猪圆环病毒2型特异性荧光定量PCR检测方法的建立与应用

Development and Application of a Specific Fluorescent Quantitative PCR Assay for the Detection of Porcine Circovirus Type 2

研究旨在建立基于SYBR Green I荧光定量PCR检测PCV 2的技术方法。根据GenBank中的猪圆环病毒2型(PCV 2)的基因序列(HQ148879),设计一对特异性引物,利用普通PCR技术扩增出PCV 2的ORF2基因702bp片段,并克隆到pVAX1载体,以纯化的质粒作为PCR检测的标准模板,以10倍梯度稀释质粒为阳性标准品,摸索荧光定量PCR试验,并绘制标准曲线,以此建立一种荧光定量PCR特异性检测猪圆环病毒的方法。结果表明,该方法对PCV 2有很好的特异性,其敏感性比常规PCR高100倍,批间与批内重复试验变异系数均小于2.5%。经临床应用表明荧光定量PCR方法的建立实现了对PCV 2特异性的快速诊断和定量检测。

A fluorescent quantitative PCR based on SYBR Green I was developed for the detection of porcine circovirus type 2.A pair of primers were designed specific for PCV 2 according to the published nucleotide sequence（GenBank accession No.HQ148879）.Using PCR assay,a 702 bp region of the PCV 2 ORF2 gene was cloned into pVAX1 vector.Serial dilutions of plasmid PCV 2 were used to quantify the virus genomic copy number,and served as standard curve in the fluorescent quantitative PCR for development of a specific assay for the detection of porcine circovirus type 2.The results showed that the fluorescence quantitative PCR assay was able to detect PCV 2.The sensibility of this assay attained 10 copies of plasmid DNA,which was 100 times higher than routine PCR.The C.V.of the intra or inter was no more than 2.5%,indicating that the developed fluorescence quantitative PCR is a stable assay.The clinical detection showed the development of the method achieved the effct of rapid diagnosis and quantitative detection of PCV 2.