猪圆环病毒Ⅱ型TaqMan实时荧光定量PCR检测方法的建立与应用

Establishment and application of TaqMan real-time fluorescent quantitative PCR for detecting porcine circovirus typeⅡ

【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-2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5.06copies/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Taq-Man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。

[Objective] The purpose of this study was to establish a TaqMan real-time fluorescence quantitative PCR method which can detect porcine circovirus type Ⅱ quickly and accurately in clinic. [Meth- od] 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 identi- lied. Recovery of PCR amplified products,connection of pMD19-T vector,transformation of E. coli DH5a, and coating of Amp＋agar plates were conducted and the positive clones were picked for PCR and sequen- cing 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 fluores- cence 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 com- pared 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 type Ⅰ ,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 estab- lished fluorescence PCR was 14.3 % higher than that of conventional PCR. [Conclusion] This study estab- lished a TaqMan real-time fluorescence quantitative PCR method which can detect PCV-2 and be used to detect the clinical PCV-2 samples.