Establishment and Preliminary Application of One-step Reverse Transcriptase Droplet Digital PCR Assay for Bovine Viral Diarrhea Virus
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.