Establishment and Preliminary Application of Droplet Digital PCR Method for Detection of Bovine viral diarrhea virus
LIU Xin-Yi1,2,*, LI Dan3,*, QIN Gang4, CHEN Jun-Zhen1, FU Qiang1,2,5,**, SHI Hui-Jun1,2,**
1 College of Veterinary Medicine, Xinjiang Agricultural University, Urumqi 830052, China; 2 Xinjiang Key Laboratory of New Drug Study and Creation for Herbivorous Animal, Urumqi 830052, China; 3 Tecon Bio-pharmaceutical Co., Ltd., Urumqi 830032, China; 4 Xinjiang Bachu Country Animal Husbandry and Veterinary Station, Bachu 843800, China; 5 Institute of Western Agriculture, the Chinese Academy of Agricultural Sciences, Changji 831100, China
Abstract：Bovine viral diarrhea virus (BVDV) is one of the main pathogens causing viral diarrhea of calves(Bos sp.), which leads to decreased immunity and increase of morbidity and mortality. At present, the commonly used detection methods for BVDV have low sensitivity and are prone to false positives, This research is based on droplet digital PCR (ddPCR) technology to establish the accurate detection technology of BVDV. Firstly, primers and probes were designed for the BVDV 5' UTR gene conserved sequence region, and the BVDV 5' UTR gene was synthesized and the plasmid standard was constructed. Real-time quantitative PCR (qPCR) and ddPCR methods were established using BVDV plasmid standards, and the reaction parameters and conditions were optimized to analyze their sensitivity, specificity and repeatability, and the clinical application was carried out. The results showed that the optimal primers and probe concentrations were 300 and 500 nmol/L, respectively. The standard curve showed a good linear relationship, and the minimum detection limit was 1 × 104 copies/μL, with good specificity and repeatability. The minimum detection limit of ddPCR was 0.64 copies/μL, and the sensitivity of ddPCR was 15 625 times higher than that of qPCR. The method had good specificity and repeatability. The detection rate of ddPCR and qPCR was 15.58% and 14.63% respectively in 82 samples. The ddPCR method established in this study was more sensitive, efficient, specific and reproducible than the qPCR method, which provides technical support for the differential diagnosis of BVDV.
刘芯怡, 李丹, 秦刚, 陈俊贞, 付强, 史慧君. 基于微滴式数字PCR检测牛病毒性腹泻病毒方法的建立及初步应用[J]. 农业生物技术学报, 2025, 33(1): 190-199.
LIU Xin-Yi, LI Dan, QIN Gang, CHEN Jun-Zhen, FU Qiang, SHI Hui-Jun. Establishment and Preliminary Application of Droplet Digital PCR Method for Detection of Bovine viral diarrhea virus. 农业生物技术学报, 2025, 33(1): 190-199.
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