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

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.

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

“…Therefore, as no reference material was available for PepMV, RT-qPCR only provided relative quantification. However, strategies are available for indirect copy-number determination in a sample, such as the use of limiting dilutions, as discussed in our previous study on PVY, for example [30]. In the present study, the data from QX100/200 was used to calibrate the RT-qPCR and, therefore, as might be expected, the quantification according to these two PCR systems was in agreement (Figure 3).…”

“…For comparisons of output data from the RT-qPCR and RT-dPCR, Cq was used to define the RNA copy numbers. For this conversion, actual copy numbers were initially determined with RT-dPCR QX100/200, as the calibrator [30]. Cq conversion was defined by the most accurately calculated PepMV RNA concentration from RT-dPCR (i.e., which showed the lowest CV across technical repetitions).…”

Transmission studies of the newly described apple chlorotic fruit spot viroid using a combined RT-qPCR and droplet digital PCR approach

“…A qualitative result can be obtained with endpoint RT-PCR, and relative quantification can be achieved by RT-qPCR. For quantification with qPCR, a standard curve with known concentrations of the target is necessary to transform the qPCR output of the quantification cycle (Cq) into absolute concentrations [ 22 , 23 ]. For culturable microorganisms such as bacteria, cell suspensions with defined concentrations can be prepared from cultures [ 24 ].…”

“…In this study, droplet digital PCR (ddPCR) was used for the absolute quantification of ACFSVd. This technology has already been used for plant pathogens such as phytoplasma [ 28 ], Erwinia amylovora , Ralstonia solanacearum [ 29 ], Xylella fastidiosa [ 30 ] potato virus Y [ 22 ], citrus yellow vein clearing virus [ 31 ] and citrus tristeza virus [ 32 ]. To our knowledge, there is no published report on the use of ddPCR for the quantification of pathogenic viroids in plant tissue.…”

Development and optimization of a simian immunodeficiency virus (SIV) droplet digital PCR (ddPCR) assay

“…A sensitive qPCR assay was previously developed and used to detect and quantify low level SIV in cART-suppressed Rhesus macaques [26]. Our initial ddPCR assay development effort was aimed at adapting this assay to the Raindance platform with no or minimal changes introduced to the primer and probe sequences, or mastermix components, as direct transfer of qPCR assays onto ddPCR platforms was described [27,28]. As it was evident from this initial attempt that the SIV assay with its existing compositions (including mastermix) did not allow sufficient cluster separation [6], various changes were introduced to the existing mastermix composition to improve the performance of the single quencher probe assay on the Raindance platform.…”