Establishment and Application of a Quantitative PCR Method for E248R Gene of African Swine Fever Virus
Abstract
:Simple Summary
Abstract
1. Introduction
2. Materials and Methods
2.1. Construct of Plasmid Containing the E248R Gene
2.2. Virus Strains and Viral Samples
2.3. Primers and Probe for the Real-Time PCR
2.4. Formulation of Standard Curve
2.5. Optimization of Reaction Conditions
2.6. Specificity, Sensitivity and Repeatability Analysis
2.7. Viral Rescue and Characteristic Analysis of Recombinant PRRSV
2.8. Detection of ASFV Gene in the Samples and Recombinant Viruses
3. Results
3.1. Establishment of Standard Curve
3.2. Optimization of Reaction Conditions
3.3. Analysis of Repeatability, Sensitivity and Specificity
3.4. Detection of the Serum Samples and Recombinant PRRSV-Expressing ASFV E248R
4. Discussion
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Conflicts of Interest
References
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Name | Primers 5′-3′ | Position |
---|---|---|
E248R-F | 5′-GGAGGCTCTACAAGCAAA-3′ | 166996–167013 |
E248R-R | 5′-CATCACCGAATACGCCTA-3′ | 167105–167122 |
E248R-P | 5′FAM-AATACGACCAACATTATCAGCAAT-3′BHQ1-3′ | 167025–167050 |
System Components | Volume |
---|---|
Probe qPCR Mix (2×) | 10 μL |
probe | 0.4 μL |
Primer-F | 0.6 μL |
Primer-R | 0.6 μL |
Template | 1 μL |
Nuclease-free water | 7.4 μL |
Total | 20 μL |
Tm (°C) | Ct | Average Value ± Standard Deviation |
---|---|---|
56 | 24.95 | 24.927 ± 0.068 |
24.98 | ||
24.85 | ||
57 | 24.70 | 24.670 ± 0.030 |
24.67 | ||
24.64 | ||
58 | 24.47 | 24.767 ± 0.270 |
25.00 | ||
24.83 | ||
59 | 24.83 | 25.097 ± 0.326 |
25.00 | ||
25.46 | ||
60 | 24.22 | 25.25 ± 0.198 |
25.11 | ||
25.39 |
Standard Plasmid | Ct | Average Value | Standard Deviation | CV% |
---|---|---|---|---|
105 | 24.82 | 25.017 | 0.174 | 0.695 |
25.15 | ||||
25.08 | ||||
106 | 21.49 | 21.520 | 0.030 | 0.139 |
21.55 | ||||
21.52 | ||||
107 | 17.83 | 17.997 | 0.223 | 1.239 |
17.91 | ||||
18.25 |
Standard Plasmid | Ct | Average Value | Standard Deviation | CV% |
---|---|---|---|---|
105 | 24.82 | 24.303 | 0.480 | 1.977 |
24.22 | ||||
23.87 | ||||
106 | 21.49 | 21.700 | 0.279 | 1.286 |
21.86 | ||||
21.79 | ||||
107 | 17.83 | 17.540 | 0.252 | 1.439 |
17.42 | ||||
17.37 |
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Li, L.; Du, N.; Chen, J.; Zhang, K.; Tong, W.; Zheng, H.; Zhao, R.; Tong, G.; Gao, F. Establishment and Application of a Quantitative PCR Method for E248R Gene of African Swine Fever Virus. Vet. Sci. 2022, 9, 417. https://doi.org/10.3390/vetsci9080417
Li L, Du N, Chen J, Zhang K, Tong W, Zheng H, Zhao R, Tong G, Gao F. Establishment and Application of a Quantitative PCR Method for E248R Gene of African Swine Fever Virus. Veterinary Sciences. 2022; 9(8):417. https://doi.org/10.3390/vetsci9080417
Chicago/Turabian StyleLi, Liwei, Nannan Du, Jinxia Chen, Kuan Zhang, Wu Tong, Haihong Zheng, Ran Zhao, Guangzhi Tong, and Fei Gao. 2022. "Establishment and Application of a Quantitative PCR Method for E248R Gene of African Swine Fever Virus" Veterinary Sciences 9, no. 8: 417. https://doi.org/10.3390/vetsci9080417