Development of Reverse Transcription Recombinase Polymerase Amplification (RT-RPA): A Methodology for Quick Diagnosis of Potato Leafroll Viral Disease in Potato
Abstract
:1. Introduction
2. Results
2.1. Evaluation of Plant Samples and RT-RPA Primers for PLRV Detection
2.2. Optimization of One-Step RT-RPA Method for the Detection of PLRV Using Optimized Primers
2.3. Detection of PLRV Using Selected Primers and Optimization of One-Step RT-RPA
2.4. Evaluation and Optimization of RNA Extraction Method
2.5. Sensitivity and Specificity Analysis
2.6. Cloning and Sequencing of Amplicons for Primer Accuracy
2.7. Validation of One-Step RT-RPA Results on Diverse Field Samples
3. Discussion
4. Materials and Methods
4.1. Source of Viral Pure Culture and Plant Materials
4.2. The Process of RNA Isolation and cDNA Synthesis
4.3. Configuring the Reaction for One-Step RT-PCR
4.4. The Designing and Synthesis of Primers
4.5. Optimization of RT-RPA and Primer Selection
4.6. The Optimization of RT-RPA in One Step for the Detection of PLRV
4.7. Evaluation and Improvement of RNA Extraction Techniques in Leaves
4.8. Analysis of the Sensitivity and Specificity of One-Step RT-PCR/RT-RPA
4.9. Elution, Cloning and Sequencing for Specificity Confirmation
4.10. Natural Infection Validation Using a Single RT-RPA Technique
5. Conclusions
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
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Target Virus | Primer Set No. | Primers | Polarity | Sequence 5′–3′ | Bases | Target Position on Coat Protein Gene | Position on Accession Number in NCBI GeneBank (NC_001747.1) | GC (%) | Tm (°C) | Amplicon Size (bp) |
---|---|---|---|---|---|---|---|---|---|---|
PLRV | A | LRRPAF1 | Sense | GTATAAGATCACAAGCATCTTACTTCAGTTC | 31 | 327–357 | 4019–4267 | 35.5 | 61.3 | 249 |
LRRPAR1 | Antisense | GTATCTGAAGATTTTCCATTTCCCTTC | 27 | 548–575 | 37 | 62.5 | ||||
B | LRRPAF2 | Sense | TAAGATCACAAGCATCTTACTTCAGTTC | 28 | 330–357 | 4022–4267 | 35.7 | 60.5 | 246 | |
LRRPAR2 | Antisense | GTATCTGAAGATTTTCCATTTCCCTTC | 27 | 548–575 | 37 | 62.5 | ||||
C | LRRPAF3 | Sense | ACTCAAGGCCTACCATGAGTATAAGAT | 27 | 309–335 | 4001–4183 | 40.7 | 60.9 | 153 * | |
LRRPAR3 | Antisense | GTAATTTGGAACTTGTTGACGTAGGACT | 28 | 434–461 | 39.3 | 63 |
Field Sample No. | One-Step RT-PCR [12] | One Step RT-RPA with Primer Pair C (LRRPAF3/R3) under Heating Block/Water Bath | One Step RT-RPA with Primer Pair C (LRRPAF3/R3) under Heating Block/Water Bath |
---|---|---|---|
Leaves | Purified RNA | Purified RNA | Crude RNA (Cellular Disc-Mediated RNA Extract with Added 1.0% Sodium Sulphite) |
1 | + | + | + |
2 | + | + | + |
3 | + | + | − |
4 | − | − | − |
5 | + | + | + |
6 | + | + | + |
7 | + | + | + |
8 | + | + | + |
9 | + | + | + |
10 | + | + | + |
11 | − | − | − |
12 | + | + | + |
13 | + | + | + |
14 | + | + | + |
15 | − | − | − |
Healthy Control | − | − | − |
Negative Control | − | − | − |
Positive Control | + | + | + |
Field Sample No. | One-step RT-PCR [12] | One-step RT-RPA with primer pair C (LRRPAF3/R3) under thermal Cycler | One step RT-RPA with primer pair C (LRRPAF3/R3) under heating block/water bath |
Tubers | Purified RNA | Purified RNA | Purified RNA |
1 | + | + | + |
2 | + | + | + |
3 | + | + | + |
4 | + | + | − |
5 | − | − | − |
6 | + | + | + |
7 | + | + | + |
8 | + | + | + |
9 | + | + | + |
10 | + | + | + |
Healthy Control | − | − | − |
Positive Control | + | + | + |
Sample No. | Location/State | Cultivar/Variety | DAS-ELISA | One-Step RT-PCR [12] | One Step RT-RPA | ||||||
---|---|---|---|---|---|---|---|---|---|---|---|
RNA | RNA | Crude RNA | |||||||||
Leaves | Dormant Tubers | Sprouted Tubers | Leaves | Dormant Tubers | Leaves | Dormant Tubers | Leaves | Dormant Tubers | |||
1 | Gujarat | Kufri Pukhraj | − | − | − | − | − | + | − | + | − |
2 | Gujarat | Kufri Jyoti | + | − | − | + | + | + | + | + | + |
3 | Punjab | Kufri Pukhraj | − | − | − | − | − | + | − | + | − |
4 | Punjab | Kufri Badshah | − | − | − | + | + | + | + | + | − |
5 | Haryana | Kufri Chandramukhi | + | − | − | + | + | + | + | + | + |
6 | Haryana | Kufri Jyoti | + | − | + | + | + | + | + | + | + |
7 | Haryana | Kufri Bahar | − | − | − | − | − | + | − | + | − |
8 | Himachal Pradesh | Kufri Jyoti | − | − | − | − | − | + | − | + | − |
9 | Himachal Pradesh | Kufri Himalini | − | − | − | − | − | + | − | + | − |
10 | Himachal Pradesh | Kufri Chandramukhi | + | − | − | + | + | + | + | + | + |
11 | Uttar Pradesh | Kufri Pukhraj | + | − | − | + | − | + | − | + | + |
12 | Uttar Pradesh | Kufri Chipsona 1 | + | − | + | + | + | + | + | + | + |
13 | Uttar Pradesh | Kufri Chipsona 4 | − | − | − | + | − | + | − | + | − |
14 | Uttar Pradesh | Kufri Bahar | − | − | − | + | − | + | + | + | + |
15 | Bihar | Kufri Sindhuri | − | − | − | − | − | + | + | + | − |
16 | Bihar | Kufri Chandramukhi | − | − | − | + | − | + | + | + | − |
17 | Bihar | Kufri Anand | − | − | − | − | − | − | − | − | − |
18 | Madhya Pradesh | Kufri Pukhraj | + | − | + | + | + | + | + | + | + |
19 | Madhya Pradesh | Kufri Lauvkar | − | − | − | − | − | + | − | + | − |
20 | Madhya Pradesh | Kufri Chandramukhi | − | − | − | − | − | + | − | + | − |
21 | West Bengal | Kufri Jyoti | + | − | − | + | + | + | + | + | + |
22 | West Bengal | Kufri Chandramukhi | − | − | − | − | − | + | + | + | − |
23 | West Bengal | Kufri Pukhraj | + | − | − | + | + | + | + | + | + |
24 | West Bengal | Kufri Ashoka | + | − | + | + | + | + | + | + | + |
25 | Meghalaya | Kufri Megha | + | − | − | + | + | + | + | + | + |
26 | Meghalaya | Kufri Giriraj | − | − | − | − | − | + | − | + | − |
27 | Meghalaya | Kufri Jyoti | + | − | − | + | + | + | + | + | + |
Total | 13 | 0 | 04 | 16 | 12 | 26 | 16 | 26 | 13 |
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Kumar, R.; Kaundal, P.; Tiwari, R.K.; Lal, M.K.; Kumari, H.; Kumar, R.; Naga, K.C.; Kumar, A.; Singh, B.; Sagar, V.; et al. Development of Reverse Transcription Recombinase Polymerase Amplification (RT-RPA): A Methodology for Quick Diagnosis of Potato Leafroll Viral Disease in Potato. Int. J. Mol. Sci. 2023, 24, 2511. https://doi.org/10.3390/ijms24032511
Kumar R, Kaundal P, Tiwari RK, Lal MK, Kumari H, Kumar R, Naga KC, Kumar A, Singh B, Sagar V, et al. Development of Reverse Transcription Recombinase Polymerase Amplification (RT-RPA): A Methodology for Quick Diagnosis of Potato Leafroll Viral Disease in Potato. International Journal of Molecular Sciences. 2023; 24(3):2511. https://doi.org/10.3390/ijms24032511
Chicago/Turabian StyleKumar, Ravinder, Priyanka Kaundal, Rahul Kumar Tiwari, Milan Kumar Lal, Hema Kumari, Rakesh Kumar, Kailash Chandra Naga, Awadhesh Kumar, Brajesh Singh, Vinay Sagar, and et al. 2023. "Development of Reverse Transcription Recombinase Polymerase Amplification (RT-RPA): A Methodology for Quick Diagnosis of Potato Leafroll Viral Disease in Potato" International Journal of Molecular Sciences 24, no. 3: 2511. https://doi.org/10.3390/ijms24032511