| 摘要: |
| 【目的】建立一种能在临床上快速、准确检测猪圆环病毒Ⅱ型 (PCV-2)的TaqMan实时荧光定量PCR方法。【方法】根据GenBank中已公布的PCV-陕西分离株的全基因序列，在PCV-2的ORF2核苷酸序列的保守区域，设计合成1条TaqMan探针和1对特异性引物；利用设计的引物扩增ORF2部分基因并鉴定后，回收PCR扩增产物，连接pMD19-T载体，转化DH5α大肠杆菌，涂布Amp+琼脂平板，挑取阳性克隆进行PCR及测序鉴定后提取质粒，作为荧光定量PCR的标准品；以标准品为模板建立检测PCV-2的TaqMan实时荧光定量PCR方法，并对该方法的灵敏度、稳定性和特异性进行评价；用建立的荧光定量方法对56份临床样品进行检测，并将检测结果与传统PCR方法进行比较。【结果】建立了PCV-2的TaqMan实时荧光定量PCR检测方法，其标准曲线的斜率为-3.383，R2=0.998，具有良好的线性关系；重复性试验结果表明，该方法循环数阈值Ct的变异系数(CV)为0.86%～1.02%，具有良好的稳定性；敏感性检测结果表明，该方法的最低检测限度为5.06 copies/μL；特异性检测结果显示，该方法对猪圆环病毒Ⅰ型(PCV-1)、伪狂犬病病毒(PRV)、猪繁殖与呼吸综合征病毒(PRRSV)和猪瘟病毒(CSFV)的检测结果均为阴性，对PCV-2标准品的检测结果为阳性，说明该方法具有较好的特异性。临床样品的检测中，所建立的TaqMan实时荧光定量PCR检测方法的检出阳性率较传统PCR方法高14.3%。【结论】成功建立了PCV-2的TaqMan实时荧光定量PCR检测方法，该方法可用于临床上对PCV-2的检测。 |
| 关键词: 猪圆环病毒Ⅱ型 TaqMan荧光定量PCR 病毒检测 |
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| 基金项目:陕西省“13115”科技创新工程重大科技专项(2010ZDKG-71)；西北农林科技大学博士科研启动费项目(2010BSJJ010) |
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| Establishment and application of TaqMan real-time fluorescent quantitative PCR for detecting porcine circovirus typeⅡ |
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| Abstract: |
| 【Objective】The purpose of this study was to establish a TaqMan real-time fluorescence quantitative PCR method which can detect porcine circovirus typeⅡ quickly and accurately in clinic.【Method】According to the published complete genome sequence of PCV-2 isolate SX-1 in GenBank,a TaqMan probe and a pair of primers were designed and synthesized for the conversed open reading frame 2 (ORF2) of PCV-2.After the gene fragment was amplified using designed primers,the amplified product was identified.Recovery of PCR amplified products,connection of pMD19-T vector,transformation of E.coli DH5α,and coating of Amp+agar plates were conducted and the positive clones were picked for PCR and sequencing identification.Then the plasmids were extracted as the standard plasmid of fluorescent quantitative PCR.The standard plasmid was used as a quantitative template to establish the TaqMan real-time fluorescence quantitative PCR method for PCV-2 detecting,and sensitivity,stability and specificity of the method were evaluated.56 clinical samples were detected with the established method and their results were compared with conventional PCR.【Result】The TaqMan real-time fluorescence quantitative PCR method was established and the correlation coefficient (R2) and the slope of the standard curve were 0.998 and -3.383 respectively,which showed a good linear relationship.Repeatability tests showed that the threshold cycle of the method had very good stability with the coefficient of variations (CV) between assays within 0.86%-1.02%.Results of sensitivity tests showed that the detection limit was 5.06 copies/μL.Specificity tests showed that the detection results of porcine circovirus typeⅠ,pseudorabies virus,porcine reproductive and respiratory syndrome virus,and class swine fever virus samples were negative while that of the standard PCV-2 sample was positive,indicating a good specificity.In clinical samples tests,detection rate of established fluorescence PCR was 14.3% higher than that of conventional PCR.【Conclusion】This study established a TaqMan real-time fluorescence quantitative PCR method which can detect PCV-2 and be used to detect the clinical PCV-2 samples. |
| Key words: porcine circovirus typeⅡ TaqMan fluorescence quantitative PCR virus detection |
