Key result
The multiplex PCR-based titration assay accurately identified and titrated all three Sabin poliovirus strains simultaneously in 2 to 3 days, showing excellent correlation with the conventional CCID50 assay.
Why the study?
Conventional poliovirus titration assays are time-consuming, laborious, unsuited for large numbers of clinical trial samples, and challenged by co-existing Sabin strains in Oral Poliovirus Vaccine.
The multiplex PCR-based titration assay provides a rapid, sensitive, and robust method for simultaneously identifying and titrating multiple infectious viruses in a mixture, significantly reducing assay time compared to conventional methods.
Answers are grounded in this paper and cite their sources.
Provides faster multiplex virus titration in preclinical work; leaves open clinical validation before broader use.
BACKGROUND: Conventional assays to titrate polioviruses usually test serial dilutions inoculated into replicate cell cultures to determine a 50% cytopathic endpoint, a process that is both time-consuming and laborious. Such a method is still used to measure potency of live Oral Poliovirus Vaccine during vaccine development and production and in some clinical trials. However, the conventional method is not suited to identify and titrate virus in the large numbers of fecal samples generated during clinical trials. Determining titers of each of the three Sabin strains co-existing in Oral Poliovirus Vaccine presents an additional challenge. RESULTS: A new assay using quantitative multiplex polymerase chain reaction as an endpoint instead of cytopathic effect was developed to overcome these limitations. In the multiplex polymerase chain reaction-based titration assay, cell cultures were infected with serial dilutions of test samples, lysed after two-day incubation, and subjected to a quantitative multiplex one-step reverse-transcriptase polymerase chain reaction. All three serotypes of poliovirus were identified in single samples and titers calculated. The multiplex polymerase chain reaction-based titration assay was reproducible, robust and sensitive. Its lower limits of titration for three Sabin strains were 1-5 cell culture 50% infectious doses per ml. We prepared different combinations of three Sabin strains and compared titers obtained with conventional and multiplex polymerase chain reaction-based titration assays. Results of the two assays correlated well and showed similar results and sensitivity. Multiplex polymerase chain reaction-based titration assay was completed in two to 3 days instead of 10 days for the conventional assay. CONCLUSIONS: The multiplex polymerase chain reaction-based titration (MPBT) is the first quantitative assay that identifies and titrates each of several different infectious viruses simultaneously in a mixture. It is suitable to identify and titrate polioviruses rapidly during the vaccine manufacturing process as a quality control test, in large clinical trials of vaccines, and for environmental surveillance of polioviruses. The MPBT assay can be automated for high-throughput implementation and applied for other viruses including those with no cytopathic effect.
No takes yet. Share an insight, caveat, or question.
Manukyan et al. (2019) studied Poliovirus titration. Multiplex PCR-based titration (MPBT) assay vs. Conventional CCID50 assay was evaluated on Correlation of virus titers between MPBT and CCID50 assays (R-squared 0.99-1.0). The multiplex PCR-based titration assay accurately identified and titrated all three Sabin poliovirus strains simultaneously in 2 to 3 days, showing excellent correlation with the conventional CCID50 assay.
Synapse has enriched 5 closely related papers on similar clinical questions. Consider them for comparative context: