The Development of a Multiplex Real-Time Quantitative PCR Assay for the Differential Detection of the Wild-Type Strain and the MGF505-2R, EP402R and I177L Gene-Deleted Strain of the African Swine Fever Virus
Abstract
:Simple Summary
Abstract
1. Introduction
2. Materials and Methods
2.1. Viral Strains
2.2. Clinical Samples
2.3. DNA Extraction
2.4. Primers and Probes
2.5. Construction of Standard Plasmids
2.6. Optimization of the Reaction Conditions of the Multiplex Real-Time qPCR
2.7. Construction of Standard Curves
2.8. Specificity Analysis of the Multiplex Real-Time qPCR
2.9. Sensitivity Analysis of the Multiplex Real-Time qPCR
2.10. Repeatability Analysis of the Multiplex Real-Time qPCR
2.11. Test of Clinical Samples by the Multiplex Real-Time qPCR
2.12. Statistical Analysis
3. Results
3.1. Construction of Standard Plasmids
3.2. Optimal Reaction Conditions of the Multiplex Real-Time qPCR
3.3. Standard Curves of the Multiplex Real-Time qPCR
3.4. Specificity of the Multiplex Real-Time qPCR
3.5. Sensitivity of the Multiplex Real-Time qPCR
3.6. Repeatability of the Multiplex Real-Time qPCR
3.7. Evaluation of Clinical Samples
4. Discussion
5. Conclusions
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
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Target Gene | Name | Sequences (5′→3′) | Amplicon (bp) |
---|---|---|---|
B646L | P72-F | GGCGTATAAAAAGTCCAGGAAATTC | 79 |
P72-R | TTCGGCGAGCGCTTTATC | ||
P72-P | FAM-TCACCAAATCCTTTTGCGATGCAAGCT-BHQ1 | ||
MGF505-2R | 505-2R-F | AGTCATGCACGGCATATACAA | 153 |
505-2R-R | GGTTTAAACCGTGCCACATCC | ||
505-2R-P | VIC-ACGCGGCCACCCAATTCAGAGAC-BHQ1 | ||
EP402R | EP402R-F | TACTACATGCGTCCCTCAACAC | 182 |
EP402R-R | AATGGCGGGATATTGGGTAGT | ||
EP402R-P | CY5-ACCGTGTCCTCCACCCAAACCAT-BHQ2 | ||
I177L | I177L-F | GGCATAATTATCAAATGCGAAGGG | 122 |
I177L-R | TGGAAAGTTAATGATCAGGGCTT | ||
I177L-P | Texas Red-AATCCTAGCTTGCCGGTAATGGCT-BHQ2 |
Multiplex Real-Time qPCR | OIE-Recommended Real-Time qPCR | ||||
---|---|---|---|---|---|
Reagent | Volume (μL) | Final Concentration (nM) | Reagent | Volume (μL) | Final Concentration (nM) |
Premix Ex Taq (2×) | 10 | 1× | Premix Ex Taq (2×) | 12.5 | 1× |
P72-F (20 μM) | 0.2 | 200 | P72-P1 (50 μM) | 1 | 2000 |
P72-R (20 μM) | 0.2 | 200 | P72-P2 (50 μM) | 1 | 2000 |
P72-P (20 μM) | 0.2 | 200 | P72-P (5 μM) | 1 | 200 |
I177L-F (20 μM) | 0.1 | 100 | / | / | / |
I177L-R (20 μM) | 0.1 | 100 | / | / | / |
I177L-P (20 μM) | 0.1 | 100 | / | / | / |
EP402R-F (20 μM) | 0.2 | 200 | / | / | / |
EP402R-R (20 μM) | 0.2 | 200 | / | / | / |
EP402R-P (20 μM) | 0.2 | 200 | / | / | / |
MGF505-2R-F (20 μM) | 0.2 | 200 | / | / | / |
MGF505-2R-R (20 μM) | 0.2 | 200 | / | / | / |
MGF505-2R-P (20 μM) | 0.3 | 300 | / | / | / |
Template | 2 | / | Template | 2.5 | / |
Distilled water | Up to 20 | / | Distilled water | Up to 25 | / |
Plasmid | Concentration (Copies/μL) | Ct Values of Intra-Assay for Repeatability | Ct Values of Inter-Assay for Reproducibility | ||||
---|---|---|---|---|---|---|---|
SD | CV (%) | SD | CV (%) | ||||
pASFV-B646L | 3.21 × 108 | 12.60 | 0.10 | 0.80 | 12.69 | 0.13 | 1.01 |
3.21 × 106 | 18.45 | 0.16 | 0.88 | 18.45 | 0.11 | 0.62 | |
3.21 × 104 | 24.53 | 0.18 | 0.71 | 24.75 | 0.30 | 1.20 | |
pASFV-I177L | 3.21 × 108 | 12.41 | 0.07 | 0.59 | 12.45 | 0.10 | 0.84 |
3.21 × 106 | 18.42 | 0.17 | 0.92 | 18.37 | 0.13 | 0.69 | |
3.21 × 104 | 24.44 | 0.23 | 0.92 | 24.37 | 0.20 | 0.83 | |
pASFV-MGF505-2R | 3.21 × 108 | 12.57 | 0.09 | 0.62 | 12.59 | 0.17 | 1.36 |
3.21 × 106 | 18.70 | 0.17 | 0.89 | 18.75 | 0.21 | 1.10 | |
3.21 × 104 | 24.81 | 0.11 | 0.43 | 24.79 | 0.16 | 0.63 | |
pASFV-EP402R | 3.21 × 108 | 13.18 | 0.07 | 0.53 | 13.29 | 0.13 | 0.99 |
3.21 × 106 | 19.05 | 0.09 | 0.46 | 19.03 | 0.30 | 1.56 | |
3.21 × 104 | 25.13 | 0.07 | 0.26 | 25.19 | 0.13 | 0.53 |
Source | Tissue | Environmental Swab | Whole Blood | Nasopharyngeal Swab | Total |
---|---|---|---|---|---|
Pig farm | / | 2/107 (1.87%) c | 11/1134 (0.97%) | 3/37 (8.11%) | 15/1278 (1.25%) d |
Slaughterhouse | 58/480 (12.08%) b | 214/752 (28.46%) a | / | / | 330/1908 (17.30%) b |
Farmer’s market | 14/175 (8.00%) b | 53/451 (11.75%) b | / | / | 67/626 (10.70%) c |
Harmless disposal site | 105/365 (28.77%) a | 16/62 (25.81%) a | / | / | 121/427 (28.34%) a |
Total | 177/1020 (17.35%) B | 285/1372 (20.77%) A | 11/1134 (0.97%) D | 3/37 (8.11%) C | 534/4239 (12.60%) |
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Zhao, K.; Shi, K.; Zhou, Q.; Xiong, C.; Mo, S.; Zhou, H.; Long, F.; Wei, H.; Hu, L.; Mo, M. The Development of a Multiplex Real-Time Quantitative PCR Assay for the Differential Detection of the Wild-Type Strain and the MGF505-2R, EP402R and I177L Gene-Deleted Strain of the African Swine Fever Virus. Animals 2022, 12, 1754. https://doi.org/10.3390/ani12141754
Zhao K, Shi K, Zhou Q, Xiong C, Mo S, Zhou H, Long F, Wei H, Hu L, Mo M. The Development of a Multiplex Real-Time Quantitative PCR Assay for the Differential Detection of the Wild-Type Strain and the MGF505-2R, EP402R and I177L Gene-Deleted Strain of the African Swine Fever Virus. Animals. 2022; 12(14):1754. https://doi.org/10.3390/ani12141754
Chicago/Turabian StyleZhao, Kang, Kaichuang Shi, Qingan Zhou, Chenyong Xiong, Shenglan Mo, Hongjin Zhou, Feng Long, Haina Wei, Liping Hu, and Meilan Mo. 2022. "The Development of a Multiplex Real-Time Quantitative PCR Assay for the Differential Detection of the Wild-Type Strain and the MGF505-2R, EP402R and I177L Gene-Deleted Strain of the African Swine Fever Virus" Animals 12, no. 14: 1754. https://doi.org/10.3390/ani12141754
APA StyleZhao, K., Shi, K., Zhou, Q., Xiong, C., Mo, S., Zhou, H., Long, F., Wei, H., Hu, L., & Mo, M. (2022). The Development of a Multiplex Real-Time Quantitative PCR Assay for the Differential Detection of the Wild-Type Strain and the MGF505-2R, EP402R and I177L Gene-Deleted Strain of the African Swine Fever Virus. Animals, 12(14), 1754. https://doi.org/10.3390/ani12141754