124 河南农业科学第 42 卷, (HPS) ;PRRSV PCV2, [4] PRRSV CSFV ;20, PRRSV,CSFV [5-9],, 1.2 主要试剂和仪器 Real-timePCR PRRSV CSFV PCV2, 3 RotorGene, RG-3000; ND-1000 Na

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1 河南农业科学,2013,42(2): JournalofHenanAgriculturalSciences PCR PRRSV CSFV PCV2,, (, ) : 根据 TaqMan 复合荧光探针设计原则, 应用 Primer5.0 和 Oligo6.0 软件设计检测猪繁殖与呼吸综合征病毒 (PRRSV) 猪瘟病毒 (CSFV) 和猪圆环病毒 2 型 (PCV2) 的特异性引物和探针, 优化反应条件, 建立了检测 PRRSV CSFV 和 PCV2 的单项和多重 Real-timePCR 方法结果表明, 建立的方法具有高度特异性, 与其他病原检测无明显交叉反应 ; 对阳性质粒和病毒的最低检测量分别为 <10 1 个拷贝和 <1TCID 50 / 反应, 检测灵敏度比常规 PCR 检测高 100 倍通过对 20 份临床样本进行检测, 多重 Real-timePCR 检测结果和单项 Real-timePCR 检测结果一致建立的多重 Real-timePCR 方法可用于同时快速检测 PRRSV CSF 和 PCV2 的混合感染, 具有灵敏特异重复性好并能对样品进行定量检测等优点 : 猪繁殖与呼吸综合征病毒 ; 猪瘟病毒 ; 猪圆环病毒 2 型 ; 多重实时定量 PCR :S828 :A : (2013) DetectionofMixedInfectionofPRRSV,CSFVandPCV2 inswineby MultiplexReal-timePCR Assay ZHAO Xu-yong,NING Yu-chang,ZHAO Li,MA Hui (ZhengzhouColegeofAnimalHusbandryEngineering,Zhengzhou450011,China) Abstract:AccordingtothereportednucleotidesequencesofPRRSV,CSFVandPCV2,threepairs ofspecificprobesweredesigned.byoptimizingtheprimersandprobes concentration,amultiplex real-timequantitativepcrassayfordetectionoftheseviruseswasestablished.thespecificityand sensitivityofthemultiplexreal-timequantitativepcrassaywereanalyzedformultipleviruses. Theresultsshowedthatthespecificity washighandnonon-specificamplification wasachieved fromotherpathogens.bothmultiplexandsingleplexassayswereconsistentlyabletodetect<10 1 copiesofplasmidtemplatesor<1tcid 50virus/reaction.Atotalof20clinicalsampleswerecom- parativelydetectedusingthemultiplexandsingleplexassays.thedatashowedthattheresultsof multiplexassaywereinaccordancewiththatofsingleplexassay.conclusively,amultiplexreal- timepcrassaywasdeveloped,whichisrapid,sensitive,specificandaccuratetodiscriminatethe PRRSV,CSFVandPCV2infectioninasingletesttube. Keywords:PRRSV;CSFV;PCV2;multiplexreal-timePCR, (classicalswinefevervirus,csfv), 2 (porcinecircovirustype2,pcv2) (porcinerepro-, ductiveandrespiratorysyndromevirus,prrsv) [1-3] PRRSV PCV2, : : (2011A230016) : (1973-),,,,, Zhaoxy6868@163.com

2 124 河南农业科学第 42 卷, (HPS) ;PRRSV PCV2, [4] PRRSV CSFV ;20, PRRSV,CSFV [5-9],, 1.2 主要试剂和仪器 Real-timePCR PRRSV CSFV PCV2, 3 RotorGene, RG-3000; ND-1000 Nanodrop 毒株与菌种 CSFV (SIV) (PRV) PCR 1.3 引物的设计与合成 GenBank PRRSV CSFV PCV2, DNAStar, TaqMan, Primer5.0 Oligo6.0 3 (PPV) (JEV), BLAST (ETEC), 1 1 (5 3 ) /bp PRRSV-F PRRSV-R PRRSV-Probe CSFV-F CSFV-R CSFV-Probe PCV2-F PCV2-R PCV2-Probe TCAGCTGTGCCAGATGCTGG AAATGGGGCTTCTCCGGGTTTT FAM -TCCCGGTCCCTTGCCTCTGGA-ECLIPSE AGCTCCCTGGGTGGTCTAAGT CCCTCGTCCACATAGCATCT ROX-AGTTCGACGTGAGCAGAAGCCCACC-ECLIPSE GGATATTGTAGTTCCTGGTCG CCACTATTGATTACTTCCAACC HEX -TCGAACGCAGTGCCGAGGCC-ECLIPSE 77 AB AH AB 标准阳性模板的制备, PCV2 DNA ~ DNA TRIzol / μ L, -20 PRRSV CSFV RNA, OligodT Primer, PrimeScriptRTase 1.5 多重实时荧光定量 PCR 反应条件及参数设定 cdna PCV2 PRSV CSFV PCV2 Real-timePCR TaqDNA DNA [10-13], DL2000 Marker TRIzol ;2 TaqMan Uni- Real-time versalmaster Mix ABI ; PCR PRRSV CSFV PCV2 (E.coli)JM109 DH5α 3, ;PRRSV CSFV PCV2 PCR, 3 PCR DNA CSFV PRRSV cdna, Real-timePCR, PCR PCR, Real-timePCR pgem-t Easy, E.coliJM109, PCR 25μL, Real-, timepcr 12.5μL2 TaqMan Uni-

3 第 2 期赵绪永等 : 多重实时定量 PCR 快速检测 PRRSV CSFV 和 PCV2 混合感染方法的建立 125 versalmastermix 0.1~0.5μmol/L 1.8 临床样本的检测 0.2~0.6μmol/L,2μL, μl Real-timePCR, 3 DNA RNA, RNA cdna, : Real-timePCR 50 2min( UNG, PCR, ),95 10 min( UNG, AmpliTaqGoldpolymerase); 95 15s,55~60 45s,40, 2.1 多重 Real-timePCR 反应条件的优化, Real-timePCR 1.6 灵敏度和重复性试验, 0.4μmol/L, 0.3 PCR, μmol/l 50 2 min, min;95 15s,60 45s,40 ( TCID 50 ), 2.2 多重 Real-timePCR 标准曲线的建立 DNA, Real-timePCR RT-PCR PCR, Real-timePCR 3,, 特异性试验 JEV SIV PRRSV CSFV cdna ( ) Real-timePCR ( ), 1 ETEC HPS PPV PRV PCV2 DNA , Real-timePCR, (R 2 ) 0.99, 2 Real-timePCR, :,,PCR,2, 2.3 敏感性试验结果 1 PRRSV CSFV PCV2 Real-timePCR,, PCR Real-timePCR 10 1 / , PCR, 2 3 PRRSV ( TCID 50 /ml) CSFV ( TCID 50 /ml) 2 PCV2 ( TCID 50 /ml) DNA 10-3, PCR 1 TCID 50, PCR 100 DNA Real-timePCR / Real-timePCR ( /%) NoCt (13) (7) NoCt (11) (6) NoCt (6) (9) (8) NoCt (12)

126 河南农业科学第 42 卷 2 DNA Real-timePCR / Real-timePCR ( /%) 5 10 2 0 10 4 5.8 10 1 (24) NoCt 8.2 10 3 (9) 6 10 5 0 10 2 7.4 10 4 (15) NoCt 7.8 10 1 (13) 7 10 3 10 1 0 8.0 10 2 (10 ) 7.

4 126 河南农业科学第 42 卷 2 DNA Real-timePCR / Real-timePCR ( /%) (24) NoCt (9) (15) NoCt (13) (10 ) (8) NoCt (15) (5) NoCt (18) (8) NoCt (18) (7) (38) (20) (25) (20) (9) (3) (15) (13) (23) (4) (28) (7) (16) 2.4 重复性试验结果, 3, 3,, (10 1 / ),, (CV) 16% 22%,, 10%,, 2.5 特异性试验结果 A-C.PCV2 PPRSV CSFV;D-I.SIV JEV HPS ETEC PPV PRV Real-timePCR 2.6 临床样本的检测结果, 2 PCV DNA CSFV PRRSV cdna Real-timePCR, 3 3, 40 PCR, Real-timePCR Real-timePCR, PRRSV CSFV PCV2 3 Real-timePCR /( / μ L) 2 Real-timePCR /( / μ L) PCR PRRSV/CSFV/PCV /-/ /-/ /-/ /+/ /-/ /-/ /-/ /-/ /+/ /+/ /+/ /+/ /-/ /-/ /+/ /-/ /+/ /-/ /+/ /-/+

5 第 2 期赵绪永等 : 多重实时定量 PCR 快速检测 PRRSV CSFV 和 PCV2 混合感染方法的建立 % 40% 70%;,, PCR,PCV2 PRRSV PCV2 CSFV PRRSV CSFV PRRSV CSFV PCV ,,PCV2, PRRSV CSFV3 2 ; 1, 3, 10 3 ~10 5 / ; :,, ( ) [1],. [M].2. : 3,1997: [2] Joaquim S,Gordon M,Alan G M.Porcinecircovirus, diseases[j].anim Health ResRev,2005,6(2):119-, 142., [3],,,. [J].,2004,34 PCV2 CSFV PRRSV, (7):40-43., [4] DroletR,LarocheleR,Morin M,etal.Detectionrates PCR ofporcinereproductiveandrespiratorysyndromevi- rus,porcinecircovirustype2andswineinfluenzavirus ELISA, inporcineproliferativeandnecrotizingpneumonia[j]. VetPathol,2003,40(2): Real-timePCR [5],,,. N, PRRSV ELISA [J].,2010,27(12):40-44.,, PCR [6],,,. RT-PCR, [J].,2009(1): [7],,,. 2 SPA [J].,, G Tm ,36(7): ,, [8],,,. NS3, ELISA [J].,2010,37(1):78-84., PCR [9],,.PCR PCV2 PRRSV CSFV [J]., FAM HEX ROX3, 2010,30(2): [10],,.,, PCR [J].,2009,31(4): Real-timePCR, [11],,. 2, 0.99,PCR PCR [J].,2011,32(2): %, [12],,,.PRRSV PCV2 PRV, SYBR Green-Ⅰ PCR < 10 1 < [J].,2008,38(6): [13] Tian H,WuJY,ShangYJ,etal.Thedevelopmentof 1TCID 50 / arapidsybronestepreal-timert-pcrfordetec- 10%, 20 tionofporcinereproductiveandrespiratorysyndrome, Real-timePCR virus[j].virologyjournal,2010,7:90. Real-timePCR [14] KleiboekerSB,SchommerSK,LeeS M,etal.Simul- taneousdetectionof North Americanand European,PCV2, 70%, porcinereproductiveandrespiratorysyndromevirus [14-15], usingreal-timequantitativereversetranscriptase-pcr, [J].Journalof Veterinary DiagnosticInvestigation, 2005,17: ,, [15],,,. 2, [J].,2006,36 (2):

荧光定量PCR

荧光定量PCR 荧光定量 PCR 荧光定量 PCR 原理荧光定量 PCR 最早称 TaqMan PCR, 后来也叫 Real-Time PCR, 是美国 PE(Perkin Elmer) 公司 1995 年研制出来的一种新的核酸定量技术该技术是在常规 PCR 基础上加入荧光标记探针或相应的荧光染料