Detection and Phylogenetic Analysis of Caprine Arthritis Encephalitis Virus Using TaqMan-based qPCR in Eastern China
Abstract
:Simple Summary
Abstract
1. Introduction
2. Materials and Methods
2.1. Sample Collection
2.2. Primers and Probes
2.3. RNA Extraction and Reverse Transcription
2.4. The TaqMan RT-qPCR Method
2.5. Standard Curve Generation
2.6. Specificity and Sensitivity Analysis of TaqMan RT-qPCR Assay
2.7. Repeatability Determination of TaqMan RT-qPCR Assay
2.8. Detecting of Clinical Samples
2.9. Construction of Phylogenetic Tree
3. Results
3.1. Designing Specific Primers and Probes
3.2. Preparation of the Standard Curve of Caprine Arthritis Encephalitis Virus
3.3. The Specificity of the RT-qPCR Assay
3.4. The Sensitivity of the RT-qPCR Assay
3.5. Intra-Assay and Inter-Assay Reproducibility of the RT-qPCR Assay
3.6. Detection of Samples from Farms in Eastern China
3.7. Phylogenetic Analyses of Caprine Arthritis Encephalitis Virus
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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Virus | Location | Primer/Probe | Sequences (5′-3′) | Length (bp) |
---|---|---|---|---|
CAEV | gag | Primer-CAEV-F | ATGTCTTTGCAGGCCACATT | 68 |
Primer-CAEV-R | TGCAATGTCTCAGGTTTCATCC | |||
Probe-CAEV | ROX-CCATTTGGCAGCACATTAGACCCGC-BHQ−1 | |||
CAEV | gag | CAEV-F | AACTGGAAAGCAGTAGAC | 571 |
CAEV-R | TACACTAGCTTGTTGCAC |
Virus Standards | Copy Number | Intra-Assay | Inter-Assay | ||
---|---|---|---|---|---|
CT Value (Mean ± SD) | CV (%) | CT Value (Mean ± SD) | CV (%) | ||
CAEV | 109 | 13.16 ± 0.06 | 0.3636 | 14.86 ± 0.19 | 0.6621 |
108 | 15.55 ± 0.14 | 0.7356 | 18.15 ± 0.07 | 0.2131 | |
107 | 18.44 ± 0.18 | 0.6902 | 21.87 ± 0.54 | 1.3212 | |
106 | 21.30 ± 0.05 | 0.1232 | 25.13 ± 0.20 | 0.4617 | |
105 | 24.23 ± 0.06 | 0.1856 | 28.29 ± 0.05 | 0.1212 | |
104 | 26.56 ± 0.23 | 0.6158 | 31.93 ± 0.49 | 0.8793 | |
103 | 29.38 ± 0.25 | 0.6095 | 34.34 ± 0.26 | 0.7571 | |
102 | 31.70 ± 0.42 | 0.9550 | 36.18 ± 0.47 | 1.2991 | |
101 | 34.34 ± 0.11 | 0.2615 | 38.56 ± 0.28 | 0.7261 | |
100 | NaN | NaN | NaN | NaN | |
NC | NaN | NaN | NaN | NaN |
Location | Farm | Farm Size | Type of Sample | Sample Collection | Clinical Symptoms | NO. of Samples | NO. of Positive Samples |
---|---|---|---|---|---|---|---|
Jiangsu | JS-1 | 500–1000 | Goat Blood | Jugular | No | 34 | 2 (JS-2023-1, JS-2023-2) |
JS-2 | >2000 | Goat Blood | No | 77 | 0 | ||
JS-3 | >2000 | Goat Blood | No | 35 | 0 | ||
Shanghai | SH-1 | >2000 | Sheep Blood | Jugular | No | 352 | 3 (SH-2023) |
Sheep Brain | Dissection | Yes | 4 | 0 | |||
SH-2 | 500–1000 | Sheep Blood | Jugular | No | 150 | 0 | |
SH-3 | 500–1000 | Sheep Blood | Jugular | No | 128 | 1 | |
Total | 780 | 6 |
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Tian, Y.; Zhang, H.; Zhang, Y.; Zhang, X.; Guan, Z.; Zhang, J.; Qiu, Y.; Li, B.; Liu, K.; Li, Z.; et al. Detection and Phylogenetic Analysis of Caprine Arthritis Encephalitis Virus Using TaqMan-based qPCR in Eastern China. Vet. Sci. 2024, 11, 138. https://doi.org/10.3390/vetsci11030138
Tian Y, Zhang H, Zhang Y, Zhang X, Guan Z, Zhang J, Qiu Y, Li B, Liu K, Li Z, et al. Detection and Phylogenetic Analysis of Caprine Arthritis Encephalitis Virus Using TaqMan-based qPCR in Eastern China. Veterinary Sciences. 2024; 11(3):138. https://doi.org/10.3390/vetsci11030138
Chicago/Turabian StyleTian, Yutong, Hailong Zhang, Yan Zhang, Xinya Zhang, Zhilei Guan, Junjie Zhang, Yafeng Qiu, Beibei Li, Ke Liu, Zongjie Li, and et al. 2024. "Detection and Phylogenetic Analysis of Caprine Arthritis Encephalitis Virus Using TaqMan-based qPCR in Eastern China" Veterinary Sciences 11, no. 3: 138. https://doi.org/10.3390/vetsci11030138