荧光定量PCR检测人巨细胞病毒的方法学建立

目的建立人巨细胞病毒（HCMV）的TaqMan MGB探针荧光定量PCR（FQ—PCR）检测方法。方法选取HCMV MIE exon4为PCR扩增靶序列，经TA克隆构建重组质粒作为定量标准品，经FQ—PCR反应条件的优化及方法学评价，再将其应用于临床检测。结果FQ—PCR最适循环参数为：95℃ 5 min；95℃ 20 s，60℃ 60 s（40 cycles），20μl最适反应体系为：2．0mmol／L Mg^2＋、0．5μmol／L引物、1．5μmol／L探针、200μmol／L dNTP、2110×buffer、1．0 U Taq酶、2．0μl DNA模板。检测批内CV（变异系数）值为1．32％，批间CV值为1．96％；特异性较好；线性范围为10^2-10^8copies／μl。结论成功地建立了检测HCMV的FQ—PCR法，完全适用于临床检测。

Methodological establishment of fluorescent quantitative PCR for detection of human cytomegalovirus

Objective To establish fluorescent quantitative PCR （FQ-PCR） with Taq Man MGB probe for detection of human cytomegalovirus （HCMV）. Method HCMV MIE exon 4 was chosen as the target gene for primer design. The gene fragment was linked with the pMD18-T vector to construct the recombinant plasmid as HCMV standard for quantitative analysis. FQ-PCR was constructed through optimizing the reaction system and cycling parameter. The new method was then evaluated. Result The optimal reaction condition of FQ-PCR was as follows：each 20 μl PCR mixture contained 2 μl of purified DNA,0.5 μmol/L concentration of each primer,and 1.5 μmol/L probe,2 μl 10 × buffer,2.0 mmol/L Mg^2＋ , 200 μmol/L dNTP and 1.0 U Taq DNA polymerase. After 5 min at 95 ℃ for predenaturation, the DNA template was subjected to 40 cycles of PCR. Each cycle was at 95 ℃ for 20 s （denaturation） and 60 ℃ for 60 s （ annealing and extension）. Intra-assay coefficient of variation was 1.32% and inter-assay coefficient of variation was 1.96% ; the range of linear assay was obtained between 10^2 copies/μl and 10^8 copies/μl. Conclusion Real-time PCR method was successfully developed for detection of HCMV, which was a suitable method for early diagnosis and monitoring therapeutic effect of HCMV disease.