Rapid, Sensitive and Simultaneous Detection of Two Wheat RNA Viruses Using Reverse Transcription Recombinase Polymerase Amplification (RT-RPA)
Abstract
:1. Introduction
2. Materials and Methods
2.1. Virus and Plant Material
2.2. Total RNA Extraction and cDNA Synthesis
2.3. PCR Assays
2.4. Primer Design
2.5. Recombinant Plasmids and Viruses
2.6. RPA Reaction
2.7. RT-RPA Reaction Optimization
2.8. Sensitivity Assay
2.9. Specificity Assay
2.10. Applicability Assay
3. Results
3.1. Samples Screened by PCR-CP
3.2. Selection of RPA Primers
3.3. Optimization of Incubation Temperature and Time
3.4. Validation of Incubation Conditions under Simulated Field Conditions
3.5. Sensitivity Determination by Comparing PCR-CP, PCR-RPA and RT-RPA
3.6. Specificity Determination of RT-RPA Method
3.7. Applicability of the RT-RPA Method
4. Discussion
5. Conclusions
Supplementary Materials
Author Contributions
Funding
Data Availability Statement
Conflicts of Interest
References
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Primer Name | Sequence 5’-3’ | Set | Targeted Region | Expected Product Length (bp) |
---|---|---|---|---|
WYMV-RPA-1-F | TTGACTTCTTTGTCCCCCGCCCGTGGATG | WYMV-S1 | WYMV coat protein | 166 |
WYMV-RPA-1-R | TGTCCATCAGTATCCAGAACCCTGCGGTTG | |||
WYMV-RPA-2-F | TGAATGGTGGCTTGAGACGGATCCTGCGAG | WYMV-S2 | WYMV coat protein | 250 |
WYMV-RPA-2-R | AGGTTGGTTGTGTCGGAGGTGAGCATGGTAT | |||
WYMV-RPA-3-F | CTTCAGACAAACACGCAGAAAACCAGACCA | WYMV-S3 | WYMV coat protein | 346 |
WYMV-RPA-3-R | TTGGTTGTGTCGGAGGTGAGCATGGTATTGT | |||
WYMV-RPA-4-F | ACCAGTCTTGGCATCACCACGGATGAGGCA | WYMV-S4 | WYMV coat protein | 445 |
WYMV-RPA-4-R | GGTTGGTTGTCTTGCGGAGGTTGGTTGTGT | |||
CWMV-RPA-1-F | CTTTTGCGACCTTGGCGGAGAGGGGGTCAGT | CWMV-S1 | CWMV coat protein | 158 |
CWMV-RPA-1-R | CAGTCTGCCCTTGTTCTTCTGTTTCTATCT | |||
CWMV-RPA-2-F | TAATTTCTTCTCACGGAGTAAACGCTTCGGT | CWMV-S2 | CWMV coat protein | 236 |
CWMV-RPA-2-R | TAGATATAGCCAACGATTGATCAGTCTGCCCTT | |||
CWMV-RPA-3-F | TGGCAGAGTACGAGGACGAGTGTGTTGTCT | CWMV-S3 | CWMV coat protein | 343 |
CWMV-RPA-3-R | AACTAACATAAGTCATCAGTTCGCCCAAAGCAT | |||
CWMV-RPA-4-F | AAGTTGAGACATGGCAGAGTACGAGGACGA | CWMV-S4 | CWMV coat protein | 437 |
CWMV-RPA-4-R | TCAACTCGAACCTTCCCACTTAAGATTATACT |
Sample | PCR | RPA | Sample | PCR | RPA | ||||
---|---|---|---|---|---|---|---|---|---|
WYMV | CWMV | WYMV | CWMV | WYMV | CWMV | WYMV | CWMV | ||
Henan-1 | + | + | + | - | Anhui-1 | + | - | + | - |
Henan-2 | + | - | + | - | Anhui-2 | + | - | + | - |
Henan-3 | + | - | + | - | Anhui-3 | + | - | + | - |
Henan-4 | + | - | + | - | Anhui-4 | + | - | + | - |
Henan-5 | + | - | + | - | Anhui-5 | + | - | + | - |
Henan-6 | + | - | + | - | Anhui-6 | + | + | + | + |
Henan-7 | - | - | - | - | Anhui-7 | + | + | + | + |
Henan-8 | - | - | - | - | Anhui-8 | + | + | + | + |
Henan-9 | - | - | - | - | Anhui-9 | - | - | - | - |
Henan-10 | + | - | + | - | Anhui-10 | - | - | - | - |
Henan-11 | + | - | + | - | Anhui-11 | - | - | - | - |
Henan-12 | + | - | + | - | Anhui-12 | - | - | - | - |
Henan-13 | + | - | + | - | Anhui-13 | - | - | - | - |
Henan-14 | + | - | + | - | Anhui-14 | - | - | - | - |
Henan-15 | + | - | + | - | Anhui-15 | - | - | - | - |
Henan-16 | + | - | + | - | Anhui-16 | - | - | - | - |
Henan-17 | - | - | - | - | Anhui-17 | - | - | - | - |
Henan-18 | - | - | - | - | Shandong-1 | + | + | + | + |
Henan-19 | - | - | - | - | Shandong-2 | + | - | + | - |
Henan-20 | - | - | - | - | Shandong-3 | + | + | + | - |
Henan-21 | - | - | - | - | Shandong-4 | - | - | - | - |
Henan-22 | - | - | - | - | Shandong-5 | - | - | - | - |
Henan-23 | - | - | - | - | Shandong-6 | + | + | + | + |
Henan-24 | - | - | - | - | Shandong-7 | + | + | + | + |
Henan-25 | - | - | - | - | Shandong-8 | + | + | + | + |
Henan-26 | + | - | + | - | Shandong-9 | + | + | + | + |
Henan-27 | + | - | + | - | Shandong-10 | + | + | + | - |
Henan-28 | + | - | + | - | Shandong-11 | + | + | + | + |
Henan-29 | + | - | + | - | Shandong-12 | + | + | + | + |
Henan-30 | + | - | + | - | Shandong-13 | + | + | + | + |
Henan-31 | + | - | + | - | Shandong-14 | + | + | + | + |
Henan-32 | + | - | + | - | Shandong-15 | + | - | + | - |
Henan-33 | + | - | + | - | Shandong-16 | + | - | + | - |
Henan-34 | + | - | + | - | Shandong-17 | + | - | + | - |
Henan-35 | + | - | + | - | Shandong-18 | + | + | + | + |
Henan-36 | + | - | + | - | Shandong-19 | + | + | + | + |
Henan-37 | + | - | + | - | Shandong-20 | + | + | + | + |
Henan-38 | + | - | + | - | Shandong-21 | + | + | + | + |
Henan-39 | + | - | + | - | Shandong-22 | + | + | + | + |
Henan-40 | + | - | + | - | Shandong-23 | + | + | + | + |
Henan-41 | + | - | + | - | Shandong-24 | + | + | + | + |
Henan-42 | + | - | + | - | Shandong-25 | + | + | + | + |
Shanxi-1 | + | - | + | - | Shandong-26 | + | + | + | + |
Shanxi-2 | - | - | - | - | Shandong-27 | + | + | + | + |
Shanxi-3 | - | - | + | - | Shandong-28 | + | - | + | - |
Shanxi-4 | - | - | - | - | Shandong-29 | + | - | + | - |
Shanxi-5 | + | - | + | - | Shandong-30 | + | - | + | - |
Shanxi-6 | + | - | + | - | Shandong-31 | + | - | + | - |
Shanxi-7 | - | - | + | - | Jiangsu-1 | - | - | + | - |
Shanxi-8 | + | - | + | - | Jiangsu-2 | + | - | + | - |
Shanxi-9 | + | - | + | - | Jiangsu-3 | + | - | + | - |
Shanxi-10 | + | - | + | - | Jiangsu-4 | + | - | + | - |
Shanxi-11 | + | - | + | - | Jiangsu-5 | + | - | + | - |
Shanxi-12 | + | - | + | - | Jiangsu-6 | + | - | + | - |
Shanxi-13 | + | - | + | - | Jiangsu-7 | + | - | + | - |
PCR-CPa | Performance Characteristics (%) | |||||
Positive | Negative | Total | Sensitivity | Specificity | ||
RPA- WYMVc | Positive | 82 | 3 | 85 | 100.0% (95.6~100.0%, 95% CI) | 89.3% (71.8~97.7%, 95% CI) |
Negative | 0 | 25 | 25 | |||
82 | 28 | 110 | ||||
Agreement Kappa value: 0.926 (0.843~1.000, 95% CI) | ||||||
PCR-CPb | Performance Characteristics (%) | |||||
Positive | Negative | Total | Sensitivity | Specificity | ||
RPA- CWMVd | Positive | 22 | 0 | 22 | 88.0%(68.8~97.5%, 95% CI) | 100% (95.8~100.0%, 95% CI) |
Negative | 3 | 81 | 84 | |||
25 | 81 | 106 | ||||
Agreement Kappa value: 0.919 (0.828~1.000, 95% CI) |
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Chen, Z.; Zhang, T.; Lei, J.; Wang, Z.; Liu, P.; Zhong, K.; Chen, J.; Liu, J. Rapid, Sensitive and Simultaneous Detection of Two Wheat RNA Viruses Using Reverse Transcription Recombinase Polymerase Amplification (RT-RPA). Life 2022, 12, 1952. https://doi.org/10.3390/life12121952
Chen Z, Zhang T, Lei J, Wang Z, Liu P, Zhong K, Chen J, Liu J. Rapid, Sensitive and Simultaneous Detection of Two Wheat RNA Viruses Using Reverse Transcription Recombinase Polymerase Amplification (RT-RPA). Life. 2022; 12(12):1952. https://doi.org/10.3390/life12121952
Chicago/Turabian StyleChen, Zhiqing, Tianye Zhang, Jiajia Lei, Ziqiong Wang, Peng Liu, Kaili Zhong, Jianping Chen, and Jiaqian Liu. 2022. "Rapid, Sensitive and Simultaneous Detection of Two Wheat RNA Viruses Using Reverse Transcription Recombinase Polymerase Amplification (RT-RPA)" Life 12, no. 12: 1952. https://doi.org/10.3390/life12121952