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Open AccessArticle

One-Step Reverse-Transcription Digital PCR for Reliable Quantification of Different Pepino Mosaic Virus Genotypes

Department of Biotechnology and Systems Biology, National Institute of Biology, Večna pot 111, 1000 Ljubljana, Slovenia
Goričko Nature Park Public Institute, Grad 191, 9264 Grad, Slovenia
Author to whom correspondence should be addressed.
Plants 2020, 9(3), 326;
Received: 14 February 2020 / Revised: 28 February 2020 / Accepted: 2 March 2020 / Published: 4 March 2020
(This article belongs to the Special Issue Diagnosis and Control of Plant Viral Diseases)
In recent years, pepino mosaic virus (PepMV) has rapidly evolved from an emerging virus to an endemic pathogen, as it causes significant loses to tomato crops worldwide. At present, the main control strategy for prevention of PepMV disease in tomato production remains based on strict hygiene measures. To prevent damage caused by PepMV, cross-protection is used in some countries. Reliable characterisation, detection and quantification of the pathogen are vital for disease control. At present, reverse-transcription real-time quantitative polymerase chain reaction (RT-qPCR) is generally used for this purpose. However, quantitative use of RT-qPCR is linked to standardised reference materials, which are not available for PepMV. In addition, many factors can influence RT-qPCR efficiencies and lead to lower accuracy of the quantification. In this study, well-characterised PepMV-genotype-specific RT-qPCR assays were transferred to two digital PCR (dPCR) platforms. dPCR-based assays allow absolute quantification without the need for standard curves, and due to the binary nature of the reaction, dPCR also overcomes many of the other drawbacks of RT-qPCR. We have shown that these newly developed and validated PepMV-genotype-specific dPCR assays are suitable candidates for higher-order methods for quantification of PepMV RNA, as they show lower measurement variability, with sensitivity and specificity comparable to RT-qPCR. View Full-Text
Keywords: digital PCR; pepino mosaic virus; quantification; genotype specific digital PCR; pepino mosaic virus; quantification; genotype specific
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Mehle, N.; Gregur, L.; Bogožalec Košir, A.; Dobnik, D. One-Step Reverse-Transcription Digital PCR for Reliable Quantification of Different Pepino Mosaic Virus Genotypes. Plants 2020, 9, 326.

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