荧光RT-PCR检测新城疫病毒及其毒力鉴别
|
摘要
新城疫(ND)是由新城疫病毒(NDV)引起的一种禽类急性、高度接触性传染病，被列为A类动物严重传染病，给世界各地养禽业带来巨大威胁。目前检测NDV的方法有血凝和血凝抑制试验、病毒分离法、免疫荧光试验、酶联免疫吸附试验等。这些方法有的存在费时、有的存在敏感性不高或特异性不强等缺点。
普通RT-PCR方法可以快速和特异性地检测很少拷贝的NDV的核酸，因此可以用于NDV的检测。但其难以控制核酸污染造成假阳性，而且需要使用剧毒的化学物。
荧光RT-PCR是近几年才发展起来的一项新技术。它既具有普通RT-PCR的高度灵敏性，又具有实时、快速、能够避免核酸污染和化学物污染等优点。SYBR Green模式的荧光RT-PCR是利用荧光染料SYBR Green Ⅰ与DNA双链结合后释放荧光的特点而建立的一种应用较广的荧光PCR技术。SYBR Green模式的荧光RT-PCR的优点是成本小，不需设计探针，只要有相关的引物就可以进行实时检测，并且适合于高度变异的基因检测。本研究建立了NDV和其毒力的SYBR Green模式的荧光RT-PCR检测技术。
本研究分析大量的NDV F蛋白的核苷酸序列，并在其裂解位点上下游保守区内附近，精心设计并筛选出一对通用引物，用于所有NDV毒株SYBR Green模式的荧光RT-PCR检测；优化检测条件，并将RT和PCR并为一步；以标准NDV毒株RNA为模板，用通用引物进行荧光RT-PCR扩增，扩增产物的Tm值一般在(88±1)℃范围内；经对10株未知毒株进行检测，并将其扩增产物进行序列测定，序列测定结果和荧光RT-PCR检测结果完全一致；实验还证明，此荧光RT-PCR比英文文献报道的普通RT-PCR方法更为敏感。
NDV毒力决定于其F蛋白的裂解位点的序列。依据这个原理，本研究还精心设计并筛选出另外两对引物，分别对应于NDV强毒毒株和弱毒毒株的F蛋白的裂解位点的序列，并依据此两对引物的SYBR Green模式的荧光RT-PCR扩增结果来判断NDV的毒力；以标准NDV强毒和弱毒毒株的RNA为模板，进行扩增，依据实验结果设立判断标准；用此判断标准对8株NDV未知毒株进行检测，结果和MDT检测结果完全一致，说明此方法可用于NDV强弱毒的鉴别。
另外，本方法可以以上述通用引物扩增的PCR产物为模板，进行毒力鉴别实验，从而继承了上述通用检测所具有的敏感性很高的特性。
本研究所建立的NDV及其毒力鉴定的荧光RT-PCR技术具有重要的兽医临床意义，在禽产品进出口上也有很大的应用价值。
Newcastle disease (ND) is a highly fatal and highly contagious avian disease caused by Newcastle disease virus (NDV). It is also one of the List A animal infectious diseases, and causes severe avian epidemics worldwide every year. There are several kinds of classical NDV detection methods, such as virus isolation, HA and HI test, immunofluencent test, ELISA, etc. These techniques some are time-consuming, some are insensitive or somehow unspecific.
Common RT-PCR can detect a few copies of nucleic acids of NDV, and therefore it is used to detect NDV for clinical or quarantine purpose. But its uses are quitely limited because it is difficult to avoid the false positive signals caused by the pollution of PCR products. Common RT-PCR also requires the uses of EB, a strong carcinogen.
Real-time RT-PCR assay is one of the new detection techniques. It has high sensitivity as common RT-PCR, and avoids both the pollution of PCR products and the uses of EB. It is also faster than common RT-PCR. SYBR Green I releases fluorescence when combining to double-stranded DNA, and based on this principle, SYBR Green I real-time RT-PCR is applied widely with lower cost and without difficult probe design. It is suitable for the detection of highly variable genes.
Here in this paper SYBR Green I real-time RT-PCR techniques are reported to be established to detect and pathotype NDV.
Firstly, many sequences of NDV F gene were analysed and a general pair of primers were designed to detect all NDV strains using SYBR Green I real-time RT-PCR technique. Then, the detection conditions were opimised, and RT and PCR were integrated into one step. The Tm values of the specific amplification products are always (88+1)C. The results of such assay and the sequencing assay for the detection of 10 unknown strains were the same, and the sensitivity of such assay were higher than a reported common RT-PCR assay.
The virulence of NDV strains are determined by the lysis site sequences of the viral F protein. Based on this principle, two pairs of primers were desgined to correspond to the sequences of the F protein lysis site of virulent NDV strains and avirulent strains, respectively. The viral RNA was detected using the two primers respectively, and the virulence of NDV strains were judged according to the amplification effects of the two
pairs, of primers. The results of such assay and MDT assay for the pathotyping of 8 unknown strains were the same, which suggested that this assay can be used to pathotype NDV strains and better than the common RT-PCR technique reported previously. The amplification products of the pair of aforementioned general primers could be used as the template of such pathotyping RT-PCR assay, and the high sensitivity of the aforementioned assay of general NDV detection was inherited thereby.
The real time RT-PCR techniques report here are of important significance in veterinary clinicals and the quarantine of avian product export and import.
普通RT-PCR方法可以快速和特异性地检测很少拷贝的NDV的核酸，因此可以用于NDV的检测。但其难以控制核酸污染造成假阳性，而且需要使用剧毒的化学物。
荧光RT-PCR是近几年才发展起来的一项新技术。它既具有普通RT-PCR的高度灵敏性，又具有实时、快速、能够避免核酸污染和化学物污染等优点。SYBR Green模式的荧光RT-PCR是利用荧光染料SYBR Green Ⅰ与DNA双链结合后释放荧光的特点而建立的一种应用较广的荧光PCR技术。SYBR Green模式的荧光RT-PCR的优点是成本小，不需设计探针，只要有相关的引物就可以进行实时检测，并且适合于高度变异的基因检测。本研究建立了NDV和其毒力的SYBR Green模式的荧光RT-PCR检测技术。
本研究分析大量的NDV F蛋白的核苷酸序列，并在其裂解位点上下游保守区内附近，精心设计并筛选出一对通用引物，用于所有NDV毒株SYBR Green模式的荧光RT-PCR检测；优化检测条件，并将RT和PCR并为一步；以标准NDV毒株RNA为模板，用通用引物进行荧光RT-PCR扩增，扩增产物的Tm值一般在(88±1)℃范围内；经对10株未知毒株进行检测，并将其扩增产物进行序列测定，序列测定结果和荧光RT-PCR检测结果完全一致；实验还证明，此荧光RT-PCR比英文文献报道的普通RT-PCR方法更为敏感。
NDV毒力决定于其F蛋白的裂解位点的序列。依据这个原理，本研究还精心设计并筛选出另外两对引物，分别对应于NDV强毒毒株和弱毒毒株的F蛋白的裂解位点的序列，并依据此两对引物的SYBR Green模式的荧光RT-PCR扩增结果来判断NDV的毒力；以标准NDV强毒和弱毒毒株的RNA为模板，进行扩增，依据实验结果设立判断标准；用此判断标准对8株NDV未知毒株进行检测，结果和MDT检测结果完全一致，说明此方法可用于NDV强弱毒的鉴别。
另外，本方法可以以上述通用引物扩增的PCR产物为模板，进行毒力鉴别实验，从而继承了上述通用检测所具有的敏感性很高的特性。
本研究所建立的NDV及其毒力鉴定的荧光RT-PCR技术具有重要的兽医临床意义，在禽产品进出口上也有很大的应用价值。
Newcastle disease (ND) is a highly fatal and highly contagious avian disease caused by Newcastle disease virus (NDV). It is also one of the List A animal infectious diseases, and causes severe avian epidemics worldwide every year. There are several kinds of classical NDV detection methods, such as virus isolation, HA and HI test, immunofluencent test, ELISA, etc. These techniques some are time-consuming, some are insensitive or somehow unspecific.
Common RT-PCR can detect a few copies of nucleic acids of NDV, and therefore it is used to detect NDV for clinical or quarantine purpose. But its uses are quitely limited because it is difficult to avoid the false positive signals caused by the pollution of PCR products. Common RT-PCR also requires the uses of EB, a strong carcinogen.
Real-time RT-PCR assay is one of the new detection techniques. It has high sensitivity as common RT-PCR, and avoids both the pollution of PCR products and the uses of EB. It is also faster than common RT-PCR. SYBR Green I releases fluorescence when combining to double-stranded DNA, and based on this principle, SYBR Green I real-time RT-PCR is applied widely with lower cost and without difficult probe design. It is suitable for the detection of highly variable genes.
Here in this paper SYBR Green I real-time RT-PCR techniques are reported to be established to detect and pathotype NDV.
Firstly, many sequences of NDV F gene were analysed and a general pair of primers were designed to detect all NDV strains using SYBR Green I real-time RT-PCR technique. Then, the detection conditions were opimised, and RT and PCR were integrated into one step. The Tm values of the specific amplification products are always (88+1)C. The results of such assay and the sequencing assay for the detection of 10 unknown strains were the same, and the sensitivity of such assay were higher than a reported common RT-PCR assay.
The virulence of NDV strains are determined by the lysis site sequences of the viral F protein. Based on this principle, two pairs of primers were desgined to correspond to the sequences of the F protein lysis site of virulent NDV strains and avirulent strains, respectively. The viral RNA was detected using the two primers respectively, and the virulence of NDV strains were judged according to the amplification effects of the two
pairs, of primers. The results of such assay and MDT assay for the pathotyping of 8 unknown strains were the same, which suggested that this assay can be used to pathotype NDV strains and better than the common RT-PCR technique reported previously. The amplification products of the pair of aforementioned general primers could be used as the template of such pathotyping RT-PCR assay, and the high sensitivity of the aforementioned assay of general NDV detection was inherited thereby.
The real time RT-PCR techniques report here are of important significance in veterinary clinicals and the quarantine of avian product export and import.
引文
1 殷震，刘景华主编．动物病毒学，北京：科技出版社，1997年
2 Office International des Epizooties (2001), Newcastle disease, Manual of standards for Diagnostic Tests and Vaccines, 4 th edi, Paris: OIE [M]
3 Kranevld F C. A poultry disease in the Duch East Indies. Ned Indisch B, Diergdneeskd, 1926, 38: 448-450
4 Kilboumeed. Influenza of molecular epidemioiogy [J] J. Infect Dis, 1973, 3: 478
5 Alexander D J. Newcastle disease and other paramyxovirus infectious In: Calnek B W, Barnes H J, Beard C W, Reid W M, Yoder H W Jr(eds). Disease of Poultry. Iowa State University Press, Ames, 1991, PP: 496-519
6 Richardson C D, Scbeid A, Choppin P W. Specific inhibition of paramyxovirus and myxovirus replication by oligopept ides with an inoacid sequences similar to those at the N-terminal of the F1 or HA2 viral peptidies [J]. Virol, 1980, 105: 205～222
7 罗俊，巩霞，王川庆校．新城疫病毒(禽Ⅰ型副粘病毒)的检测和鉴别[J]．上海畜牧兽医通讯．2002，3，10-13
8 Schaper U M, Fuller F J, Ward M D W, et al. Nucleotide sequence of the envolop protein genes of a highly virulent neuotropic strains of Newcastle disease virus[J]. Virol, 1988, 165: 291～295
9 E. W. Aldons & D. J. Alexander Avian Virology, VLA Weybridge, Addlestone, Surrey KT15 3NB, UK Avian Pathology, 2001, 30(2): 117～128
10 Peeters B P, de Leeuw O S, Koch G. Rescue of Newcastle disease virus from cloned cDNA: Evidence that cleavability of the Fusion protein is a major determinant for virulence [J]. J Virol, 1999, 73(6): 5001～5009
11 Millar N S, Chambers P, Emmerson P T. Neculotide sequence analysis of the bemagglutin in-neuram in idase geue of Newcastle disease vine[J]. J Gen Virol, 1986, 67: 1917～1927
12 Morrison T G, Simpson D. Structure, function and intracellular processing of para-myxovirus membrance proteins[J]. VirusRes,1988,10:113～136
13 Liu X, Zhang R, Yu S. Development of polyethylene Glyool medicated ELISA based on monoclonal antibodies against NDV for the detecting of viral antigens in checken specimen [J]. J G Viral, 1997, 62～72
14 Morrison T, McQuain C, and McGinnes L. Complementation between avirulent Newcastle disease virus and a fusion protein gene expressed from a retrovirus vector: requirements for membrane fusion: Journal of Virlolgy, 1991, 65: 813-822
15 Nagy E, Oerbyshire J B, Dobos P, et al. Cloning and expression of NDV HN cDNA in a baculovirus expression vector system [J]. Virol, 1990, 176: 426～438
16 Mori A H, Tawara H, Nakazawa H, et al. Expression of the NDV fusion glycol protein and vaccination against NDV challenge with a recombiant baculo virus [J]. Avian Dis, 1994, 38: 772～777
17 Morgan R W, Gelb J J, Pope C R, et al. Efficacy in chickens of Herpesvirus of turkey recombinant vaccine containing the fusion gene of NDV: On set of protection and effect of maternal antibodies[J]. AvianDis, 1988, 37: 1032～1040
18 Toyoda T, Gotoh B, Sakaguchi T, et al. Identification of am ino acid relevant three anti-gennic determinants on the fusion protein of NDV that are involved in fusion inhibition and neutralization [J]. J Girol, 1988, 62: 4427～4430
19 Sakaguchi T, Toyoda T, Gotoh B, et al. Newcastle disease virus evolutionl, Multiply lineages defined by sequence variability of the hemagglutinin-neuram in idase gene[J]. Virol, 1989, 169: 260～272
20 Giuffer R M, Tovell D R, Kay C M, et al. Evidence for an interaction between the membrance protein of a paramyoxvirus and action[J]. J Virol, 1982,42:963～968
21 Wu R, Grossman L. Recombiant DNA Methodology [M], 1989
22 Iorio R M, Bratt M A. Monocloned antibodies as functional probes of the Hnglycoprotein of NDV: Antigenic separation of the hemagglutin in and neuram inidase sites [J]. J Immunol, 1984, 133: 2215～2219
23 McMillar B C, Hanson R P. RNA oligonucleotide ingerpdnting: Aproposed method of identifying strains of Newcastle disease virus [J]. AvianDis,1980, 24:1016～1020
24 Schcvalbe J C, Hihhtower L E. Maturation of the envelope glycol protein of NDV on cellular membrances [J]. J Virol,1982, 41: 947～957
25 Toyoda T M, Hamaguchi M, Nagai Y. Detection of polycistronic transcrips in Newcastle disease virus infected cells and identification of the sequence content [J]. Arch Virol, 1987, 95: 97～110
26 Collins P L, Hightower L E and BallL A. Transcriptional map for Newcastle disease virus [J]. J Virol, 1980, 35: 682～693
27 Yoshida T, Nagai Y, Maeno K, et al. Studies on the role of M protein in assembly using a ts mutant of HVJ (Sendai virus) [J]. Virol, 1979, 92: 139～154
28 King D J, Avian Dis.,1985, 29(2): 297～311
29 Stone-Hulslander J and Morridon T G. Detection of an interaction between the HN and F proteins in Newcastle disease virus-infected cell. Journal of Virlolgy, 1997, 71:6287-6295
30 Jestin V, Jestin A. Detection of Newcastle disease virus RNA in infected allantoic fluids by in vitro enzymatic amplification (PCR) [J]. Arch. Virology., 1991, 118: 151～161
31 Espion D S, Henau D, Letellier C, et al. Expression at the cellsurface of native fusion protein of the NDV strains Italien from cloned Cdna [J]. Arch. Viol,1987,95:79～95
32 Westcott D G, King D P, Drew T W, et al. Use of an internal standard in a closed one-tube RT-PCR for the detection of equine arteritis virus RNA with fluorescent probes[J]. Vet Res, 2003, 34: 165-176
33 Le L, Brasseur R, Weners C, et al. Quantitive basic residue requirements in the cleavage-activation site of the fusion glycoprotein as a determinant of virulence for Newcastle disease virus. Journal of Virlogy, 1988, 62: 354-356
34 Toyoda T, Sakaguchi T, Imai K, etal. Structural comparison of the cleavage-activation site of the fusion glycoprotein between virulent and avirulent strains of Newcastle disease virus. Virology, 1987, 158: 242-247
35 Boursnell M E G, Green P F, Samson A C R, et al. A recombinant fow l pox virus expressing the HN gene of NDV protects chickens against challenge by NDV [J]. Virol, 1990, 178: 297～300
36 张秀根．新城疫病毒融合蛋白基因的扩增、克隆及在火鸡庖疹病毒中的表达与应用[D]．南京：南京农业大学动物医学院，1999
37 Pringle. Virology-A Practical Approach[M]. IRLPress. Oxford, UnitedKingdom. 1985. 95～117
38 Collins M S, Strong I, Alexander D J, 1994, Evaiution of the molecular basis of pathogenicity of the variant Newcastle disease viruses termed pigeon PMV-1 viruses Arch.Virol., 134:403-411
39 Collins M S, Alexander D J, Brokman S etal, 1989, Evaluation of mouse monoclonal antibodies raised against an isolate of the variant avian paramyxovirus type 1 responsible for the current panzootic in pigeons. Arch, Virol., 104: 53-61
40 Fulton R E, Wong J P, Siddiqui Y M, et al. Virological Methods, 1988, 22: 149～164
41 Russell, P H, Griffithe, P C, Goswami, K K, et al.The characterization of monoclonal antibiodies to Newcastle dieease virus. J Gen Virol, 1983, 64: 2069～2072
42 Meulemans G C, Letellier C, Gonze M, et al. NDV F glycol protein expressed from a recombiant vaccine virus vector protects chickens against live-virus-challenge [J]. Avian Dis, 1988,17:821～827
43 Alexander D J, Manvell R J, Kemp P A, et al. Use of monoclonal antibodies in the characterization of avian paramyxovirus type 1(Newcastle disease virus) isolates submitted to an international reference laboratory. Avian Pathology. 1987, 16: 553-565
44 Judit Erdei等．鸡新城疫疫苗(LaSota)特异性单克隆抗体，国外兽医学．畜禽传染病，1989，9(1)：
20-21，谷登峰译
45 曹殿军，卢景良，王莉林，刘春丽，李一经，张莹．应用单克隆抗体鉴别新城疫病毒强＼弱毒株的研究，中国预防兽医学报，1996，5：36-38
46 Hodder A N, Liu Z Y, Selleck P W, et al. Characterization of field isolates of Newcastle disease virus using antipeptide antibodies[J].AvianDis, 1994, 38: 103～118
47 王树双，孙淑华，吴时友，等．新城疫病毒分子生物学水平上的致病性分析．用抗病毒囊膜蛋白特异性结构多肽抗体进行强弱毒株鉴别研究[C]．1994年中国兽医微生物学与病毒学学术研讨会论文集．1994．116
48 Jarecki-Black J C, Benntt J D, Palmieri S. A novel oligonucleotide probe for the detection of Newcastle disease virus[J]. AvianDis, 1992, 36: 134～138
49 Jarechi-Black J C, King D J, An oligonucleotide probe that distinggushes isolates of low virulent from the more pathogeins strains of Newcastle disease virus, Avian Disease, 1993,37:724～730
50 Angela. Newcastle disease virus detection and characterization by PCR of recent German isolates different in pathogenicity [J]. AvianPathol, 1998, 27: 237～243
51 贺东生，宋长绪，刘福安，等．用核酸探针检测新城疫病毒的研究，中国畜禽传染病，1997，(4)：3-4
52 贺东生，刘福安，宋长绪，NDA-RNA杂交法鉴别新城疫强毒株和弱毒株，中国兽医科技，1998，28(7)：22-23
53 刘伟忠，姜焱，吴艳涛，等．新城疫病毒特异性糖蛋白基因探针的制备及应用，畜牧兽医学报，1999，30(1)：44-49
54 E. W. Aldous, M. S. Collins, et al. Rapid pathotyping of Newcastle disease virus(NDV) using fluorogenic probes in a PCR assay [J]. Veterinary Microbiology 80(2001)201-212
55 Palmier S, Michael L, Perdue. An alternative method of oligonueleotide fingerprinting for resolving NDV specific RNA fragments. Avian Disease,1989, 33: 345-350
56 Ballagi-Pordany A, Wehmann E, Herezeg J, et al.,1998,27:237-243
57 宋长绪，刘福安，用PCR检测鸡NDV的初步试验[J]．中国兽医科技．1995，25(10)：20～22
58 曹殿军，刘培欣，等．鸡新城疫病毒强弱毒株RT-PCR鉴别诊断方法[J]．中国预防兽医学报，2001，6：415～417
59 Kant A G, Koch D J. Differentiation of virulent and non-virulent strains of NDV within 24 hours by PCR [J]. AvianPathol, 1997, 26: 837～849.
60 Z. wang, F T, Vreede, J O, Mitchell etal. Rapid detection and differentiation of Newcastle disease virus isolates by a triple one-step RT-PCR[J]. Veterinary Reasearch, 2001, 68: 131-134
61 阎玉河，邓瑞广，赵传壁，等．用反转录-聚合酶链反应快速鉴别新城疫病毒．中国兽医科
技，2000，30(1)：3-5
62 黄庚明，辛朝安．应用逆转录套式PCR检测新城疫病毒核酸．中国预防兽医学报．2001(23)4：295-299
63 谢芝勋，谢志勤，庞珊耀，等．应用三重PCR同时检测NDV、IBV、MG(C)的研究[J]．中国动物检疫，2000，17(11)：20—22
64 谢芝勋，谢志勤，庞耀珊，等．应用多重聚合酶链反应同时检测鉴别鸡新城疫病毒、传染性支气管炎病毒、鸡传染性喉气管炎病毒、鸡毒霉形体的研究[J]．中国预防兽医学报，2000，22(6)：443—446
65 Kant A, Koch G, Van Roozelaar D J, et al. Avi pathol., 1997,26: 837～849
66 Marras, S.A., Kramer, F.R. & Tyagi, S. Multiplex detection of single-nucleotide variations using molecular beacons[J]. Genetic Analysis, 1999,14, 151-156
67 Wittwer, C.T., Herrmann, M.G., Moss, A.A. & Rasmussen, R.P. Continuous Fluorescence Monitoring of Rapid Cycle DNA Amplification [J]. BioTecimiques, 1997, 22, 130-138 63. Willard M F, Stephen J W, Kent E V. Quantitative RT-PCR: Pitfall and Potential. Bio Techniques, 1999, 26(1): 112-125
68 Wise M G, Suarez D L,Seal B S,Pedersen J C,Senne D A,King D J,Kapczynski D R.Spackman E. Development of a real-time reverse-trandcription PCR for detection of Newcastle disease virus RNA in clinical samples. J Clin Microbiol.2004 Jan: 42(1): 329-38
69 Puig M, Mihalik K. Yu MY, etal. Sensitivity and reproducibility of HCV quantitation in chimpanzee sera using TaqMan real-time PCR assay[J]. J Virol Methods, 2002, 105:253-263
70 Klein S A, Karsten S, Ruster B, et al. Comparison of TaqMan real-time PCR and p24 Elisa for quantification of in vitro HIV-1 replication[J]. J Virol Methods, 2003, 107: 169-175
71 Weidmann M, Meyer-Konig U, Hufert FT. Rapid detection of herpes simplex virus and varicella-zoster virus infections by real-time PCR [J]. J Clin Microbiol, 2003, 41: 1565-1568
72 Singer VL, Lawlor TE, Yue S. Comparison of SYBR Green 1 nucleic acid gel mutagenicity and ethidium bromide mutagenicity in the Salmonella/mammalian microsome reverse mutation assay Mutat Res, 1999,439(1):37-47
73 Morrison, T.B., Weis, J.J. & Wittwer, C.T. (1998). Quantification of Low-Copy Transcripts by Continuous SYBR Green I Monitoring during Amplification[J]. Bio Techniques, 24(6), 954-962
74 Tyagi, S. & Kramer, F.R. (1996). Molecular beacons: probes that fluoresce upon hybridization[J]. Nature Biotechnology, 14, 303-308
75 Zanella I, Rossini A, Domenighini D, et al. Real-time quantitation of hepatitis B virus (HBV) DNA in tumorous and surrounding tissue from patients with hepatocellular carcinoma[J]. J Med Virol,
2002, 68: 494-499
76 WOO T H, Patel B K, Cinco M, Smythe L D, Norris M A, Symonds M L, Dohnt M F, Piispanen J. Identification of Leptospira biflexa by real-time homogeneous detection of rapid cycle PCR product. J Microbiol Methods. 1999, 35 (1): 23-30
77 van Elden L J, Ni J huis M, Schipper P, et al. Simultaneous detection of influenza viruses A and B using real-time quantitative PCR[J]. J Clin Microbioi, 2001, 39: 196-200
78 Nasci RS, Gottfried KL, Burkhalter KL, et al. Comparison of vero cell plaque assay, TaqMan reverse transcriptase polymerase chain reaction RNA assay, and VecTest antigen assay for detection of West Nile virus in field-collected mosquitoes[J]. J Am Mosq Control Assoc, 2002, 18: 294-300
79 Lambert A J, Martin DA, Lanciotti RS. Detection of North American eastern and western equine encephalitis viruses by nucleic acid amplification assays[J]. J Clin Microbiol, 2003, 41: 379-385
80 Weidmann M, Meyer-Konig U, Hufert FT. Rapid detection of herpes simplex virus and varicella-zoster virus infections by real-time PCR[J]. J Clin Microbiol, 2003, 41: 1565-1568.
81 Monpoeho S, Coste-Burel M, Costa-Mattioli M, et al. Application of a real-time polymerase chain reaction with internal positive control for detection and quantification of enterovirus in cerebrospinal fluid. Eur[J]. J Clin Microbiol Infect Dis, 2002, 21: 532-536
82 Spackman E, Senne DA, Myers T J, etal. Development of a real-time reverse transcriptase PCR assay for type A influenza virus and the avian H5 and H7 hemagglutinin subtypes[J]. J Clin Microbiol, 2002, 40:3256-3260
83 Gibb T R, Norwood D A Jr, Woollen N, Henchal EA. Development and evaluation of a fluorogenic 5'-nuclease assay to identify Marburg virus[J]. Mol Cell Probes, 2001, 15: 259-266
84 King DP, Reid S M, Hutchings G H, etal. Development of a TaqMan PCR assay with internal amplification control for the detection of African swine fever virus[J]. J Virol Methods, 2003, 107: 53-61
85 Smith G D, Solenberg P J, Koenig M C, et al. Use of TaqMan reverse transcriptase-polymerase chain reaction analysis and serologic testing to eliminate an enzootie infection of mouse hepatitis virus[J]. Comp Med, 2002, 52: 456-460
86 Fabre F, Kervarrec C, Mieuzet L, Riault G, Vialatte A, Jacquot E. Improvement of Barley yellow dwarf virus-PAV detection in single aphids using a fluorescent real-time RT-PCR[J]. J Virol Methods. 2003 Jun; 110(1): 51-60
87 olland P, Abramson R, Watson R, et al. Detection of specific polymerase chain reaction product by utilizing the 5' to 3' exonuclease activity of Thermus aquaticus DNA polymerase[J]. Proc Natl Acad Sci U S A, 1991, 88: 7276-7280
88 Wall S J, Edward D R. Quantitative reverse transcription-polynerase chain reaction (TR-PCR):A comparison of prime-dropping, and real-time RT-PCRs. Anal Biochem,2002,300(2):269-273
2 Office International des Epizooties (2001), Newcastle disease, Manual of standards for Diagnostic Tests and Vaccines, 4 th edi, Paris: OIE [M]
3 Kranevld F C. A poultry disease in the Duch East Indies. Ned Indisch B, Diergdneeskd, 1926, 38: 448-450
4 Kilboumeed. Influenza of molecular epidemioiogy [J] J. Infect Dis, 1973, 3: 478
5 Alexander D J. Newcastle disease and other paramyxovirus infectious In: Calnek B W, Barnes H J, Beard C W, Reid W M, Yoder H W Jr(eds). Disease of Poultry. Iowa State University Press, Ames, 1991, PP: 496-519
6 Richardson C D, Scbeid A, Choppin P W. Specific inhibition of paramyxovirus and myxovirus replication by oligopept ides with an inoacid sequences similar to those at the N-terminal of the F1 or HA2 viral peptidies [J]. Virol, 1980, 105: 205～222
7 罗俊，巩霞，王川庆校．新城疫病毒(禽Ⅰ型副粘病毒)的检测和鉴别[J]．上海畜牧兽医通讯．2002，3，10-13
8 Schaper U M, Fuller F J, Ward M D W, et al. Nucleotide sequence of the envolop protein genes of a highly virulent neuotropic strains of Newcastle disease virus[J]. Virol, 1988, 165: 291～295
9 E. W. Aldons & D. J. Alexander Avian Virology, VLA Weybridge, Addlestone, Surrey KT15 3NB, UK Avian Pathology, 2001, 30(2): 117～128
10 Peeters B P, de Leeuw O S, Koch G. Rescue of Newcastle disease virus from cloned cDNA: Evidence that cleavability of the Fusion protein is a major determinant for virulence [J]. J Virol, 1999, 73(6): 5001～5009
11 Millar N S, Chambers P, Emmerson P T. Neculotide sequence analysis of the bemagglutin in-neuram in idase geue of Newcastle disease vine[J]. J Gen Virol, 1986, 67: 1917～1927
12 Morrison T G, Simpson D. Structure, function and intracellular processing of para-myxovirus membrance proteins[J]. VirusRes,1988,10:113～136
13 Liu X, Zhang R, Yu S. Development of polyethylene Glyool medicated ELISA based on monoclonal antibodies against NDV for the detecting of viral antigens in checken specimen [J]. J G Viral, 1997, 62～72
14 Morrison T, McQuain C, and McGinnes L. Complementation between avirulent Newcastle disease virus and a fusion protein gene expressed from a retrovirus vector: requirements for membrane fusion: Journal of Virlolgy, 1991, 65: 813-822
15 Nagy E, Oerbyshire J B, Dobos P, et al. Cloning and expression of NDV HN cDNA in a baculovirus expression vector system [J]. Virol, 1990, 176: 426～438
16 Mori A H, Tawara H, Nakazawa H, et al. Expression of the NDV fusion glycol protein and vaccination against NDV challenge with a recombiant baculo virus [J]. Avian Dis, 1994, 38: 772～777
17 Morgan R W, Gelb J J, Pope C R, et al. Efficacy in chickens of Herpesvirus of turkey recombinant vaccine containing the fusion gene of NDV: On set of protection and effect of maternal antibodies[J]. AvianDis, 1988, 37: 1032～1040
18 Toyoda T, Gotoh B, Sakaguchi T, et al. Identification of am ino acid relevant three anti-gennic determinants on the fusion protein of NDV that are involved in fusion inhibition and neutralization [J]. J Girol, 1988, 62: 4427～4430
19 Sakaguchi T, Toyoda T, Gotoh B, et al. Newcastle disease virus evolutionl, Multiply lineages defined by sequence variability of the hemagglutinin-neuram in idase gene[J]. Virol, 1989, 169: 260～272
20 Giuffer R M, Tovell D R, Kay C M, et al. Evidence for an interaction between the membrance protein of a paramyoxvirus and action[J]. J Virol, 1982,42:963～968
21 Wu R, Grossman L. Recombiant DNA Methodology [M], 1989
22 Iorio R M, Bratt M A. Monocloned antibodies as functional probes of the Hnglycoprotein of NDV: Antigenic separation of the hemagglutin in and neuram inidase sites [J]. J Immunol, 1984, 133: 2215～2219
23 McMillar B C, Hanson R P. RNA oligonucleotide ingerpdnting: Aproposed method of identifying strains of Newcastle disease virus [J]. AvianDis,1980, 24:1016～1020
24 Schcvalbe J C, Hihhtower L E. Maturation of the envelope glycol protein of NDV on cellular membrances [J]. J Virol,1982, 41: 947～957
25 Toyoda T M, Hamaguchi M, Nagai Y. Detection of polycistronic transcrips in Newcastle disease virus infected cells and identification of the sequence content [J]. Arch Virol, 1987, 95: 97～110
26 Collins P L, Hightower L E and BallL A. Transcriptional map for Newcastle disease virus [J]. J Virol, 1980, 35: 682～693
27 Yoshida T, Nagai Y, Maeno K, et al. Studies on the role of M protein in assembly using a ts mutant of HVJ (Sendai virus) [J]. Virol, 1979, 92: 139～154
28 King D J, Avian Dis.,1985, 29(2): 297～311
29 Stone-Hulslander J and Morridon T G. Detection of an interaction between the HN and F proteins in Newcastle disease virus-infected cell. Journal of Virlolgy, 1997, 71:6287-6295
30 Jestin V, Jestin A. Detection of Newcastle disease virus RNA in infected allantoic fluids by in vitro enzymatic amplification (PCR) [J]. Arch. Virology., 1991, 118: 151～161
31 Espion D S, Henau D, Letellier C, et al. Expression at the cellsurface of native fusion protein of the NDV strains Italien from cloned Cdna [J]. Arch. Viol,1987,95:79～95
32 Westcott D G, King D P, Drew T W, et al. Use of an internal standard in a closed one-tube RT-PCR for the detection of equine arteritis virus RNA with fluorescent probes[J]. Vet Res, 2003, 34: 165-176
33 Le L, Brasseur R, Weners C, et al. Quantitive basic residue requirements in the cleavage-activation site of the fusion glycoprotein as a determinant of virulence for Newcastle disease virus. Journal of Virlogy, 1988, 62: 354-356
34 Toyoda T, Sakaguchi T, Imai K, etal. Structural comparison of the cleavage-activation site of the fusion glycoprotein between virulent and avirulent strains of Newcastle disease virus. Virology, 1987, 158: 242-247
35 Boursnell M E G, Green P F, Samson A C R, et al. A recombinant fow l pox virus expressing the HN gene of NDV protects chickens against challenge by NDV [J]. Virol, 1990, 178: 297～300
36 张秀根．新城疫病毒融合蛋白基因的扩增、克隆及在火鸡庖疹病毒中的表达与应用[D]．南京：南京农业大学动物医学院，1999
37 Pringle. Virology-A Practical Approach[M]. IRLPress. Oxford, UnitedKingdom. 1985. 95～117
38 Collins M S, Strong I, Alexander D J, 1994, Evaiution of the molecular basis of pathogenicity of the variant Newcastle disease viruses termed pigeon PMV-1 viruses Arch.Virol., 134:403-411
39 Collins M S, Alexander D J, Brokman S etal, 1989, Evaluation of mouse monoclonal antibodies raised against an isolate of the variant avian paramyxovirus type 1 responsible for the current panzootic in pigeons. Arch, Virol., 104: 53-61
40 Fulton R E, Wong J P, Siddiqui Y M, et al. Virological Methods, 1988, 22: 149～164
41 Russell, P H, Griffithe, P C, Goswami, K K, et al.The characterization of monoclonal antibiodies to Newcastle dieease virus. J Gen Virol, 1983, 64: 2069～2072
42 Meulemans G C, Letellier C, Gonze M, et al. NDV F glycol protein expressed from a recombiant vaccine virus vector protects chickens against live-virus-challenge [J]. Avian Dis, 1988,17:821～827
43 Alexander D J, Manvell R J, Kemp P A, et al. Use of monoclonal antibodies in the characterization of avian paramyxovirus type 1(Newcastle disease virus) isolates submitted to an international reference laboratory. Avian Pathology. 1987, 16: 553-565
44 Judit Erdei等．鸡新城疫疫苗(LaSota)特异性单克隆抗体，国外兽医学．畜禽传染病，1989，9(1)：
20-21，谷登峰译
45 曹殿军，卢景良，王莉林，刘春丽，李一经，张莹．应用单克隆抗体鉴别新城疫病毒强＼弱毒株的研究，中国预防兽医学报，1996，5：36-38
46 Hodder A N, Liu Z Y, Selleck P W, et al. Characterization of field isolates of Newcastle disease virus using antipeptide antibodies[J].AvianDis, 1994, 38: 103～118
47 王树双，孙淑华，吴时友，等．新城疫病毒分子生物学水平上的致病性分析．用抗病毒囊膜蛋白特异性结构多肽抗体进行强弱毒株鉴别研究[C]．1994年中国兽医微生物学与病毒学学术研讨会论文集．1994．116
48 Jarecki-Black J C, Benntt J D, Palmieri S. A novel oligonucleotide probe for the detection of Newcastle disease virus[J]. AvianDis, 1992, 36: 134～138
49 Jarechi-Black J C, King D J, An oligonucleotide probe that distinggushes isolates of low virulent from the more pathogeins strains of Newcastle disease virus, Avian Disease, 1993,37:724～730
50 Angela. Newcastle disease virus detection and characterization by PCR of recent German isolates different in pathogenicity [J]. AvianPathol, 1998, 27: 237～243
51 贺东生，宋长绪，刘福安，等．用核酸探针检测新城疫病毒的研究，中国畜禽传染病，1997，(4)：3-4
52 贺东生，刘福安，宋长绪，NDA-RNA杂交法鉴别新城疫强毒株和弱毒株，中国兽医科技，1998，28(7)：22-23
53 刘伟忠，姜焱，吴艳涛，等．新城疫病毒特异性糖蛋白基因探针的制备及应用，畜牧兽医学报，1999，30(1)：44-49
54 E. W. Aldous, M. S. Collins, et al. Rapid pathotyping of Newcastle disease virus(NDV) using fluorogenic probes in a PCR assay [J]. Veterinary Microbiology 80(2001)201-212
55 Palmier S, Michael L, Perdue. An alternative method of oligonueleotide fingerprinting for resolving NDV specific RNA fragments. Avian Disease,1989, 33: 345-350
56 Ballagi-Pordany A, Wehmann E, Herezeg J, et al.,1998,27:237-243
57 宋长绪，刘福安，用PCR检测鸡NDV的初步试验[J]．中国兽医科技．1995，25(10)：20～22
58 曹殿军，刘培欣，等．鸡新城疫病毒强弱毒株RT-PCR鉴别诊断方法[J]．中国预防兽医学报，2001，6：415～417
59 Kant A G, Koch D J. Differentiation of virulent and non-virulent strains of NDV within 24 hours by PCR [J]. AvianPathol, 1997, 26: 837～849.
60 Z. wang, F T, Vreede, J O, Mitchell etal. Rapid detection and differentiation of Newcastle disease virus isolates by a triple one-step RT-PCR[J]. Veterinary Reasearch, 2001, 68: 131-134
61 阎玉河，邓瑞广，赵传壁，等．用反转录-聚合酶链反应快速鉴别新城疫病毒．中国兽医科
技，2000，30(1)：3-5
62 黄庚明，辛朝安．应用逆转录套式PCR检测新城疫病毒核酸．中国预防兽医学报．2001(23)4：295-299
63 谢芝勋，谢志勤，庞珊耀，等．应用三重PCR同时检测NDV、IBV、MG(C)的研究[J]．中国动物检疫，2000，17(11)：20—22
64 谢芝勋，谢志勤，庞耀珊，等．应用多重聚合酶链反应同时检测鉴别鸡新城疫病毒、传染性支气管炎病毒、鸡传染性喉气管炎病毒、鸡毒霉形体的研究[J]．中国预防兽医学报，2000，22(6)：443—446
65 Kant A, Koch G, Van Roozelaar D J, et al. Avi pathol., 1997,26: 837～849
66 Marras, S.A., Kramer, F.R. & Tyagi, S. Multiplex detection of single-nucleotide variations using molecular beacons[J]. Genetic Analysis, 1999,14, 151-156
67 Wittwer, C.T., Herrmann, M.G., Moss, A.A. & Rasmussen, R.P. Continuous Fluorescence Monitoring of Rapid Cycle DNA Amplification [J]. BioTecimiques, 1997, 22, 130-138 63. Willard M F, Stephen J W, Kent E V. Quantitative RT-PCR: Pitfall and Potential. Bio Techniques, 1999, 26(1): 112-125
68 Wise M G, Suarez D L,Seal B S,Pedersen J C,Senne D A,King D J,Kapczynski D R.Spackman E. Development of a real-time reverse-trandcription PCR for detection of Newcastle disease virus RNA in clinical samples. J Clin Microbiol.2004 Jan: 42(1): 329-38
69 Puig M, Mihalik K. Yu MY, etal. Sensitivity and reproducibility of HCV quantitation in chimpanzee sera using TaqMan real-time PCR assay[J]. J Virol Methods, 2002, 105:253-263
70 Klein S A, Karsten S, Ruster B, et al. Comparison of TaqMan real-time PCR and p24 Elisa for quantification of in vitro HIV-1 replication[J]. J Virol Methods, 2003, 107: 169-175
71 Weidmann M, Meyer-Konig U, Hufert FT. Rapid detection of herpes simplex virus and varicella-zoster virus infections by real-time PCR [J]. J Clin Microbiol, 2003, 41: 1565-1568
72 Singer VL, Lawlor TE, Yue S. Comparison of SYBR Green 1 nucleic acid gel mutagenicity and ethidium bromide mutagenicity in the Salmonella/mammalian microsome reverse mutation assay Mutat Res, 1999,439(1):37-47
73 Morrison, T.B., Weis, J.J. & Wittwer, C.T. (1998). Quantification of Low-Copy Transcripts by Continuous SYBR Green I Monitoring during Amplification[J]. Bio Techniques, 24(6), 954-962
74 Tyagi, S. & Kramer, F.R. (1996). Molecular beacons: probes that fluoresce upon hybridization[J]. Nature Biotechnology, 14, 303-308
75 Zanella I, Rossini A, Domenighini D, et al. Real-time quantitation of hepatitis B virus (HBV) DNA in tumorous and surrounding tissue from patients with hepatocellular carcinoma[J]. J Med Virol,
2002, 68: 494-499
76 WOO T H, Patel B K, Cinco M, Smythe L D, Norris M A, Symonds M L, Dohnt M F, Piispanen J. Identification of Leptospira biflexa by real-time homogeneous detection of rapid cycle PCR product. J Microbiol Methods. 1999, 35 (1): 23-30
77 van Elden L J, Ni J huis M, Schipper P, et al. Simultaneous detection of influenza viruses A and B using real-time quantitative PCR[J]. J Clin Microbioi, 2001, 39: 196-200
78 Nasci RS, Gottfried KL, Burkhalter KL, et al. Comparison of vero cell plaque assay, TaqMan reverse transcriptase polymerase chain reaction RNA assay, and VecTest antigen assay for detection of West Nile virus in field-collected mosquitoes[J]. J Am Mosq Control Assoc, 2002, 18: 294-300
79 Lambert A J, Martin DA, Lanciotti RS. Detection of North American eastern and western equine encephalitis viruses by nucleic acid amplification assays[J]. J Clin Microbiol, 2003, 41: 379-385
80 Weidmann M, Meyer-Konig U, Hufert FT. Rapid detection of herpes simplex virus and varicella-zoster virus infections by real-time PCR[J]. J Clin Microbiol, 2003, 41: 1565-1568.
81 Monpoeho S, Coste-Burel M, Costa-Mattioli M, et al. Application of a real-time polymerase chain reaction with internal positive control for detection and quantification of enterovirus in cerebrospinal fluid. Eur[J]. J Clin Microbiol Infect Dis, 2002, 21: 532-536
82 Spackman E, Senne DA, Myers T J, etal. Development of a real-time reverse transcriptase PCR assay for type A influenza virus and the avian H5 and H7 hemagglutinin subtypes[J]. J Clin Microbiol, 2002, 40:3256-3260
83 Gibb T R, Norwood D A Jr, Woollen N, Henchal EA. Development and evaluation of a fluorogenic 5'-nuclease assay to identify Marburg virus[J]. Mol Cell Probes, 2001, 15: 259-266
84 King DP, Reid S M, Hutchings G H, etal. Development of a TaqMan PCR assay with internal amplification control for the detection of African swine fever virus[J]. J Virol Methods, 2003, 107: 53-61
85 Smith G D, Solenberg P J, Koenig M C, et al. Use of TaqMan reverse transcriptase-polymerase chain reaction analysis and serologic testing to eliminate an enzootie infection of mouse hepatitis virus[J]. Comp Med, 2002, 52: 456-460
86 Fabre F, Kervarrec C, Mieuzet L, Riault G, Vialatte A, Jacquot E. Improvement of Barley yellow dwarf virus-PAV detection in single aphids using a fluorescent real-time RT-PCR[J]. J Virol Methods. 2003 Jun; 110(1): 51-60
87 olland P, Abramson R, Watson R, et al. Detection of specific polymerase chain reaction product by utilizing the 5' to 3' exonuclease activity of Thermus aquaticus DNA polymerase[J]. Proc Natl Acad Sci U S A, 1991, 88: 7276-7280
88 Wall S J, Edward D R. Quantitative reverse transcription-polynerase chain reaction (TR-PCR):A comparison of prime-dropping, and real-time RT-PCRs. Anal Biochem,2002,300(2):269-273