EVALUATION OF A REAL-TIME PCR ASSAY FOR DETECTION AND QUANTIFICATION OF RUBELLA VIRUS IN MMR VACCINE

Background and Aim:Rubella virus is the causative agent of rubella, a mild illness that may lead to fetal death or congenital defects when the virus infects pregnant women in early pregnancy. Rubella vaccination has been safe and really effective. Potency of rubella-containing vaccine (MMR) is conventionally measured by two assays: CCID50 and plaque forming unit (pfu). These two are labor intensive and time consuming. Quantitative Real-time PCR has been introduced as an easily available and fast method to estimate the potency of live-attenuated viral vaccines.Methods:Primers and probe were designed for RT-qPCR system. A 400-base pair fragment was amplified using another set of primers. The fragment was then cloned into pTZ57R/T plasmid by T/A cloning technique. Serial 10-fold and 2-fold dilutions of recombinant plasmids were used to generate standard curves. RK-13 cells were inoculated with vaccine virus. After 2 days of virus inoculation, rubella virus RNA was extracted and RT-qPCR was performed. The results of Real-Time PCR were compared with those of CCID50.Results:Standard curves were evaluated for parameters such as linearity and precision. Inter-assay CV and intra-assay CV were determined to be below 2%. Moreover, limit of detection was defined as 35 copies/reaction and quantification as 70 copies/reaction for the test. The results indicated that the titers estimated by RT-qPCR were comparable to CCID50 and statistical analysis showed promising results with p-value> 0.05.Conclusion:Real-Time PCR has the ability to estimate virus titer in vaccine preparations. It is relatively inexpensive, rapid and repeatable.

کلیدواژه ها:

Rubella virus ، MMR vaccine ، Real-Time PCR