Comparative Assessment of Different Nucleic Acid Templates for Use as Standards in the qPCR‐Based Quantification of Virus Genomes

Quantitative PCR (qPCR) employing either non-specific dyes or gene-specific dye-labelled probes is a commonly used technique for the quantification of viral nucleic acids. While commercial kits often come equipped with their own proprietary standards, many research endeavours necessitate the preparation of bespoke standards that suit the specific needs of the research. This study aimed to assess the appropriateness of various nucleic acid standards, namely circular plasmids, linear plasmids, PCR products, and transcribed RNA, in virus nucleic acid quantification. The evaluation of these standards was conducted through the implementation of qPCR and qRT-PCR methods utilising two different targeted gene-specific primer and probe sets. The findings of this investigation revealed notable differences between the standards. Notably, the study emphasises the necessity to choose standards carefully because their behaviour in PCR is not equal.