Establishment of real-time quantitative TaqMan PCR assay for the detection of tick-borne encephalitis virus / 解放军医学杂志

ABSTRACT

Objective To develop a real-time quantitative PCR(RQ-PCR) assay based on TaqMan technology for rapid detection and quantification of tick-born encephalitis virus (TBEV) RNA. Methods According to all the TBEV genome sequences in GenBank, RQ-PCR primers and probes were designed in the conservative regions of TBEV C gene and NS5 gene. In addition, primers for conventional PCR were designed using E gene as target. The detective system was established and validated by using TBEV MDJ01 strain. In order to examine the specificity of the system, other viruses of flavivirus were assayed with the RQ-PCR simultaneously. The TBEV standard curve was drawn respectively by measuring TCID_ 50 titre and copy number. The sensitivity of RQ-PCR and the conventional PCR assays were compared, and TBEV infected mice model was reproduced for evaluation. Results The sensitivity of RQ-PCR assay was 100copies/reaction or 1 TCID_ 50 , which was 10 fold higher than conventional PCR. The results were all negative when used to detect other flavivirus including the yellow fever virus, dengue virus type 1, 2, 3 and 4, Japanese encephalitis virus, West Nile virus. The coefficient of variability was less than 5% from inter- and intra-assay showing that both the repeatability and stability of the system were good. Conclusion A sensitive, specific and convenient RQ-PCR method has been established, which is valuable for early detection of TBEV.