ABC Assay: Method Development and Application to Quantify the Role of Three DWV Master Variants in Overwinter Colony Losses of European Honey Bees
Abstract
:1. Introduction
2. Materials and Methods
2.1. Primer Design
2.2. Viral Master Variant Plasmid Standards
2.3. Primer Optimisation
2.4. Viral Master Variant cRNA Standards
2.5. Method Validation
2.6. Application of the RT-qPCR (ABC Assay)
2.7. HRM Assay
2.8. Sequencing
3. Results
3.1. PCR and RT-PCR Sensitivities
3.2. Competitive PCR
3.3. PCR and RT-PCR Efficiencies
3.4. Robustness of the RT-qPCR (ABC Assay)
3.5. Detection of DWV in Honey Bees Using the ABC Assay
3.6. DWV Master Variants Implicated in OCL
4. Discussion
Supplementary Materials
Acknowledgments
Author Contributions
Conflicts of Interest
References
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Target | Primer Name | Sequence (5′–3′) | Genome Target (NC004830.2) | Size of Product (bp) | Reference |
---|---|---|---|---|---|
DWV | DWVnew-F1 | TACTAGTGCTGGTTTTCCTTT | 8653–8673 | This study | |
DWV Type A | DWVA-R1 | CTCATTAACTGTGTCGTTGAT | 8808–8788 | 155 | This study |
DWV Type B | DWVB-R1 | CTCATTAACTGAGTTGTTGTC | 8808–8788 | 155 | This study |
DWV Type C | DWVC-R1 | ATAAGTTGCGTGGTTGAC | 8805–8788 | 152 | This study |
DWV q | DWVq-R1 | CTGTGTCGTTGATAATTGAATCTC | 8656–8676 | 145 | [25] |
DWVq-F1 | TAGTGCTGGTTTTCCTTTGTC | 8800–8777 | |||
M13 | M13F | GTAAAACGACGGCCA | Na | 361 | [25] |
M13R | CAGGAAACAGCTATG | ||||
Actin | ActinR1 | AAGAATTGACCCACCAATCCATAC | Na | 120 | [25] |
ActinF1 | CCTGGAATCGCAGATAGAATGC |
Target | Insert |
---|---|
DWV A | TATCTTGGAATACTAGTGCTGGTTTTCCTTTGTCTTCATTAAAGCCACCTGGAACATCAGGTAAGCGATGGTTGTTTGATATTGAGCTACAAGATTCGGGATGTTATCTCTTGCGTGGAATGCGTCCCGAACTTGAGATTCAATTATCAACGACACAGTTAATGAGGAAAAAGGGA |
DWV B | TATCCTGGAATACTAGTGCTGGTTTTCCTTTATCTTCATTAAAACCGCCAGGCTCTTCTGGTAAGCGATGGTTGTTTGATATTGAATTACAAGATTCAGGATGTTATCTTTTGAGAGGGATGAGACCTGAACTTGAGATACAGTTGACAACAACTCAGTTAATGAGGAAGAAGGGA |
DWV C | TTTCGTGGAATACTAGTGCTGGTTTTCCTTTATCCTCACTGAAACCAGCTGGAACATCAGGAAAAAGGTGGTTATTTGATATTGAATTGCAAGATTCGGGATGTTATCTTTTACGAGGTATGCGTCCCGAATTAGAAATACAATTGTCAACCACGCAACTTATGAGGAAAAAGGGA |
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Kevill, J.L.; Highfield, A.; Mordecai, G.J.; Martin, S.J.; Schroeder, D.C. ABC Assay: Method Development and Application to Quantify the Role of Three DWV Master Variants in Overwinter Colony Losses of European Honey Bees. Viruses 2017, 9, 314. https://doi.org/10.3390/v9110314
Kevill JL, Highfield A, Mordecai GJ, Martin SJ, Schroeder DC. ABC Assay: Method Development and Application to Quantify the Role of Three DWV Master Variants in Overwinter Colony Losses of European Honey Bees. Viruses. 2017; 9(11):314. https://doi.org/10.3390/v9110314
Chicago/Turabian StyleKevill, Jessica L., Andrea Highfield, Gideon J. Mordecai, Stephen J. Martin, and Declan C. Schroeder. 2017. "ABC Assay: Method Development and Application to Quantify the Role of Three DWV Master Variants in Overwinter Colony Losses of European Honey Bees" Viruses 9, no. 11: 314. https://doi.org/10.3390/v9110314
APA StyleKevill, J. L., Highfield, A., Mordecai, G. J., Martin, S. J., & Schroeder, D. C. (2017). ABC Assay: Method Development and Application to Quantify the Role of Three DWV Master Variants in Overwinter Colony Losses of European Honey Bees. Viruses, 9(11), 314. https://doi.org/10.3390/v9110314