Development and Application of a TaqMan RT-qPCR for the Detection of Foot-and-Mouth Disease Virus in Pigs
Simple Summary
Abstract
1. Introduction
2. Materials and Methods
2.1. Samples and Materials
2.2. Nucleic Acid Extraction and Reverse Transcription
2.3. Primers and Probes
2.4. Construction and Characterization of Standard Plasmids
2.5. Preliminary Establishment of FDMV RT-qPCR
2.6. Optimization of RT-qPCR Reaction System
2.7. Establishment of the Standard Curve of RT-qPCR
2.8. Limit of Detection Determination
2.9. Specificity of FMDV RT-qPCR
2.10. Repeatability Test
2.11. Interference Assay
2.12. Detection of Clinical Samples
3. Results
3.1. Identification of Recombinant Plasmids
3.2. Primer Probe Selection
3.3. RT-qPCR Reaction System Optimization
3.4. Establishment of the Standard Curve
3.5. Sensitivity Test of FMDV RT-qPCR
3.6. Specificity of the Detection System
3.7. Repeatability Test of FMDV RT-qPCR
3.8. Interference Test of FMDV RT-qPCR
3.9. Clinical Sample Results
4. Discussion
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Conflicts of Interest
References
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Primer/Probe | Sequences (5′-3′) | Length (bp) | Product Length (bp) |
---|---|---|---|
FMVD-F1 | CCTCAAAAGACACTTCCACATGG | 23 | 99 |
FMVD-R1 | CTCTCCTTTGCACGCCGTG | 19 | |
FMVD-F2 | GAAGGACCTTACGAGGGACC | 20 | 124 |
FMVD-R2 | CGACTTGCAAAAGATGGTCATGGG | 24 | |
FMVD-P1 | HEX-CGAGGGTCTTCGAGGCCATCACAGGT-TAMRA | 26 | 99 |
FMVD-P2 | CY5-CGAGGGTCTTCGAGGCCATCACAGGT-BHQ3 |
Factor | Parameters | Plasmid Concentration (Copies/µL) | ||
---|---|---|---|---|
6.43 × 104 | 6.43 × 105 | 6.43 × 106 | ||
Primer concentration (µM) | 50 | 33.69 ± 0.55 | 29.76 ± 0.45 | 25.43 ± 0.32 |
70 | 33.34 ± 0.41 | 30.11 ± 0.05 | 25.15 ± 0.24 | |
90 | 34.74 ± 0.69 | 31.24 ± 0.16 | 25.82 ± 0.25 | |
100 | 33.61 ± 1.48 | 30.09 ± 0.20 | 25.59 ± 0.44 | |
Probe concentration (µM) | 10 | 33.61 ± 0.62 | 28.63 ± 0.33 | 24.43 ± 0.23 |
20 | 34.20 ± 0.94 | 29.78 ± 0.29 | 25.81 ± 0.78 | |
30 | 34.20 ± 0.94 | 29.78 ± 0.29 | 25.81 ± 0.78 | |
50 | 33.54 ± 0.34 | 29.77 ± 0.66 | 24.69 ± 0.13 | |
Annealing temperature (℃) | 58 | 24.98 ± 0.03 | 31.58 ± 0.99 | 35.12 ± 0.89 |
60 | 25.15 ± 0.07 | 30.88 ± 0.73 | 34.21 ± 0.35 | |
0.35 | 62 | 24.80 ± 0.01 | 30.36 ± 0.66 | 34.09 ± 0.39 |
Concentrations of Standard Plasmid | Within-Group Variance | Between-Group Variance | ||
---|---|---|---|---|
Mean ± SD | CV (%) | Mean ± SD | CV (%) | |
6.43 × 107 copies/µL | 14.74 ± 0.26 | 1.78 | 14.85 ± 0.14 | 0.97 |
6.43 × 105 copies/µL | 24.82 ± 0.27 | 1.07 | 24.99 ± 0.19 | 0.75 |
6.43 × 103 copies/µL | 32.65 ± 0.24 | 0.74 | 32.32 ± 0.23 | 0.70 |
Interference Reagents | Name | Ct (Mean ± SD) | CV (%) |
---|---|---|---|
Exogenous and endogenous interference with material | Blood of sheep | 25.42 ± 1.44 | 5.67 |
Liver tissues | 23.44 ± 0.79 | 4.45 | |
Intestinal contents | 21.42 ± 0.72 | 0.55 | |
Lung tissues | 22.18 ± 0.62 | 1.71 | |
Milk | 23.84 ± 0.94 | 3.35 | |
Mucin | 21.94 ± 0.45 | 3.38 | |
Oral swab | 22.48 ± 1.00 | 2.79 | |
Manure | 24.31 ± 0.42 | 3.94 | |
Animal feed | 21.01 ± 0.12 | 2.06 | |
Reagent 1 a | 21.93 ± 0.73 | 3.40 | |
Reagent 2 b | 22.49 ± 0.65 | 2.90 | |
Reagent 3 c | 23.81 ± 0.67 | 2.81 | |
Reagent 4 d | 22.81 ± 1.22 | 5.35 | |
Positive control | Standard plasmid | 22.59 ± 1.39 | 6.13 |
Negative control | ddH2O | N/A e | N/A |
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Dong, C.; Xiao, X.; Wang, M.; Sun, Y.; Jin, H.; Zhang, Y.; Zhao, H.; Cao, Q.; Yang, Y.; Yin, R. Development and Application of a TaqMan RT-qPCR for the Detection of Foot-and-Mouth Disease Virus in Pigs. Vet. Sci. 2024, 11, 541. https://doi.org/10.3390/vetsci11110541
Dong C, Xiao X, Wang M, Sun Y, Jin H, Zhang Y, Zhao H, Cao Q, Yang Y, Yin R. Development and Application of a TaqMan RT-qPCR for the Detection of Foot-and-Mouth Disease Virus in Pigs. Veterinary Sciences. 2024; 11(11):541. https://doi.org/10.3390/vetsci11110541
Chicago/Turabian StyleDong, Changying, Xingyu Xiao, Meiqi Wang, Yajuan Sun, Hui Jin, Yongzhe Zhang, Hongri Zhao, Qianyue Cao, Yanran Yang, and Rui Yin. 2024. "Development and Application of a TaqMan RT-qPCR for the Detection of Foot-and-Mouth Disease Virus in Pigs" Veterinary Sciences 11, no. 11: 541. https://doi.org/10.3390/vetsci11110541
APA StyleDong, C., Xiao, X., Wang, M., Sun, Y., Jin, H., Zhang, Y., Zhao, H., Cao, Q., Yang, Y., & Yin, R. (2024). Development and Application of a TaqMan RT-qPCR for the Detection of Foot-and-Mouth Disease Virus in Pigs. Veterinary Sciences, 11(11), 541. https://doi.org/10.3390/vetsci11110541