Establishment of a cell‐based quantitative reverse transcription‐polymerase chain reaction (RT‐qPCR) assay for detection of multivalent rotavirus vaccine

Abstract

Background Because of deficiencies of traditional potency tests in rotavirus detection, a one‐step TaqMan probe‐based quantitative RT‐qPCR assay combined with cell‐based method was established to determine the infectious potency of the target virus in multivalent live rotavirus vaccines in vitro . Methods Series dilutions of rotavirus samples were inoculated into Vero cells and cultured for 24 hours. The cells were lysed and the potency was detected by RT‐qPCR. The reference standards with a known titer (lgCCID₅₀/ml) were assayed in parallel, and the potencies of each sample were determined using parallel line method. The specificity, precision and accuracy of the assay were evaluated, respectively. Results The results showed that mRNA produced during rotavirus replication was the primary template of RT‐qPCR and the primers and probes were specific to each strain. The coefficient of variation of different wells and different working days did not exceed 6% and the results of the assay were proved to be concordant with those of CCID₅₀ with a relative deviation less than 5%. Conclusion This assay is a more rapid, cost‐effective and high‐throughput way for detecting multivalent rotavirus vaccine, and will be a valuable tool in the quality control and stability monitoring of live multivalent rotavirus vaccine.