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Establishment of a cell‐based quantitative reverse transcription‐polymerase chain reaction (RT‐qPCR) assay for detection of multivalent rotavirus vaccine
Author(s) -
Wang Yunjin,
Liu Yueyue,
Wang Mingqiang,
Yu Li,
Ma Chao,
Li Xiongxiong,
Guo Tai,
Bao Hong,
Kou Kuiying,
Chen Yueru,
Gong Hanbo,
Zhou Xu
Publication year - 2020
Publication title -
journal of medical virology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.782
H-Index - 121
eISSN - 1096-9071
pISSN - 0146-6615
DOI - 10.1002/jmv.26128
Subject(s) - rotavirus , taqman , virology , serial dilution , titer , potency , biology , reverse transcriptase , rotavirus vaccine , vero cell , real time polymerase chain reaction , plaque forming unit , reverse transcription polymerase chain reaction , polymerase chain reaction , microbiology and biotechnology , virus , cell culture , in vitro , messenger rna , gene , medicine , biochemistry , alternative medicine , genetics , pathology
Abstract Because of deficiencies of traditional potency tests in rotavirus detection, a one‐step TaqMan probe‐based quantitative reverse transcription‐polymerase chain reaction (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. 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 (lgCCID 50 /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. The results showed that messenger RNA 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 cell culture infective dose 50% with a relative deviation less than 5%. 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.