Development and Validation of Three Triplex Real-Time RT-PCR Assays for Typing African Horse Sickness Virus: Utility for Disease Control and Other Laboratory Applications
Abstract
:1. Introduction
2. Materials and Methods
2.1. Clinical Samples and Virus Isolates
2.2. Virus Cloning by End Point Dilution of AHSV Suspensions Containing More than One AHSV Serotype (Polyvalent Commercial LAV and Original AHSV Reference Strains)
2.3. Nucleid Acid Extraction
2.4. Primers and Probes Design
2.5. Optimisation of rRT-PCRs in Triplex
2.6. Validation Parameters Evaluated
2.7. Ethical Statement
3. Results
3.1. Analytical Sensitivity
3.2. Analytical Specificity
- Exclusivity
- Exclusivity among serotypes
3.3. Diagnostic Sensitivity
3.4. Diagnostic Specificity
3.5. Intra-Assay and Intra-Laboratory Repeatability
4. Discussion
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
Correction Statement
References
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AHSV | Primers (F/R) and Probe | Sequence 5′-3′ |
---|---|---|
Serotype 1 | AHS-1F | GCAAGCGCTGGCACTTG |
AHS-1R | TTCGAACTCATTCCTTACATCAACA | |
AHS1P | FAM-AATGTCTTAGATCGTCAACT-MGB | |
Serotype 2 | AHS-2F | CGGAAACTYTGTATTGCCAAA |
AHS-2R | TTGTCRTCCTGATCAACCCTAA | |
AHS-2P | Cy5-TGAAGGTGCTTACCCGATCTTTCCACA-BBQ | |
Serotype 3 | AHS-3F | AATTATTACAGCGGAGAATGCAGTT |
AHS-3R | GGTTATGAGTGGGGTGCGA | |
AHS-3P | FAM-AGAGTTGAGGTTGCGGGA-MGB | |
Serotype 4 | AHS-4F | TGAGGTGGAACACGAYATGTC |
AHS-4R | GATATGCCCCCTCACAYCTGA | |
AHS-4P | VIC-TATCGGRATTTATGTACAATGAG-MGB | |
Serotype 5 | AHS-5F | GAAGAGACAGGCGATTCAAATGA |
AHS-5R | AAAGCCACCCTTTTTGGTACAAA | |
AHS-5P | NED-TGTTGARATGCTGAGGC-MGB | |
Serotype 6 | AHS-6F | AGCCAGGGCTTCTTTGCA |
AHS-6R | CTCATGTTCAACCCACTGTACATTAA | |
AHS-6P | VIC-GTCATCACCGTAAGCG-MGB | |
Serotype 7 | AHS-7F | AGCCAGGGCTTCTTTGCA |
AHS-7R | CTCATGTTCAACCCACTGTACATTAA | |
AHS-7P | VIC-GTCATCACCGTAAGCG-MGB | |
Serotype 8 | AHS-8F | GAAATTATCAGCGGACTGACTAAGAA |
AHS-8R | AAACATCTACCTTTTGCGAATCTTG | |
AHS-8P | NED-ACGTGATTCTTTTCCC-MGB | |
Serotype 9 | AHS-9F | TACTGTGTCGGTGAGGGATTTT |
AHS-9R | GCCACGACCGGATATGA | |
AHS-9P | FAM-AAACAAACGAAATGTGAA-MGB |
Strain | Positive Serotypes in Triplex TS-rRT-PCRs Assays (Ct Value) * | Specific Antisera Used in the Cloning Process | |
---|---|---|---|
Before Cloning | After Cloning | ||
AHSV1 29/62 reference strain | 1 (14.7) and 4 (26.4) | 1 (18.3) | without antiserum |
AHSV2 82/61 reference strain | 2 (15.2) | 2 (18.9) | without antiserum |
AHSV3 13/63 reference strain | 1 (24.8) and 3 (15.0) | 3 (19.4) | without antiserum |
AHSV4 32/62 reference strain | 4 (13.5) | 4 (18.1) | without antiserum |
AHSV5 30–62 reference strain | 3 (15.6) and 5 (18.9) | 5 (17.2) | without antiserum |
AHSV6 39/62 reference strain | 6 (19.8) and 7 (21.3) | 6 (19.7) | without antiserum |
AHSV7 62/31 reference strain | 3 (15.5) and 7 (18.3) | 7 (22.7) | without antiserum |
AHSV8 62/10 reference strain | 5 (28.2) and 8 (15.5) | 8 (22.1) | without antiserum |
AHSV9 90/61 reference strain | 9 (15.0) | 9 (17.4) | without antiserum |
AHSV commercial vaccine OBP vial 1 (serotypes 1, 3 and 4) | 1 (17.2), 3 (15.5) and 4 (17.9) | 1 (14.0) | serotypes 3 and 4 |
3 (13.9) | serotypes 1 and 4 | ||
4 (14.7) | serotypes 1 and 3 | ||
AHSV commercial vaccine OBP vial 2 (serotypes 2, 6, 7 and 8) | 2 (22.0), 6 (21.8), 7 (23.9) and 8 (21.7) | 2 (17.4) | serotypes 6, 7 and 8 |
6 (18.4) | serotypes 2, 7 and 8 | ||
7 (17.7) | serotypes 2, 6 and 8 | ||
8 (13.6) | serotypes 2, 6 and 7 |
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Villalba, R.; Tena-Tomás, C.; Ruano, M.J.; Valero-Lorenzo, M.; López-Herranz, A.; Cano-Gómez, C.; Agüero, M. Development and Validation of Three Triplex Real-Time RT-PCR Assays for Typing African Horse Sickness Virus: Utility for Disease Control and Other Laboratory Applications. Viruses 2024, 16, 470. https://doi.org/10.3390/v16030470
Villalba R, Tena-Tomás C, Ruano MJ, Valero-Lorenzo M, López-Herranz A, Cano-Gómez C, Agüero M. Development and Validation of Three Triplex Real-Time RT-PCR Assays for Typing African Horse Sickness Virus: Utility for Disease Control and Other Laboratory Applications. Viruses. 2024; 16(3):470. https://doi.org/10.3390/v16030470
Chicago/Turabian StyleVillalba, Rubén, Cristina Tena-Tomás, María José Ruano, Marta Valero-Lorenzo, Ana López-Herranz, Cristina Cano-Gómez, and Montserrat Agüero. 2024. "Development and Validation of Three Triplex Real-Time RT-PCR Assays for Typing African Horse Sickness Virus: Utility for Disease Control and Other Laboratory Applications" Viruses 16, no. 3: 470. https://doi.org/10.3390/v16030470