| 刊名 | Animal Husbandry and Feed Science |
| 作者 | Bing Liyuan1, Ye Jingfei2,Liu Jiwei3, Wang Shuai1, Zheng Dingcheng1, Meng Tingting1, Shang Yumo1, Ciren Qiongda1, Sun Liang2*, Guo Li1* |
| 作者单位 | 1. Jilin Agricultural Science and Technology University, Jilin 132109, China; 2. Jilin Provincial Animal Disease Prevention and Control Center, Changchun 130000, China; 3. Jilin Academy of Agricultural Sciences, Changchun 130000, China |
| DOI | 10.19578/j.cnki.ahfs.2023.01-06.007 |
| 年份 | 2023 |
| 刊期 | 1 |
| 页码 | 30-35 |
| 关键词 | Bovine viral diarrhea virus; One-step procedure; Droplet digital PCR; Quantitative detection |
| 摘要 | [Objective] The paper was to establish a one-step reverse transcriptase droplet digital PCR (RT-ddPCR) assay for bovine viral diarrhea virus (BVDV). [Method] Based on one-step real-time quantitative PCR (RT-qPCR) assay, BVDV-specific primers and probes were designed in this study. The reverse transcriptase, annealing temperature, primer and probe concentrations and reaction conditions of RT-ddPCR assay were optimized. Meantime, the specificity, sensitivity and repeatability of RT-ddPCR assay were evaluated. [Result] The optimal reverse transcription system for the established RT-ddPCR assay was as follows: commercial one-step reverse transcriptase droplet digital PCR kit with matching reagents, a final primer concentration of 900 nmol/L, a final probe concentration of 250 nmol/L and an optimal annealing temperature of 57 ℃. The results were negative when the method was used to detect other common epidemic viruses; the minimum detection limit was 3.2 copies/μL with good repeatability, and the coefficient of variation was less than 5%. RT-ddPCR and RT-qPCR assays were used to test 24 bovine swab samples and the test results showed that the established RT-ddPCR assay was superior to RT-qPCR assay. [Conclusion] The RT-ddPCR assay established in this study has strong specificity, high sensitivity and good repeatability, and is suitable for nucleic acid detection of clinical samples. This study provides a technical support for early detection and quantitative diagnosis of BVDV infection. |
