猪圆环病毒2型TaqMan实时荧光定量PCR检测方法的建立

Establishment of TaqMan Real-time Quantitative PCR Method for Detection of PCV2

本研究以猪圆环病毒2型(PCV2)核衣壳蛋白ORF2基因为目的基因,设计合成特异性引物和探针。PCR扩增得到目的基因,并克隆到pMD18-T载体,筛选得到标准阳性质粒。通过对荧光定量PCR反应条件的优化,建立了PCV2的TaqMan实时荧光定量PCR检测方法。试验结果表明,该方法特异性强,对猪伪狂犬病病毒(PRV)、猪繁殖与呼吸综合征病毒(PRRSV)、猪瘟病毒(CSFV)等猪常见病原的检测结果均为阴性;与普通PCR方法相比,其敏感性高出100倍,可达100拷贝/μL;对临床样品的检测证明,该方法可有效检测出淋巴结、肺脏等组织中的PCV2。

The research with porcine circovirus type 2 （PCV2） of the ORF2 capsid protein gene for the purpose, design and synthesis of specific primers and probes. Gene was amplified by PCR and cloned into the pMD18-T vector, screened positive plasmid standards. By quantitative PCR optimization of reaction conditions, establishment of a real-time detection of PCV2 Taq Man quantitative PCR method. The results showed that the method was specificity, the pseudorabies virus （PRV）, porcine reproductive and respiratory syndrome virus （PRRSV）, swine fever virus （CSFV） and other common diseases of the original pig detection results were negative. Compared with ordinary PCR method, the sensitivity was 100 times higher, up to 100 cop- ies/μL; for the detection of clinical samples showed that the method can effectively detect lymph nodes, lungs and other tissues of PCV2.