Method Validation for Determination of Porcine Parvovirus in Cell Lines used for Vaccine Production by Real-time Polymerase Chain Reaction (Real-time PCR)
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Abstract
Background: Cell lines are crucial for the production of vaccines and other biological products. Detecting adventitious viruses in these cell lines is a critical requirement set by the World Health Organization (WHO) to prevent misidentification and cross-species contamination.
Objectives: This study aimed to develop and validate a method for detecting Porcine parvovirus (PPV) contamination in cell cultures used for vaccine production using Real-time PCR and to implement this method as a standard procedure for quality control of cell cultures in vaccine production.
Methods: This experimental research studied from November 2021 to October 2022 that investigated the optimal conditions for a technique to analyze the quantity of virus from pigs in cell cultures used for vaccine production using Real-time PCR. It also examined the method's accuracy in terms of parameters such as linearity and analytical range, specificity, precision, accuracy, minimum detectable amount, and sensitivity.
Results: The results showed that the designed primers and probes effectively amplified the target genes of PPV without cross-reacting with other viruses. The standard curve demonstrated linearity with a working range between 8 and 4 log10 copies/µl and an R² value exceeding 0.9996. The coefficient of variation (CV) for intra-day, inter-day, and inter-analyst reproducibility was within the acceptable range of less than 25%. Accuracy analysis showed %CV of 0.510% and %recovery ranging from 101.39% to 100.66%. The limit of detection (LOD95%) was established at 1,857 copies/µl. The Real-time PCR assays exhibited high sensitivity, detecting PPV at a concentration of 7.5 TCID50/ml or 4 log10 copies/µl in PPV-infected ST cells.
Conclusions: The findings indicated that the developed method was effective and reliable for detecting Porcine parvovirus in cell lines used for vaccine production.
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