Establishment and Implementation of the Point-of-Care RT-RAA-CRISPR/Cas13a Diagnostic Test for Foot-And-Mouth Disease Virus Serotype O in Pigs
Abstract
1. Introduction
2. Materials and Methods
2.1. Design of RT-RAA Primers, CRISPR crRNAs, Digital Droplet PCR Primers, Probes, and RNA Reporters
2.2. Nucleic Acids and Samples
2.3. Screening of RT-RAA Primers and the Optimal Temperature for RT-RAA
2.4. Preparation of crRNAs
2.5. Screening of the crRNAs and the RT-RAA-CRISPR/Cas13a Reaction System
2.6. Lateral Flow Assay
2.7. RT-RAA-CRISPR/Cas13a Detection Method Specificity Test
2.8. Calibration of the Copy Number of FMDV Serotype O RNA by Digital PCR
2.9. Sensitivity of the RT-RAA-CRISPR/Cas13a Detection Method
2.10. RT-RAA-CRISPR/Cas13a Detection Method Repeatability Test
2.11. Simulated Clinical Samples Testing
2.12. Data Analysis
3. Results
3.1. Screening of RT-RAA Primers
3.2. Optimal Reaction Temperature Selection for RT-RAA
3.3. crRNA Selection for RT-RAA-CRISPR/Cas13a
3.4. Specificity Testing for RT-RAA-CRISPR/Cas13a Assay
3.5. Sensitivity Testing for RT-RAA-CRISPR/Cas13a Assay
3.6. Repeatability Testing for RT-RAA-CRISPR/Cas13a Assay
3.7. Simulated Clinical Sample Detection for RT-RAA-CRISPR/Cas13a Assay
4. Discussion
5. Conclusions
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Conflicts of Interest
References
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Primer Name | Primer Sequence (5′ → 3′) |
---|---|
FMDV-O-F1 | GAAATTAATACGACTCACTATAGGGGACCCAMCATGTGTGCAACCCC |
FMDV-O-R1 | CCGGTCRCCTATTCAGGCATAGAAGC |
FMDV-O-F2 | GAAATTAATACGACTCACTATAGGGCTATGAGAACAAACGCATYACMGTTGARG |
FMDV-O-R2 | GTAGATGTTGTTCARAATTGTGTTGATGAT |
FMDV-O-F3 | GAAATTAATACGACTCACTATAGGGCTTYAAATCYCTTGGYCAAACCATYACTCC |
FMDV-O-R3 | TTTGTAAAACCCAGTTCCATARTCCATGTG |
FMDV-O-F4 | GAAATTAATACGACTCACTATAGGGAACAACAARTTGGACATMAYCAAAGCTC |
FMDV-O-R4 | GAACAYRTCTTGTTGCATTCTCTTCATTTC |
FMDV-O-F5 | GAAATTAATACGACTCACTATAGGGTCGAAGACCCTCGARGCYATCCTCTC |
FMDV-O-R5 | CGTTCACCCADCGCAGGTAAAGTGA |
FMDV-O-F6 | GAAATTAATACGACTCACTATAGGGCAAACCATYACTCCAGCTGACAAAAGC |
FMDV-O-R6 | ATCACAGGTTTRTAAAACCCAGTTCCRTA |
crRNA Template 1 | CARTAYGACTGTGCCCTTCTCAACGGCAGTTTTAGTCCCCTTCGTTTTTGGGGTAGTCTAAATCCCCTATAGTGAGTCGTATTAATTTC |
crRNA Template 2 | AATAYGACTGTGCCCTTCTCAACGGMATGTTTTAGTCCCCTTCGTTTTTGGGGTAGTCTAAATCCCCTATAGTGAGTCGTATTAATTTC |
crRNA Template 3 | ATAYGACTGTGCCCTTCTCAACGGMATGGTTTTAGTCCCCTTCGTTTTTGGGGTAGTCTAAATCCCCTATAGTGAGTCGTATTAATTTC |
crRNA Template 4 | TAYGACTGTGCCCTTCTCAACGGMATGGGTTTTAGTCCCCTTCGTTTTTGGGGTAGTCTAAATCCCCTATAGTGAGTCGTATTAATTTC |
crRNA Template 5 | AYGACTGTGCCCTTCTCAACGGMATGGCGTTTTAGTCCCCTTCGTTTTTGGGGTAGTCTAAATCCCCTATAGTGAGTCGTATTAATTTC |
crRNA Template 6 | YGACTGTGCCCTTCTCAACGGMATGGCCGTTTTAGTCCCCTTCGTTTTTGGGGTAGTCTAAATCCCCTATAGTGAGTCGTATTAATTTC |
T7-oligo | GAAATTAATACGACTCACTATAGGG |
Fluorescence RNA reporter | 5′-FAM/UUUUUUUUUUU/BHQ1-3′ |
LFS RNA reporter | 5′-FAM/UUUUUUUUUUU/Biotin-3′ |
FMDV 3D F | GCGAGTCCTGCCACGGA |
FMDV 3D R | ACTGGGTTTTACAAACCTGTGA |
FMDV 3D probe | 5′-FAM/TCCTTTGCACGCCGTGGGAC/BHQ1-3′ |
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Meng, P.; Ni, B.; Li, C.; Sha, Z.; Liu, C.; Ren, W.; Wei, R.; Liu, F.; Li, J.; Wang, Z. Establishment and Implementation of the Point-of-Care RT-RAA-CRISPR/Cas13a Diagnostic Test for Foot-And-Mouth Disease Virus Serotype O in Pigs. Viruses 2025, 17, 721. https://doi.org/10.3390/v17050721
Meng P, Ni B, Li C, Sha Z, Liu C, Ren W, Wei R, Liu F, Li J, Wang Z. Establishment and Implementation of the Point-of-Care RT-RAA-CRISPR/Cas13a Diagnostic Test for Foot-And-Mouth Disease Virus Serotype O in Pigs. Viruses. 2025; 17(5):721. https://doi.org/10.3390/v17050721
Chicago/Turabian StyleMeng, Ping, Bo Ni, Chenyu Li, Zhou Sha, Chunju Liu, Weijie Ren, Rong Wei, Fuxiao Liu, Jinming Li, and Zhiliang Wang. 2025. "Establishment and Implementation of the Point-of-Care RT-RAA-CRISPR/Cas13a Diagnostic Test for Foot-And-Mouth Disease Virus Serotype O in Pigs" Viruses 17, no. 5: 721. https://doi.org/10.3390/v17050721
APA StyleMeng, P., Ni, B., Li, C., Sha, Z., Liu, C., Ren, W., Wei, R., Liu, F., Li, J., & Wang, Z. (2025). Establishment and Implementation of the Point-of-Care RT-RAA-CRISPR/Cas13a Diagnostic Test for Foot-And-Mouth Disease Virus Serotype O in Pigs. Viruses, 17(5), 721. https://doi.org/10.3390/v17050721