ObjectiveTo establish reference substance for virus titer detection of oral hexavalent reconstituted rotavirus attenuated live vaccines (Vero cell), and to determine the range and stability of the reference.

MethodsReference substances for virus titer detection of 6 rotavirus serotypes were prepared with rotavirus stock solution validated in phase Ⅲ clinical trials. A comprehensive evaluation of new and old reference substances was conducted using fluorescence focus assay, and the stability of each serotype reference under different conditions was monitored.

ResultsThe viral titer calibration ranges for serotypes G1, G2, G3, G4, G8, and G9 rotavirus working references were 7.5-8.7, 6.4-7.8, 6.6-7.8, 6.4-7.6, 7.1-8.3, and 7.2-8.4 lg fluorescent focus units/mL, respectively. The coefficients of variation（CVs）of the titer results for each serotype of the same sample were <5% and not statistically significantly different when titered against the old and new references. The working reference samples for all 6 serotypes of rotavirus had good stability when stored at 2-8 ℃ and 25 ℃ for 1, 3, 7 days, or after 2 freeze-thaw cycles. Storage at 37 ℃ for 3-7 days had no impact on the virus titer. The stability was good after storage at -60 ℃ for 12 months with CV between tests <5%.

ConclusionThe established and range-determined reference material for rotavirus vaccine titer has good stability under different conditions and can ensure the consistency of test results.