Development and Validation of a Multiplex Reverse Transcription PCR Assay for Simultaneous Detection of Three Papaya Viruses
Abstract
:1. Introduction
2. Materials and Methods
2.1. Viruses
2.2. Primer Design
Virus | Primer a | Sequence (5’-3’) | Size (bp) | Target Gene | Location (nt) b |
---|---|---|---|---|---|
PRSV | PRSV953-F | GCGATGCTCATAAACATACCTGA | 953 | HC-Pro/P3 | 2774–2796 |
PRSV953-R | TGTACACAGTACTTCGGTGAGAAGTCGTA | 3698–3726 | |||
PRSV613-F | TTGTGTACGACTTCTCACCGAA | 613 | P3 | 3693–3714 | |
PRSV613-R | CGAATGTCATCCAAAGACTGATGATAAAC | 4277–4305 | |||
PRSV515-F | TTGTGTACGACTTCTCACCGAA | 515 | P3 | 3693–3714 | |
PRSV515-R | CACATCAATCATCATCAAAATTAATGT | 4181–4207 | |||
PapMV | PapMV476-F | ATGGTAGCTGCTAAGGTTCCAGC | 476 | CP | 6024–6046 |
PapMV476-R | GACCCAGAAATTTGGCCTTTGGTGATG | 6473–6499 | |||
PapMV205-F | CCAAATTTGCCGCGTTCGACT | 205 | CP | 6295–6315 | |
PapMV205-R | GACCCAGAAATTTGGCCTTTGGTGATG | 6473–6499 | |||
PLDMV | PLDMV355-F | GGCATGTGGTTTATGATGCAAGGG | 355 | CP | 9528–9551 |
PLDMV355-R | GCTCCGTGTTCTCAGTCGCATT | 9861–9882 |
2.3. Nucleic Acid Extraction
2.4. Reverse Transcription
2.5. The Uniplex PCR
2.6. Optimization of the Multiplex PCR Conditions
2.7. The Sensitivity of the Uniplex PCR and Multiplex PCR Assays
2.8. The Detection of Viruses in Field Samples by Uniplex RT-PCR and Multiplex RT-PCR
3. Results
3.1. Specificity and Compatibility of Primer Pairs
3.2. Optimization of Multiplex RT-PCR
3.3. Sensitivities of the Uniplex and Multiplex RT-PCR
3.4. Evaluation of Multiplex RT-PCR Using Field Samples
Geographical Region | No. of Samples | PRSV Only | PLDMV Only | PapMV Only | PRSV and PLDMV (Mixed Infection) |
---|---|---|---|---|---|
Haikou | 30 | 15(50%) | 8(26.7%) | 1(3.3%) | 6(20%) |
Sanya | 28 | 17(60.7%) | 8(28.6%) | 0(0%) | 3(10.7%) |
Wenchang | 16 | 10(62.5%) | 3(18.8%) | 0(0%) | 3(18.8%) |
Qionghai | 13 | 7(53.8%) | 2(15.4%) | 0(0%) | 4(30.8%) |
Wanning | 9 | 6(66.7%) | 2(22.2%) | 0(0%) | 1(11.1%) |
Wuzhishan | 13 | 8(61.5%) | 3(23.1%) | 0(0%) | 2(15.4%) |
Dongfang | 34 | 9(26.5%) | 13(38.2%) | 1(2.9%) | 11(32.4%) |
Danzhou | 12 | 8(66.7%) | 3(25%) | 0(0%) | 1(8.3%) |
Lingao | 15 | 11(73.3%) | 4(26.7%) | 0(0%) | 0(0%) |
Chengmai | 13 | 6(46.2%) | 4(30.8%) | 0(0%) | 3(23.1%) |
Dingan | 16 | 16(100%) | 0(0%) | 0(0%) | 0(0%) |
Tunchang | 14 | 10(71.4%) | 2(14.3%) | 0(0%) | 2(14.3%) |
Changjiang | 20 | 14(70%) | 4(20%) | 0(0%) | 2(10%) |
Baisha | 12 | 9(75%) | 1(8.3%) | 0(0%) | 2(16.7%) |
Qiongzhong | 18 | 8(44.4%) | 8(44.4%) | 0(0%) | 2(11.1%) |
Lingshui | 19 | 11(57.9%) | 7(36.8%) | 0(0%) | 1(5.3%) |
Baoting | 21 | 10(47.6%) | 6(28.6%) | 0(0%) | 5(23.8%) |
Ledong | 38 | 11(28.9%) | 15(39.5%) | 1(2.6%) | 11(28.9%) |
Total | 341 | 186(54.5%) | 93(27.3%) | 3(0.9%) | 59(17.3%) |
4. Discussion
Supplementary Files
Supplementary File 1Acknowledgments
Author Contributions
Conflicts of Interest
References and Notes
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Tuo, D.; Shen, W.; Yang, Y.; Yan, P.; Li, X.; Zhou, P. Development and Validation of a Multiplex Reverse Transcription PCR Assay for Simultaneous Detection of Three Papaya Viruses. Viruses 2014, 6, 3893-3906. https://doi.org/10.3390/v6103893
Tuo D, Shen W, Yang Y, Yan P, Li X, Zhou P. Development and Validation of a Multiplex Reverse Transcription PCR Assay for Simultaneous Detection of Three Papaya Viruses. Viruses. 2014; 6(10):3893-3906. https://doi.org/10.3390/v6103893
Chicago/Turabian StyleTuo, Decai, Wentao Shen, Yong Yang, Pu Yan, Xiaoying Li, and Peng Zhou. 2014. "Development and Validation of a Multiplex Reverse Transcription PCR Assay for Simultaneous Detection of Three Papaya Viruses" Viruses 6, no. 10: 3893-3906. https://doi.org/10.3390/v6103893