One-Step Reverse-Transcription Digital PCR for Reliable Quantification of Different Pepino Mosaic Virus Genotypes
Abstract
:1. Introduction
2. Results and Discussion
2.1. Optimisation of the Reverse-Transcription Digital Polymerase Chain Reaction (RT-dPCR) Assays on the QX100/200 Platform
2.2. Optimisation of the RT-dPCR Assays on the Naica Platform
2.3. Validation of the RT-dPCR Assays
2.3.1. Specificity, Repeatability and Reproducibility of the RT-dPCR Assays
2.3.2. Sensitivity and Linear Range for Different PepMV Genotypes by RT-dPCR Compared to Reverse-Transcription Real-Time Quantitative Polymerase Chain Reaction (RT-qPCR)
3. Materials and Methods
3.1. Samples and Sample Preparation
3.2. Assays Used
3.3. RT-qPCR
3.4. Optimisation of RT-dPCR: QX100/200
3.5. Optimisation of RT-dPCR: Naica
3.6. Validation of RT-dPCR Assays for Quantification of Different PepMV Genotypes
3.7. Data Analysis
4. Conclusions
Supplementary Materials
Author Contributions
Funding
Acknowledgments
Conflicts of Interest
References
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Genotype | Simplex | Duplex | Simplex vs. Duplex | |||
---|---|---|---|---|---|---|
Input RNA (mean; copies/µL) | CV (%) | Input RNA (mean; copies/µL) | CV (%) | Input RNA (Δ; copies/µL) | Δ Duplex (%Simplex) | |
Mix a | 20,573 | 1.1 | 22,144 | 3.8 | 1571 | 7.6 |
Ch2 | 7021 | 1.4 | 7292 | 7.9 | 271 | 3.9 |
Eur | 2398 | 3.1 | 2253 | 7.1 | 145 | 6.0 |
US1 | 16,717 | 1.6 | 17,027 | 2.3 | 310 | 1.9 |
Sample | Runs (n) | Replicates (n) | QX100/200 | Naica | ||
---|---|---|---|---|---|---|
Input RNA (mean; copies/µL) | CV (%) | Input RNA (mean; copies/µL) | CV (%) | |||
PepMV-Ch2-M | 3 | 8 | 5483 | 7.9 | 7552 | 5.6 |
PepMV-Eur-M | 3 | 8 | 1616 | 8.2 | 2165 | 6.1 |
PepMV-US1-M | 3 | 8 | 12,905 | 4.1 | 16,896 | 3.0 |
PepMV-Ch2-L | 3 | 8 | 51 | 14.3 | 71 | 20.0 |
PepMV-Eur-L | 3 | 7–8 a | 14 | 21.2 | 21 | 21.8 |
PepMV-US1-L | 3 | 6–8 b | 110 | 13.9 | 152 | 15.1 |
PepMV-Ch2-zero | 3 | 8 | 0 | NA | 0 c | NA |
PepMV-Eur-zero | 3 | 5 | 0 | NA | 0 c | NA |
PepMV-US1-zero | 3 | 5 | 0 | NA | 0 | NA |
Sample | RNA Dilution | RT-qPCR Cq | Input RNA (copies/µL) | ||
---|---|---|---|---|---|
RT-qPCR a | QX100/200 b | Naica b | |||
PepMV-Ch2 | 10−3 | 21.1 ± 0.1 | 46,737 ± 1648 | 51,907 ± 2337 e | 71,818 ± 2502 |
10−4 | 24.4 ± 0.1 | 5350 ± 186 | 5031 ± 90 | 7495 ± 588 | |
10−5 | 28.2 ± 0.1 | 465 ± 30 | 500 ± 14 | 727 ± 58 | |
10−6 | 31.5 ± 0.1 | 58 ± 3.5 | 44 ± 2.4 | 57 ± 3.4 | |
10−7 | 35.4 ± 1.0 | 5 ± 2.7 | 6 ± 0.9 | 7 ± 3.1 | |
10−8 | Negative c | 0 c | 1 ± 0.2 | 1 ± 1.4 e | |
PepMV-Eur | 10−3 | 22.4 ± 0.1 | 16,485 ± 1005 | 17,254 ± 603 | 21,777 ± 610 |
10−4 | 25.6 ± 0.1 | 1822 ± 86 | 1713 ± 23 | 2315 ± 164 | |
10−5 | 29.1 ± 0.3 | 170 ± 35 | 175 ± 9 | 235 ± 15 | |
10−6 | 38.7 ± 0.8 | 0 ± 0.1 | 14 ± 3.8 | 18 ± 4.2 | |
10−7 | Negative | 0 | 1 ± 1.1 | 1 ± 0.7 d | |
10−8 | Negative | 0 | 0 c | 0 ± 0.4 d | |
PepMV-US1 | 10−3 | 20.5 ± 0.1 | 109,074 ± 8848 | NA | 115,505 ± 19,153 e |
10−4 | 23.8 ± 0.1 | 13,888 ± 676 | 12,495 ± 384 | 17,500 ± 396 | |
10−5 | 27.6 ± 0.2 | 1288 ± 196 | 1215 ± 33 | 1616 ± 20 | |
10−6 | 31.1 ± 0.4 | 142 ± 30 | 94 ± 6.4 | 135 ± 6 | |
10−7 | 35.1 ± 0.6 | 12 ± 3.9 | 10 ± 1.7 | 12 ± 2.3 | |
10−8 | Negative | 0 | 0 ± 0.3d | 1 ± 0.8 d | |
PepMV-Ch2 (in RNA from healthy plants) | 10−4 | 25.1 ± 0.1 | 4297 ± 184 | 4608 ± 175 | 7273 ± 127 |
10−5 | 28.4 ± 0.1 | 440 ± 34 | 459 ± 6 | 716 ± 15 e | |
10−6 | 31.8 ± 0.2 | 45 ± 5.0 | 44 ± 5.8 | 70 ± 2.2 | |
10−7 | 34.8 ± 0.4 | 6 ± 1.6 | 5 ± 1.2 | 9 ± 0.8 e | |
10−8 | 37.8 ± 0.6 d | 1 ± 0.4 d | 0 | 1 ± 0.9 | |
10−9 | Negative | 0 | 0 ± 0.4 d | 1 ± 0.5 d |
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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. https://doi.org/10.3390/plants9030326
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(3):326. https://doi.org/10.3390/plants9030326
Chicago/Turabian StyleMehle, Nataša, Larisa Gregur, Alexandra Bogožalec Košir, and David Dobnik. 2020. "One-Step Reverse-Transcription Digital PCR for Reliable Quantification of Different Pepino Mosaic Virus Genotypes" Plants 9, no. 3: 326. https://doi.org/10.3390/plants9030326