Development of a Multiplex RT-PCR Detection for Six Viruses Infecting Strawberry
Abstract
1. Introduction
2. Materials and Methods
2.1. Plant Materials and the Positive RNA Sample
2.2. RNA Extraction and Reverse Transcription
2.3. Design of Virus-Specific Primers
2.4. Evaluation of Primer Specificity
2.5. Optimization of Multiplex RT-PCR Assay
2.6. Sensitivity of Multiplex RT-PCR Assay
2.7. Survey of Strawberry Viruses Using Multiplex RT-PCR Assay
3. Results
3.1. Specificity of Primer Pairs
3.2. Optimization of Multiplex RT-PCR Assay
3.3. Sensitivities of Uniplex RT-PCR and Multiplex RT-PCR Assays
3.4. Application of Multiplex RT-PCR Assay in Survey of Strawberry Viruses
4. Discussion
5. Conclusions
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Conflicts of Interest
References
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Virus | Primer | Sequence (5′-3′) | Product Size (bp) | Melting Temperature (°C) |
---|---|---|---|---|
SMYEV | Multi-SMYEV-F1 | TGGCGGATTCCAATACTTGCC | 1263 | 60 |
Multi-SMYEV-R1 | CTCATCCAGTCTTGGAGGCG | 60 | ||
SVBV | Multi-SVBV-F | TTGAAGCCTTCGCAGAAGCC | 931 | 60 |
Multi-SVBV-R | AGCTTGAGTAGCTCTTCGATCTGAG | 60 | ||
SMoV | Multi-SMoV-F | CGTGAGAGGACCCGAGCTTATG | 792 | 62 |
Multi-SMoV-R | AGAGATAGTCGAACCTTTGGTGGTC | 60 | ||
SPV-1 | Multi-SPV1-F | AACGCGGAGTGGAGAGTGAC | 635 | 61 |
Multi-SPV1-R | CATTTGTTCCGCCGCTTCTC | 59 | ||
SPaV | Multi-SPaV-F | CCATGGAGAATGTGGAGAGCAG | 449 | 59 |
Multi-SPaV-R | CACCACGTTCTCTTCCTCAGG | 59 | ||
SCrV-4 | Multi-SCrV4-F | ATAGGCGCGAAATCCAAACTTCC | 230 | 60 |
Multi-SCrV4-R | TCAATGATAGGTTCGCTGTTCGC | 60 |
Viral Primer | Final Primer Concentrations (μmol/L) | |||||
---|---|---|---|---|---|---|
1 | 2 | 3 | 4 | 5 | 6 | |
Multi-SMYEV-F1/R1 | 0.2 | 0.3 | 0.3 | 0.3 | 0.4 | 0.4 |
Multi-SVBV-F/R | 0.2 | 0.15 | 0.3 | 0.3 | 0.3 | 0.3 |
Multi-SMoV-F/R | 0.2 | 0.15 | 0.1 | 0.075 | 0.075 | 0.075 |
Multi-SPV1-F/R | 0.2 | 0.3 | 0.3 | 0.4 | 0.4 | 0.4 |
Multi-SPaV-F/R | 0.2 | 0.2 | 0.25 | 0.25 | 0.25 | 0.2 |
Multi-SCrV4-F/R | 0.2 | 0.2 | 0.1 | 0.075 | 0.075 | 0.075 |
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Wang, Y.; Zeng, X.; Xiao, G.; Zhang, D.; Wen, X.; Zhou, X.; Wang, Z.; Deng, J.; Han, Y. Development of a Multiplex RT-PCR Detection for Six Viruses Infecting Strawberry. Viruses 2024, 16, 1858. https://doi.org/10.3390/v16121858
Wang Y, Zeng X, Xiao G, Zhang D, Wen X, Zhou X, Wang Z, Deng J, Han Y. Development of a Multiplex RT-PCR Detection for Six Viruses Infecting Strawberry. Viruses. 2024; 16(12):1858. https://doi.org/10.3390/v16121858
Chicago/Turabian StyleWang, Yong, Xiangguo Zeng, Guilin Xiao, Dongmei Zhang, Xin Wen, Xinxin Zhou, Zexian Wang, Jiangli Deng, and Yongchao Han. 2024. "Development of a Multiplex RT-PCR Detection for Six Viruses Infecting Strawberry" Viruses 16, no. 12: 1858. https://doi.org/10.3390/v16121858
APA StyleWang, Y., Zeng, X., Xiao, G., Zhang, D., Wen, X., Zhou, X., Wang, Z., Deng, J., & Han, Y. (2024). Development of a Multiplex RT-PCR Detection for Six Viruses Infecting Strawberry. Viruses, 16(12), 1858. https://doi.org/10.3390/v16121858