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

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.

Application of One-Step Reverse Transcription Droplet Digital PCR for Dengue Virus Detection and Quantification in Clinical Specimens

“…In addition, RT-ddPCR consistently reported more DENV RNA copies in samples at low concentrations compared to qRT-PCR ( Figure 4 A). The superior detection and quantification performance of RT-ddPCR over qRT-PCR with environmental and clinical samples containing low target concentrations was also reported in previous studies on rotavirus [ 13 ], pepper mild mottle virus [ 35 ], pepino mosaic virus [ 36 ], and severe acute respiratory syndrome coronavirus 2 [ 37 ]. The results from three out of eight DENV samples detected by RT-ddPCR, but not qRT-PCR, were above LLOQ making the quantification results reportable.…”

Current Developments and Challenges in Plant Viral Diagnostics: A Systematic Review

“…Various studies have successfully detected and identified different genotypes of PepMV in tomato crops. Using a newly developed one-step RT digital PCR, reliable quantification of different Pepino mosaic virus genotypes has been reported and found more suitable than only RT-PCR [ 82 ]. In another study, a RT-PCR was developed for the simultaneous detection and identification of three groups of Pepino mosaic virus (PepMV): European/Peruvian, Chilean 1/US1, and Chilean 2/US2 groups in routine analysis of field tomato samples [ 83 ].…”

Moving from qPCR to Chip Digital PCR Assays for Tracking of some Fusarium Species Causing Fusarium Head Blight in Cereals

“…Digital PCR, on the contrary, gives an absolute quantification of the molecular target as output and can, therefore, be proposed for characterization of the calibrators needed for standard curves in qPCR analyses. As suggested by other authors [ 25 ], dPCR can hypothetically be exploited for the production of calibrators.…”