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Quantitation of intracellular viral genomes is critical in both clinical and fundamental virology. Quantitative real time PCR (qPCR) is currently the gold standard to detect and monitor virus infections, due to its high sensitivity and reproducibility. The reliability of qPCR data depends primarily on the technical process. Normalization, which corrects inter-sample variations related to both pre-analytical and qPCR steps, is a key point of an accurate quantitation. Total DNA input and qPCR-measured standards were evaluated to normalize intracellular Vaccinia virus (VACV) genomes. Three qPCR assays targeting either a single-copy chromosomic gene, a repeated chromosomic DNA sequence, or a mitochondrial DNA sequence were compared. qPCR-measured standards, unlike total DNA input, allowed for accurate normalization of VACV genome, regardless of the cell number. Among PCR-measured standards, chromosomic DNA and mitochondrial DNA were equivalent to normalize VACV DNA and multi-copy standards displayed lower limits of quantitation than single-copy standards. The combination of two qPCR-measured standards slightly improved the reliability of the normalization. Using one or two multi-copy standards must be favored for relative quantitation of intracellular VACV DNA. This concept could be applied to other DNA viruses. |
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