Type inactivated bivalent hantavirus vaccines using dual fluorescence quantitative real-time PCR: Establishment and evaluation.

To establish a dual fluorescence quantitative real-time polymerase chain reaction (DFqRT-PCR) method for typing inactivated bivalent vaccines of Hantaan virus (HTNV, type I) and Seoul virus (SEOV, type II) found in China. Primers and probes were designed based on S segment of the Hantavirus. FAM and HEX were added to the probes for the type I and type II vaccines, respectively. The S fragments were cloned into the pMD18-T vector to create pMD18-T/HTNV and pMD18-T/SEOV plasmids, respectively. The DFqRT-PCR reaction conditions were optimized. The specificity, sensitivity, accuracy, and reproducibility of the DFqRT-PCR method within and among batches of the samples and labs were evaluated. The sensitivity of the DFqRT-PCR method for detecting type I and II vaccines was 4.68 × 101 copies/µL and 5 × 101 copies/µL of pMD18-T/HTNV and pMD18-T/SEOV, respectively. This method distinguished the hantavirus vaccines from several other virus genus and distinguished the type I and II vaccines from each other with coefficients of variation (CVs) of the intra- and inter-batch assay of 0.19-0.66% and 0.09-0.69%, respectively. For bivalent inactivated vaccine, the CVs of the intra- and inter-batch assays, and the intra- and inter-laboratory assays were 0.06-1.82% for the samples from the same batches and different batches from the same manufacturer or the samples from the different manufacturers. The DFqRT-PCR method was simple, fast, and sensitive for identifying and determining the inactivated hantavirus vaccine with high accuracy and reproducibility. Therefore, it can be used for quality control of hantavirus vaccines. J. Med. Virol. 89:10-16, 2017. © 2016 Wiley Periodicals, Inc.