POCT technology has shown promise for rapid diagnosis. POCT nanoparticle-based immunoassays can be used to detect different targets with high sensitivity, selectivity, and affordability.

Background: Because of the deficiencies of traditional methods in multivalent rotavirus vaccine potency detection, a cell‐based quantitative RT‐qPCR assay (C‐QPA) was established and validated for specificity, precision, and accuracy.Methods: In order to further validate the robustness of this method in actual titer detection, the linear range and the practical application under different conditions were tested using monovalent and trivalent rotavirus samples and standards.Results: Results showed that the linear range was 2.0–6.5, 3.9–8.3, and 3.5–8.1 UI (unit of infectivity) for G2, G3, and G4, respectively. Besides, unknown sample with high titer exceeding the linear range can be calculated by dilution. The UIs of serotypes G2, G3, and G4 in monovalent and trivalent rotavirus samples showed a relative deviation ≤4.10%, and the monovalent samples of the same serotype with or without protective agents showed a relative deviation ≤4.28%; the coefficient of variation (CV) of at least 176 tests (548 individual runs) of 3 in vitro‐transcribed RNA standards with certain concentrations was not higher than 6.50%; the results of the trivalent samples tested by more than 149 times in 5 years (467 individual runs) showed the CVs lower than 12.66%; 15 samples detected by one laboratory showed a CV lower than 9.83%, while other three samples tested by two independent laboratories showed a CV lower than 6.90%.Conclusion: In summary, the C‐QPA has good linearity, durability, repeatability, and reproducibility in practical application and has been proved by the authority to be widely used in the production, quality control and release of the recently licensed trivalent vaccine in China.