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

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Abstract

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.

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Acknowledgements

This study was supported by the Slovenian Research Agency (grant number P4-0165) and by Euphresco Project VirusCollectII (2015-F-132), financed by the Ministry of Agriculture, Forestry and Food through the Administration of the Republic of Slovenia for Food Safety, Veterinary and Plant Protection. The study was performed using ddPCR equipment financed by the Metrology Institute of the Republic of Slovenia (MIRS), with financial support from the European Regional Development Fund. The equipment is wholly owned by the Republic of Slovenia. We would like to thank Bio-Rad Laboratories, Inc. for providing the RT-ddPCR reagents. We would like to thank INRA, France, for providing the PVY-O139 and PVY-N605 isolates. We also thank Vildan Bolat (student at Nigde University, Turkey, on a collaboration through an ERASMUS internship) for technical help. The scientific language revision was carried out by Dr. Christopher Berrie.

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Authors and Affiliations

  1. National Institute of Biology, Večna pot 111, 1000, Ljubljana, Slovenia

    Nataša Mehle, David Dobnik, Maja Ravnikar & Maruša Pompe Novak

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  1. Nataša Mehle
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  2. David Dobnik
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  3. Maja Ravnikar
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  4. Maruša Pompe Novak
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Correspondence to Nataša Mehle.

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Conflict of interest

The authors declare the following competing financial interest: the reagents for this study were provided by Bio-Rad, which sells and markets the QX100/200 droplet digital PCR system. The authors declare that they have no other conflict of interest.

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Mehle, N., Dobnik, D., Ravnikar, M. et al. Validated reverse transcription droplet digital PCR serves as a higher order method for absolute quantification of Potato virus Y strains. Anal Bioanal Chem 410, 3815–3825 (2018). https://doi.org/10.1007/s00216-018-1053-3

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  • DOI: https://doi.org/10.1007/s00216-018-1053-3

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