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
AbstractBovine 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.
LIU Xin-Yi,LI Dan,QIN Gang, et al. Establishment and Preliminary Application of Droplet Digital PCR Method for Detection of Bovine viral diarrhea virus[J]. 农业生物技术学报, 2025, 33(1): 190-199.
[1] 拜小强. 2021. 牛病毒性腹泻病毒结构蛋白克隆表达及核酸检测方法的建立[D]. 硕士学位论文, 西北民族大学, 导师: 魏锁成, pp. 1-67. (Bai X Q.2021. Clone and expression of structural protein of Bovine viral diarrhea virus and establishment of nucleic acid detection method[D]. Thesis for M.S., Northwest Minzu University, Supervisor: Wei S C, pp. 1-67.) [2] 陈瑞红, 林俊, 杨立新, 等. 2020. 牛病毒性腹泻病毒重组p80蛋白的间接ELISA方法的建立[J]. 中国预防兽医学报, 2020, 42(3): 252-257. (Chen H R, Lin J, Yang L X, et al.2020. An indirect ELISA for detection of antibody against Bovine viral diarrhea virus using recombinant p80 protein expressed in E. coli[J]. Chinese Journal of Preventive Veterinary Medicine, 42(3): 252-257.) [3] 陈亚娜, 王静, 訾占超, 等. 2017. 伪狂犬病病毒微滴数字PCR定量检测方法的建立[J]. 畜牧兽医学报, 48(09): 1705-1710. (Chen Y N, Wang J, Zi Z C, et al.2017. Development of droplet digital PCR for the detection of Pseudorabies virus[J]. Acta Veterinaria et Zootechnica Sinica, 48(09): 1705-1710.) [4] 丁金花. 2017. 牛病毒性腹泻病毒(BVDV)纳米抗体生物学特性研究及ELISA检测方法的建立[D]. 硕士学位论文, 石河子大学, 导师: 陈创夫, pp. 1-71. (Ding J H.2017. Biological characteristics of Nanobody to Bovine viral diarrhea virus (BVDV) and the establishment of ELISA detection methods[D]. Thesis for M.S., Shihezi University, Supervisor: Chen C F, pp. 1-71.) [5] 黄杰, 岳丰雄. 2017. 牛病毒性腹泻病毒间接免疫荧光检测方法的建立及应用[J]. 甘肃畜牧兽医, 47(3): 69-71. (Huang J, Yue F X.2017. Establishment and application of indirect immunofluorescence detection method for Bovine viral diarrhea virus[J]. Gansu Animal Husbandry and Veterinary, 47(3): 69-71.) [6] 贾开文. 2023. 新疆集约化牛场犊牛BVDV、BCoV、BNoV和BToV的流行病学调查及BCoV的分离鉴定[D].硕士学位论文, 石河子大学, 导师: 周霞, pp. 1-81. (Jia K W.2023. Epidemiological investigation of BVDV, BCoV, BNoV and BToV in calves of intensive cattle farms in Xinjiang and isolation and identification of BCoV[D]. Thesis for M.S., Shihezi University, Supervisor: Zhou X, pp. 1-81.). [7] 宋晓兵, 黄峰, 崔一平, 等. 2024. 柑橘黄龙病菌亚洲种微滴数字PCR检测方法的建立[J]. 农学学报, 14(01): 39-43. (Song X B, Huang F, Cui Y P, et al.2024. Establishment of droplet digital PCR detection method of Candidatus liberibacter asiaticus[J]. Journal of Agriculture, 14(01): 39-43.) [8] 王勤, 何博, 刘小可, 等. 2023. 川东北地区腹泻牛群中牛病毒性腹泻病毒的分子流行病学调查及病毒分离鉴定[J]. 家畜生态学报, 44(06): 60-64, 91. (Wang Q, He B, Liu X K, et al.2023. Molecular epidemiological investigation and virus isolation and identification of Bbovine viral diarrhea virus in diarrhea cattle in north east Si chuan[J]. Acta Ecologiae Animals Domastici, 44(06): 60-64, 91.) [9] 王亚新, 陈萌萌, 王露, 等. 2023. 牛病毒性腹泻病毒TaqMan荧光定量PCR检测方法的建立[J/OL]. 中国动物传染病学报, 1-10. (Wang Y X, Chen M M, Wang L, et al.2023. Establishment of TaqMan fluorescence quantitative PCR method for detection of Bovine viral diarrhea virus[J]. Chinese Journal of Animal Infectious Diseases, 1-10.) [10] 王妍瑾. 2021. 牛病毒性腹泻病毒HB-1株的分离鉴定及间接ELISA检测方法的建立[D]. 硕士学位论文,河北科技师范学院, 导师: 马增军. pp. 1-66. (Wang Y J.2021. Isolation and identification of Bovine viral diarrhea virus HB-1 strain and establishment of indirect ELISA for detection[D]. Thesis for M.S., Hebei Normal University of Science&Technology, Supervisor: Ma Z J. pp. 1-66.) [11] 赵成莹, 张娅娣, 袁静, 等. 2023. 牛病毒性腹泻病毒通用型及分型RT-PCR检测方法的建立及应用[J]. 黑龙江畜牧兽医,(07): 66-70, 77. (Zhao C Y, Zhang Y D, Yuan J, et al.2023. Establishment and application of RT-PCR detection method for universal and differential types of Bovine viral diarrhea virus[J]. Heilongjiang Animal Science and Veterinary Medicine,(07): 66-70, 77.) [12] 郑如雯, 黄涛, 吴道义, 等. 2023. 牛病毒性腹泻病毒LAMP-LFD检测方法的建立及初步应用[J]. 畜牧兽医学报, 54(11): 4745-4753. (Zheng R W, Huang T, Wu D Y, et al.2023. Establishment and preliminary application of LAMP-LFD detection method for Bovine viral diarrhea virus[J]. Acta Veterinaria et Zootechnica Sinica, 54(11): 4745-4753.) [13] 邹宏, 夏应菊, 李玲, 等. 2023. 猪瘟病毒和牛病毒性腹泻病毒双重荧光定量PCR检测方法的建立和初步应用[J]. 畜牧兽医学报, 54(10): 4422-4427. (Zou H, Xia Y J, Li L, et al.2023. Establishment and preliminary application of a dual real-time RT-PCR assay for CSFV and BVDV[J]. Acta Veterinaria et Zootechnica Sinica, 54(10): 4422-4427.) [14] Beaudeau F, Vermesse R, Maurin L, et al.2023. Assessing the reliability of innovative criteria to certify that cattle are non-persistently infected (non-PI) with the Bovine viral diarrhoea virus (BVDV)[J]. Veterinary Microbiology, 286: 109893. [15] Chicoski L M, Fritzen J T T, Lorenzetti E, et al.2023. Serological profile of respiratory viruses in unvaccinated steers upon their arrival at Brazilian feedlot facilities[J]. Brazilian Journal of Microbiology, 54(4): 3237-3244. [16] Deregt D, Loewen K G.1995. Bovine viral diarrhea virus: Biotypes and disease[J]. Canadian Veterinary Journal, 36(6): 371-378. [17] Falkenberg S M, Dassanayake R P, Neill J D, et al.2018. Evaluation of Bovine viral diarrhea virus transmission potential to nave calves by direct and indirect exposure routes[J]. Veterinary Microbiology, 2018: 144-148. [18] Gomez-Romero N, Ridpath J F, Basurto-Alcantara F J, et al.2021. Bovine viral diarrhea virus in cattle from Mexico: Current status[J]. Frontiers in Veterinary Science, 8: 673577. [19] Jokar M, Rahmanian V, Farhoodi M, et al.2021. Seroprevalence of Bovine viral diarrhea virus (BVDV) infection in cattle population in Iran: A systematic review and meta-analysis[J]. Tropical Animal Health and Production, 53(5): 449. [20] Lanyon S R, Hill F I, Reichel M P, et al.2014. Bovine viral diarrhoea: Pathogenesis and diagnosis[J]. Veterinary Journal, 199(2): 201-209. [21] Li H, Bai R, Zhao Z, et al.2018. Application of droplet digital PCR to detect the pathogens of infectious diseases[J]. Bioscience Reports, 38(6): BSR20181170. [22] Walz P H, Grooms D L, Bell T G, et al.2005. Platelet function and association of Bovine viral diarrhea virus with platelets of persistently infected cattle[J]. American Journal of Veterinary Research, 66(10): 1738-1742. [23] Zheng D, Ye X, Zhang M Z, et al.2016. Plasma EGFR T790M ctDNA status is associated with clinical outcome in advanced NSCLC patients with acquired EGFR-TKI resistance[J]. Scientific Reports, 6: 20913.
