Microsoft Word 王乃福-二校出466

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1 6 2 Vol. 6 No Journal of Food Safety and Quality Feb., 2015 RT-PCR 王乃福 *, 黄晨, 吴冬雪, 赵祥平, 陈本龙, 董志珍 (, ) 摘要 : 目的 RT-PCR 方法 3D N VP1, 3 RT-PCR 结果,,, 结论,, 关键词 : ; ; ; RT-PCR Establishment of simultaneous detection of foot-and-mouth disease virus, vesicular stomatitis virus and swine vesicular disease virus by multiplex real-time RT-PCR WANG Nai-Fu *, HUANG Chen, WU Dong-Xue, ZHAO Xiang-Ping, CHEN Ben-Long, DONG Zhi-Zhen (Animal, Plant and Foodstuffs Inspection Center, Tianjin Entry-Exit Inspection and Quarantine Bureau, Tianjin , China) ABSTRACT: Objective To establish a multiplex real-time RT-PCR method for simultaneous detection of foot and mouth disease virus, vesicular stomatitis virus and swine vesicular disease virus. Methods According to the gene encoding 3D protein of foot-and-mouth disease virus, vesicular stomatitis virus N protein coding gene and swine vesicular disease virus VP1 protein encoding gene, specific primers and probe were designed, and 3 kinds of animal viruses were amplified by multiplex real-time RT-PCR. Results The FMDV, VSV and SVDV could be simultaneously detected after amplification, and other tested pathogens were no amplification signal, displaying the good specificity. The minimum detection limits of foot and mouth disease virus, vesicular stomatitis virus and swine vesicular disease virus were 10 1, 10 2, and 10 2 plasmid copy concentration, respectively. Conclusion This method has a good specificity and sensitivity for simultaneous detection of FMDV, VSV and SVDV at the same time. KEY WORDS: foot and mouth disease virus; vesicular stomatitis virus; swine vesicular disease virus; multiplex real-time RT-PCR 基金项目 : (TK ) Fund: Supported by the Scientific and Technological Project of Tianjin Entry-Exit Inspection and Quarantine Bureau (TK ) * 通讯作者 :,, wangnf@tjciq.gov.cn *Corresponding author: WANG Nai-Fu, Veterinarian, Animal, Plant and Foodstuffs Inspection Center, Tianjin Entry-Exit Inspection and Quarantine Bureau, NO.158, Jingmen Road, Tanggu Distruct, Tianjin , China. wangnf@tjciq.gov.cn

2 2, : RT-PCR 引言,,,, (OIE) A [1-3] 3, [4-6], 3,, PCR, 3 [7,8], 3, ( foot and mouth disease virus, FMDV) (vesicular stomatitis virus, VSV) (swine vesicular disease virus, SVDV),,, RT-PCR 2 材料与方法 2.1 毒株和质粒 (FMDV)O A AsiaⅠ, ;, ; ; ; 试剂与仪器 RNA DNA DNA Qiagen ; M-MULV Taq DNA RNA NEB ; TOP10 Takara ; PCR SuperMix-UDG with ROX Invitrogen ;, ABI Prism 7000HT ( ); ( EPPENDORF ); PCR ( BIO-RAD ); ( SIGMA ) 2.3 实验方法引物设计与合成 GenBank (O A AsiaⅠ ) ( ), Clustalx, 3D N VP1, ( 1), Table 1 表 1 试验引物及探针 The primers and probes in the study (5'-3') P1-3D CATGGATTATGGAACTGGGTT FMDV P2-3D TACTCGTCAGGTCCAGAGTGG 13~156 bp Probe-3D P3-N FAM-TTACAAACCTGTGATGGCCTCGAAG-BHQ1 ATGTGAGCACTAAAGTAGCCCT VSV P4-N GTTCTTCCAAAGCTAACCCAGT 135~294 bp Probe-N P5-VP1 ROX-TATGTCTACCAGGGAATCAAATCCG-BHQ1 GGCATGTGAAGAATTACCACTC SVDV P6-VP1 GTTGATGACCCAATAAGCGAAG 149~282 bp Probe-VP1 HEX-AGAGTCGACCGTGGAGAACTTCCTAT-BHQ2

3 样品 RNA 的提取 DEPC EP, 1 ml TRIzol 200 L, 5 min; 200 L, 30 s, 3 min, 4 15 min, ; EP, 500 L, min, 4 15 min; ; 1 ml 75 DEPC,, 4 10 min;, 5 min; 50 L DEPC, 多重荧光定量 RT-PCR 方法的建立 3 3, RNA RNA RNA, dntp Mg 2+ Taq DNA, RT-PCR 标准曲线的建立 3D N VP1, RNA 50 L PCR, PCR pgem-t easy TOP10,,, OD260, 10, RT-PCR, 特异性试验 RNA RNA, RNA,, RNA cdna RNA DNA RT-PCR 多重荧光定量 RT-PCR 的灵敏度试验 RNA RNA RNA, RT-PCR, 样本检测 480, ELISA, (O A AsiaⅠ ) 1, ( ) VP1 ( ) 3 结果与分析 3.1 多重荧光 RT-PCR 检测方法的建立 3 3, dntp Mg 2+ Taq, 50 µl, 5 L PCR (10 ), 5 L (25 mmol/l), 5 L dntps(10 mmol/l), 0.75 L FMDV-3D (15 mol/l), 1 L VSV-N (10 mol/l), 1 L SVDV-VP1 (15 mol/l), 1 L, 1 L Taq DNA (5 U/ L), 1 L AMV (5 U/ L), 4 L, 12.5 L ddh 2 O 50 2 min, 1 ; 95 2 min, 1 ; s s, 标准曲线的建立基因拷贝数测定 3D N VP1, OD260,, pgem T-3D copies/µl, pgem T-N copies/µl, pgem T-VP copies/µl 标准曲线 10, 5 RT-PCR, Y= 2.993lgX ( 1), Y= 2.840lgX , Y= 2.69lgX 特异性试验 RNA RNA

4 2, : RT-PCR 469 RNA, RNA,, RNA cdna RNA DNA RT-PCR, FMDV VSV SVDV, ( 2) 多重荧光定量 RT-PCR 方法的灵敏度试验 10, ~ copies/µl,, RT-PCR 10 1 copies/µl 10 2 copies/µl 10 2 copies/µl, RT-PCR 样本检测 480 ( 400, 80 ), ELISA 480 ELISA, RT-PCR, ELISA 100% (O A AsiaⅠ ) 1, VP1 ( ), Fig. 1 1 Standard curve of FMDV recombinant plasmid

5 : FMDV VSV SVDV Fig. 2 The specificity test (Samples from left to right: FMDV copies VSV copies SVDV copies, respectively) 3 ( : ) Fig. 3 Sensitivity test of FMDV (Samples from left to right: copies, respectively) 4 讨论,,, PCR,, [9-12], 3 RT-PCR PCR

6 2, : RT-PCR 471, PCR,,, PCR, [13-14] PCR,,,, [15] RT-PCR, 4 h, PCR PCR,, RT-PCR 参考文献 [1] Huang H, Yang Z, Xu Q. Recombinant fusion protein and DNA vaccines against foot and mouth disease virus infection in guinea pig and swine [J].Viral Immunol,1999,12(1): 1 8. [2] Cao SN, MousaAA, Aboge GO. Prime-boost vaccination with plasmid DNA followed by recombinant vaccinia virus expressing BgGARP induced a partial protective immunity to inhibit Babesia gibsoni proliferation in dogs [J]. Acta Para/Witold Stefański Inst Para, Warszawa, Poland, 2013, 58(4): [3],,,. RT-PCR [J]., 2006, 23(3): Qi HC, Gong ZH, Yang ZQ, et al. Simultaneous detection of foot-and-mouth disease virus, swine vesicular disease virus and vesicular stomatitis virus by multiplex RT-PCR [J]. China J Anim Quar, 2006, 23(3): [4] Zheng M, Jin N, Zhang H. Construction and immunogenicity of a recombinant fowlpox virus containing the capsid and 3C protease coding regions of foot-and-mouth disease virus [J]. J Virol Method, 2006, 136(1-2): [5] Reid SM, Ferris NP, Hut Chings GH, et al. Evaluation of real-time reverse transcription polymerase chain reaction assays for the detect ion of swine vesicular disease virus [J]. J Virol Method, 2004, 116: [6] Carrillo C, Tulman ER, Delhon G, et al. Comparative genomics of foot- and-mouth disease virus [J]. J Virol, 2005, 79(10): [7]. PCR [J]., 2011, (8): Han CL. Establishment of multiplex real-time method for detection of three foodborne pathogens [J]. Poul Sci, 2011, (8): [8],,,. PCR [J]., 2011, 44: Wang H, Zhu RL, Tan YL, et al. Establishment and application of multiple PCR for diagnosing proteus mirabilis,salmonella and listeria monocytogenes [J]. Scientia Agric Sinica, 2011, 44: [9] Fang X, Zhang S, Sun X. Evaluation of attenuated VSVs with mutated M or/and G proteins as vaccine vectors [J]. Vaccine, 2012, 30(7): [10],,,. RT-PCR [J]., 2009, 19(1): Xu JM, Duan XY, Zhang NZ, et al. Triplex real-time RT-PCR assay for simultaneous detection of three fish rhabdoviridae viruses [J]. Insp Quar Sci, 2009, 19(1): [11] Tomás G, Hernández M, Marandino A, et al. Development and validation of a TaqMan-MGB real-time RT-PCR assay for simultaneous detection and characterization of infectious bursal disease virus [J]. J Virol Method, 2012, 185(1): [12] Lomakina NF, Fallacara F, Pacciarini M, et al. Application of universal primers for identification of Foot-and-mouth disease virus and swine vesicular disease virus by PCR and PCR-ELISA [J]. Arch Virol, 2004, 149(6): [13] Rasmussen TB, Uttenthal A, Agüero M. Detection of three porcine vesicular viruses using multiplex real-time primer-probe energy transfer [J]. J Virol Method, 2006, 134(1-2): [14] Martín-Acebes MA, González-Magaldi M, Rosas MF, et al. Subcellular distribution of swine vesicular disease virus proteins and alterations induced in infected cells: a comparative study with foot-and-mouth disease virus and vesicular stomatitis virus [J]. Virology, 2008, 374(2): [15] McMenamy MJ, McKillen J, Reid SM, et al. Development of a minor groove binder assay for real-time one-step RT-PCR detection of swine vesicular disease virus [J]. J Virol Method, 2011, 171(1): 作者简介 ( 责任编辑 : 杨翠娜 ) 王乃福, 兽医师, 主要研究方向为动物疫病诊断技术 wangnf@tjciq.gov.cn

Microsoft Word 万超_二校 75

Microsoft Word 万超_二校 75 8 1 Vol. 8 No. 1 2017 1 Journal of Food Safety and Quality Jan., 2017 PCR 万超 1, 蒋丹 1*, 吕泉 2, 丁健 1, 林长军 1 (1., 116001; 2., 115000) 摘要 : 目的 SYBR Green PCR SYBR Green PCR 方法 CoxⅠ, SYBR Green PCR PCR, DNA

Microsoft Word 王乃福-二校 出466

Microsoft Word 王乃福-二校出466