Establishment And Application Of Real-time PCR For Detection Of HSV-2

Objective: To develop a Real-time PCR for detecting herpes simplex virus-2(HSV-2) DNA based on TaqMan technology using a new MGB probe.Methods and Results: 1). Plasmid containing the sequence of glycoprotein G (gG) gene wasconstructed as HSV-2 DNA standard for quantitative analysis. Since theHSV gG gene has the least homology between HSV-1 and HSV-2, the gGgene was chosen as target gene and we selected a highly conversationalfraction of gG gene in HSV-2 for primer design. Then a 160bp fragmentwas amplified by PCR technique. After purification, the gene fragment waslinked with the pMD18-T-vector to construct the recombinant plasmidpMD18-T-gG. By restriction enzyme digestion, PCR identification andDNA sequencing analysis confirmed that the construction of pMD18-T-gGwas successful. 2). we established a new detecting method and the reaction systemwere optimized. The better reaction condition was as follows: each30μl-PCR mixture contain 2μl of purified DNA, 600nM concentration ofeach primer, and 300nM probe, 3ul reaction buffer, 5.0mM Mg2+, 200μMdNTP. After 2 min of incubation at 50℃ and 10 min of incubation at 95℃for denaturation, the standard templet was subjected to 40 cycles ofPCR .Each cycle was 95℃ for 15 sec and 60℃ for 60 sec. 3). We have evaluated the assay through some experiments. Intra-assay coefficient of variation was 2.29% and inter-assay coefficient of variation was 4.76%, indicating good reproducibility of this method. A linear standard curve was obtained between4.75 × 101copies/μl and 4.75×106 copies/μl DNA (R>0.99). The assay has better specificity and can distinguish HSV-2 from other closely related pathogenic microorganism. 4). We conducted experiment to evaluate the Real-time PCR withELISA method. The Real-time PCR has higher sensitivity and specificitythan serodiagnosis method.Conclusion: 1).The recombinant plasmid pMD18-T-gG was successfullyconstructed. 2).We successfully developed a Real-time PCR for detection ofHSV-2, which was a suitable method for diagnosis of HSV-2.