犬细小病毒TaqMan MGB荧光定量PCR检测方法的建立及应用

Establishment and Application of TaqMan MGB Fluorescent-Quantitative PCR Assay for Detection of Canine Parvovirus

为建立一种特异、灵敏、快速检测犬细小病毒(CPV)的TaqMan MGB荧光定量PCR(FQ-PCR)方法,本研究根据GenBank中CPV的VP2基因保守区域序列设计1对特异性引物和1条TaqMan MGB荧光探针,经反应条件优化,建立了CPV TaqMan MGB FQ-PCR方法。对建立的TaqMan MGB FQ-PCR检测方法进行了灵敏度、特异性、重复性试验,对疑似CPV感染临床样品进行了应用检测,并与常规PCR方法进行了比对。结果表明,成功建立了检测CPV的TaqMan MGB FQ-PCR方法,标准曲线的循环阈值(Ct值)与模板浓度有良好的线性关系,相关系数(R2)为0.9978;最低检出限为1×101拷贝/μL,是常规PCR的100倍;特异性高,对pGEM-T/CPV重组质粒扩增呈现阳性反应曲线,而对5个对照病原的扩增曲线均呈现阴性反应;对不同浓度的pGEM-T/CPV重组质粒分别重复检测3次结果良好;对46份临床疑似CPV感染样品进行了应用检测,阳性检出率为50%,高于常规PCR方法。本研究成功建立了CPV的TaqMan MGB FQ-PCR检测方法,可用于临床上犬细小病毒病的早期快速诊断。

In order to establish a TaqMan MGB fluorescent-quantitative PCR （FQ-PCR） assay for detecting canine parvovirus （CPV） specifically, sensitively and rapidly, a highly sensitive and specific TaqMan MGB FQ-PCR assay was developed using the specific primers and TaqMan MGB probe designed basing on the conservative sequences of VP2 gene of CPV in GenBank. The sensitivity, specificity and repetition assay of FQ-PCR assay were tested, and 46 clinic suspicious CPV infected samples were detected by the FQ-PCR assay in contrast to the routine PCR method. The results indicated that the FQ-PCR was successfully established. The developed FQ-PCR assay was able to detect as little as 1 × 10^1 copies/μL of recombinant pGEX-T/ CPV plasmid DNA, and the sensitivity of which was 100 times more than that of the routine PCR. The specificity assay exhibited that positive signals could be obtained from recombinant pGEM-T/CPV plasmid, but not from the genomic DNA or total cDNA of the other 5 kinds of pathogenic microorganism acting as the controls. The repetition tests were carried out by detection repeated 3 times for 3 different concentrations of recombinant pGEX-T/CPV plasmid, and the results indicated that the FQ-PCR was reproducible. Twenty-three positive results from 46 clinic suspicious CPV infected samples were obtained, which showed the better sensitivity than that of the routine PCR, with 19 positive samples from the same 46 suspected samples. The study suggested that the CPV FQ-PCR method was successfully established, and suitable for clinic rapid diagnosing of CPV and early detection of latent infection.