河南农业科学,2013,42(2):123-127 JournalofHenanAgriculturalSciences PCR PRRSV CSFV PCV2,, (, 450011) : 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 :1004-3268(2013)02-0123-05 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, :2012-09-19 : (2011A230016) : (1973-),,,,, E-mail:Zhaoxy6868@163.com
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 1 1.1 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 AB546125 93 AH012834 131 AB246193 1.4 标准阳性模板的制备, PCV2 DNA 0.5 10 7 ~ DNA TRIzol 0.5 10 1 / 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-
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, 25 20 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,95 3 10min;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,, 3 1.7 特异性试验 JEV SIV PRRSV CSFV cdna ( ) Real-timePCR ( ), 1 ETEC HPS PPV PRV PCV2 DNA 2-3.32, 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 / 10-5 10-4 10-5, PCR, 2 3 PRRSV (10-4.45 TCID 50 /ml) CSFV (10-3.78 TCID 50 /ml) 2 PCV2 (10-4.83 TCID 50 /ml) DNA 10-3, PCR 1 TCID 50, PCR 100 DNA Real-timePCR / Real-timePCR ( /%) 1 0 10 2 10 5 NoCt 1.2 10 2 (13) 8.4 10 4 (7) 2 0 10 1 10 7 NoCt 4.0 10 0 (11) 1.5 10 6 (6) 3 0 10 7 10 2 NoCt 8.5 10 6 (6) 7.0 10 1 (9) 4 10 4 0 10 1 7.5 10 3 (8) NoCt 1.2 10 1 (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 ) 7.8 10 0 (8) NoCt 8 10 6 10 3 0 1.2 10 6 (15) 8.8 10 2 (5) NoCt 9 10 1 10 4 0 6.0 10 0 (18) 7.4 10 3 (8) NoCt 10 10 1 10 6 10 3 7.0 10 0 (18) 9.2 10 5 (7) 3.5 10 3 (38) 11 10 2 10 6 10 6 7.5 10 1 (20) 7.0 10 5 (25) 7.5 10 5 (20) 12 10 5 10 3 10 1 7.8 10 4 (9) 9.0 10 2 (3) 1.1 10 1 (15) 13 10 2 10 1 10 4 7.0 10 1 (13) 9.0 10 0 (23) 8.8 10 3 (4) 14 10 1 10 6 10 1 5.0 10 0 (28) 6.8 10 5 (7) 7.0 10 0 (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 PCV2 20 3 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/PCV2 1 1.2 10 3-2.4 10 5 1.4 10 5-2.6 10 5 +/-/+ 2 - - 3.4 10 6 - - 4.0 10 6 -/-/+ 3 7.6 10 4 - - 7.1 10 4 - - +/-/- 4-3.1 10 5 6.2 10 2-3.6 10 5 6.8 10 2 -/+/+ 5 2.8 10 1-7.5 10 5 4.0 10 1-7.9 10 5 +/-/+ 6 - - 5.2 10 4 - - 5.1 10 4 -/-/+ 7 4.7 10 5-6.4 10 1 5.6 10 5-1.1 10 2 +/-/+ 8 6.3 10 3 - - 3.4 10 3 - - +/-/- 9-5.7 10 4 2.1 10 2-7.5 10 4 4.7 10 2 -/+/+ 10 5.3 10 3 4.5 10 4 6.5 10 2 6.3 10 3 4.6 10 4 6.8 10 2 +/+/+ 11-7.4 10 3 - - 8.3 10 3 -/+/- 12 5.9 10 3 6.4 10 5-5.6 10 3 7.4 10 5 - +/+/- 13 - - 9.7 10 4 - - 1.1 10 5 -/-/+ 14 1.8 10 4-7.7 10 3 2.2 10 4-6.9 10 3 +/-/+ 15-2.4 10 3 - - 3.1 10 3 - -/+/- 16 - - - - - - -/-/- 17 5.7 10 3 7.4 10 3 5.5 10 2 6.8 10 3 5.7 10 3 6.1 10 2 +/+/+ 18 1.0 10 1-8.5 10 4 2.5 10 1-7.9 10 4 +/-/+ 19-3.8 10 4 2.6 10 2-4.6 10 4 3.3 10 2 -/+/+ 20 - - 8.4 10 3 - - 1.1 10 4 -/-/+
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