Validated reverse transcription droplet digital PCR serves as a higher order method for absolute quantification of Potato virus Y strains

• Published in: Analytical and bioanalytical chemistry Vol. 410; no. 16; pp. 3815 - 3825
• Main Authors: Mehle, Nataša, Dobnik, David, Ravnikar, Maja, Pompe Novak, Maruša
• Format: Journal Article
• Language: English
• Published: Berlin/Heidelberg Springer Berlin Heidelberg 01-06-2018; Springer; Springer Nature B.V
• Subjects: Analytical Chemistry; Biochemistry; Biological evolution; Characterization and Evaluation of Materials; Chemistry; Chemistry and Materials Science; Diagnostic systems; Food Science; Genetic aspects; Genetic variability; Health aspects; Laboratory Medicine; Monitoring/Environmental Analysis; Observations; Pathogenesis; Plant viruses; Polymerase chain reaction; Potatoes; Recombination; Reference materials; Research Paper; Reverse transcription; Ribonucleic acid; RNA; RNA viruses; Strains (organisms); Transcription (Genetics); Viruses; Quantification; PVY; Droplet digital PCR; Potato virus Y
• ISSN: 1618-2642; 1618-2650
• DOI: 10.1007/s00216-018-1053-3

RNA viruses have a great potential for high genetic variability and rapid evolution that is generated by mutation and recombination under selection pressure. This is also the case of
Potato virus Y
(PVY), which comprises a high diversity of different recombinant and non-recombinant strains. Consequently, it is hard to develop reverse transcription real-time quantitative PCR (RT-qPCR) with the same amplification efficiencies for all PVY strains which would enable their equilibrate quantification; this is specially needed in mixed infections and other studies of pathogenesis. To achieve this, we initially transferred the PVY universal RT-qPCR assay to a reverse transcription droplet digital PCR (RT-ddPCR) format. RT-ddPCR is an absolute quantification method, where a calibration curve is not needed, and it is less prone to inhibitors. The RT-ddPCR developed and validated in this study achieved a dynamic range of quantification over five orders of magnitude, and in terms of its sensitivity, it was comparable to, or even better than, RT-qPCR. RT-ddPCR showed lower measurement variability. We have shown that RT-ddPCR can be used as a reference tool for the evaluation of different RT-qPCR assays. In addition, it can be used for quantification of RNA based on in-house reference materials that can then be used as calibrators in diagnostic laboratories.