Digital PCR Methods Potent but Not Sufficiently Robust for RNA Virus Detection

JRC scientists have assessed the influence of different primer and probe chemistries on the accuracy of digital PCR (dPCR) methods for the quantification of viral Ribonucleic Acid (RNA) using an influenza model. The study demonstrated that innovative reverse transcription digital PCR (RT-dPCR) requires careful evaluation of method performance and quality control, but has, under the condition that it is sufficiently well optimised, large potential for accurate quantification of viral RNA.

RNA virus
Digital PCR methods potent but not sufficiently robust for RNA virus detection ©vitstudio,

Claims on the higher accuracy and robustness of digital PCR methods versus quantitative real-time PCR methods are based on the theoretical assumptions that each target nucleic acid molecule present in a partition of digital PCR (dPCR) systems will undergo successful amplification and that the high amplification cycle number overcomes potentially present inhibition effects. These assumptions have been assessed in a study investigating the potential of dPCR. A significant bias became visible when using different chemistries and analysing different templates. The application of more sophisticated primer and probe chemistries, such as Zip Nucleic Acid ( ZNA) and Locked Nucleic Acid (LNA), had pronounced advantages. Further, it could be seen that the type of RNA template had a significant influence on the outcome of the RNA quantification. Two different template types, both of which are typical calibration materials for real-time PCR, were not equally affected by applying different assay chemistries. This can lead to systematic errors when RNA is e.g. quantified in more complex clinical samples.

Overall, the study results reinforce the importance of careful evaluation of the RT– dPCR method performance and quality control measures. Highly accurate DNA and RNA quantification methods are required for standardisation purposes.  The outcome of this study is therefore of major importance for the microbiology community and beyond when using dPCR for DNA/RNA quantification and detection as well as for the characterisation of calibration or quality control materials.