].NucleicAcids Res, 1998, 12(12):5837.
[143]Teng I J, Hsueh P R, Huang Y H, et al. Identification of Bacteroides thetaiotaomicron on the basis of an unexpected specific amplicon of universal 16S ribosomal DNA PCR[J].Jclin Mcrobio, 2004, 42(4):1727-1730.
[144]Tajama K. Rumen bacterial divefial diversity as determined by sequence analysis of 16S rDNA librasies[J].FEMS Microbiol Ecol, 1999, 29(3):159-169.
[145]Shang S, Chen Z, Yu X. Detection of bacterial DNA and reverse hyridizatiotln in the 16S rRNA gene with particular reference to neonatal septicemia [J].Acta Paediatr, 2007, 90(2):179-183.
[146]Greisen K, Loeffeholz M, Purohit A, et al. PCR primers and probes for the 16S rRNA gene of most species of pathogenic bacteria, including bacteria found in cerebrospinal fluid [J].J Clin Microbio, 1994, 32(2):335.
[147]闫志勇,王斌,苏维奇,等.多重半套式PCR检测脑脊液中细菌16S rRNA基因.中华生物与免疫学杂志,2001, 21(4):316.
[148]Radstrom P, Backman A, Kragsbjerg, et al. Detection of bacterial DNA in cerebrospinal fluid by assay for simultaneous detection of Neiseria menginitidis heamophilus influenzae,and streptococci using a seminested PCR strategy[J]. Clin Microbiol, 1994, 32(11):2738.
[149]Jiang J L, Chreng L P, Shi Y L, et al. Use of PCR with universal primers and restriction ndonuclease digestions for detection and identification of common bacterial pathogens incerebrospinal fluid[J].J Clin Microbiol, 2000, 38(6):2076.
[150]温杰,刘毅. 16S rRNA基因RFLP用于不同科细菌区分[J].大连医科大学学报,2005,27(3):237-239.
[151]Turenne C Y, Daryl E W, Hoban J ,et al. Identification of Bacteroides thetaiotaomicron on the basis of an unexpected specific amplicon [J]. Clin Microbiol, 2000, 38(2):513-520.
[152]Gillman I M, Gunton J, Turenne C Y, et al. Gene specific assay to differentiate strains of pseudorabies virus [J]. Clin Microbiol, 2001, 39(9):3085-3091.
[153]沈永才,袁佩娜. 16S rRNA荧光定量PCR法检测双歧杆菌[J].中国微生态学杂志,2001,13(2):66-72.
[154]颜世敢,朱丽萍. 16S rRNA PCR鉴定嗜酸乳杆菌鸡源分离株[J].中国微生态杂志,2005,17(2):115-116.
[155]温燕,翁小满,陈小华. RT-PCR检测16S rRNA基因片段对马风菌活性的评价[J].中国麻风皮肤病杂志,2007, 23(9):745-748.
[156]温杰,刘毅. 16S rRNA基因RFLP用于不同科细菌区分[J].大连医科大学学报,2005,27(4):276-279.