Abstract
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.
Highlights
Pepino mosaic virus (PepMV) belongs to the genus Potexvirus and has virions that are non-enveloped flexuous rods of 508 nm in length [1]
We have shown that these newly developed and validated PepMV-genotype-specific digital PCR (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 real-time quantitative polymerase chain reaction (RT-quantitative PCR (qPCR))
The following assays were transferred from RT-qPCR to reverse-transcription dPCR (RT-dPCR) with the aim of absolute quantification without the need for standard curves: (i) for detection of all PepMV genotypes; (ii) for detection of Peruvian and European PepMV genotypes; (iii) for detection of Ch2 and US2 PepMV genotypes; and (iv) for detection of US1 PepMV genotype
Summary
Pepino mosaic virus (PepMV) belongs to the genus Potexvirus and has virions that are non-enveloped flexuous rods of 508 nm in length [1]. The PepMV genome is a 6410-nucleotide-long single strand of RNA [2]. It was first isolated in 1974 in Peru from pepino plants (Solanum muricatum) [1], and was reported as a pathogen of tomato (Lycopersicon esculentum) in 1999 in greenhouses in The. Netherlands [3]. PepMV became a major pathogen for tomato worldwide, with the first reports of its epidemic expansion in Europe, and later in North and South America [4]. PepMV can cause a wide range of symptoms that reduce the economic value of tomato crops. The development and intensity of the symptoms are affected by various factors, including virus genotype, climate and tomato cultivar [6,8]
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