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 Mg2+,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 102 copies/μl and 108 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.