Establishment And Application Of Real-Time PCR For Detection Of HCMV

Objective:To develop a Real-time PCR method for detecting human cytomegalovirus(HCMV)DNA based on TaqMan technology using a new TaqMan MGB probe.Methods and Results:1)Recombinant plasmid containing the amplified fraction of HCMV MIE exon 4 gene was constructed as HCMV standard for quantitative analysis.Since the MIE exon 4 gene of HCMV is highly conservative,the HCMV MIE exon 4 was chosen as target gene for primer design.Then a 201bp fragment was amplified by PCR technique.After purification, the gene fragment was linked with the pMD18-T vector to construct the recombinant plasmid(TA clone ).By PCR identification and DNA sequencing analysis confirmed that the construction of recombinant plasmid was successful.2)we established a new real-time PCR method and the reaction system was optimized.The optimal reaction condition was as follows:each 20μl PCR mixture contained 2μl of purified DNA,0.5μM concentration of each primer, and 1.5μM probe,2ul 10×buffer,2.0mM Mg²⁺,200μM dNTP and 1.0U Taq DNA...