Abstract：Moschus chrysogaster (sifanicus) viral hemorrhagic disease (McVHD) is an acute and highly lethal viral infectious disease, and can be a serious threat to the health and safety of musk deer population in China. McVHD is caused by Moschus chrysogaster hemorrhagic disease virus (McHDV) whose genome is highly homologous with Rabbit hemorrhagic disease virus (RHDV) which belongs to Caliciviridae Lagovirus. To develop a rapid and simple diagnostic method for McVHD, a pair of specific primers McVHD-F/R were designed and synthesized based on the comparative analysis of whole genome sequence of McHDV and RHDV in GenBank. Based on the optimization of reaction condition and analysis of specificity and sensitivity, the reverse transcription PCR (RT-PCR) method for McVHD diagnosis was established. The results showed that a specific 448 bp fragment was amplified from McHDV genome using the established RT-PCR method. The RT-PCR method was highly specific and hypersensitive, and had no cross-reaction?with?other 10 pathogenic microorganisms such as Salmonella, Pasteurella multocida, Klebsiella pneumoniae. The RT-PCR was?able?to?detect?as?low?as?10-5 ng/μL (2.38×; 103 copies/μL) of?McHDV RNA.?The detection results?of?clinical?samples?revealed?that?the?diagnostic method was able to effectively and quickly detect viral nucleic acid in clinical samples, and the results were completely consistent with the traditional pathogen separation identification. The RT-PCR method established in this study could provide technical support for diagnosis and molecular epidemiological survey of McVHD.
[1] 包世俊, 邢小勇, 丁小琴, 等. 2018. 一种新的动物传染病—马麝病毒性出血症[J]. 中国农业科学, 51(5): 1012. (Bao S J, Xing X Y, Ding X Q, et al.2018. A new animal infectious disease, Moschus chrysogaster viral hemorrhagic disease[J]. Scientia Agricultura Sinica, 51(5): 1012.) [2] 蒋应文. 1998. 驯养马麝的繁殖与行为观察[J]. 动物学杂志, 33(4): 39-41. (Jiang Y W.1998. Observation on the reproduction and behavior of Moschues chryschdster[J]. Chinese Journal of Zoology, 33(4): 39-41.) [3] 靳巍, 刘峰, 任锐, 等. 2017. 兔瘟常用检测方法[J]. 吉林畜牧兽医, 2017, 38(08): 51, 53.(Jin W, Liu F, Ren R, et al. Common detection methods of rabbit hemorrhagic disease[J]. Jilin Animal Husbandry and Veterinary Medicine, 2017, 38(08): 51, 53.) [4] 李明, 盛和林. 1996. 中国麝科动物分类的研究现状[J]. 动物学杂志, 31(02): 56-58. (Li M, Sheng H L.1996. Research situation of on the musk deer in China[J]. Chinese Journal of Zoology, 31(02): 56-58.) [5] 刘文华, 佟建明. 2005. 中国的麝资源及其保护与利用现状分析[J]. 中国农业科技导报, 7(4): 28-32. (Liu W H, Tong J M.2005. Analysis on protection and utilization of musk deer resources in China[J]. Journal of Agricultural Science and Technology, 7(4): 28-32.) [6] 路浩, 蒋应文, 黄有德. 2004. 甘肃兴隆山马麝呼吸系统疾病流行病初步调查[J]. 动物学杂志, 39(6): 62-64. (Lu H, Jiang Y W, Huang Y D.2004. Primary epidemiological investigation of respiratory disease of Moschus sifanicus in Xinglong Mountain of Gansu province[J]. Chinese Journal of Zoology, 2004,39(6) :62-64.) [7] 路浩, 黄有德, 刘宗平. 2008. 马麝呼吸系统疾病病原学研究[J]. 中国兽医学报, 28(1): 45-47. (Lu H, Huang Y D, Liu Z P.2008. Etiologic study on respiratory disease in Moschus sifanicus[J]. Chinese Journal of Veterinary Science, 28(1): 45-47.) [8] 路浩, 胡俊杰, 黄有德, 等. 2010. 马麝出血性肺炎的病理学观察[J].中国兽医杂志, 46(06): 38-39, 97.(Lu H, Hu J J, Huang Y D, et al. 2010. Pathological observation on hemorrhagic pneumonia in Moschus sifanicus[J]. Chinese Journal of Veterinary Medicine, 46(06): 38-39, 97.) [9] 吕向辉, 乔继英, 吴晓民, 等. 2010. 青海马麝肠道寄生虫感染情况调查[J]. 中国兽医杂志, 46(12): 56-57. (Lv X H, Qiao J Y, Wu X M, et al.2010. Investigation on intestinal parasitic infection in Moschus sifanicus (chrysogaster) of Qinghai[J]. Chinese Journal of Veterinary Medicine, 46(12): 56-57.) [10] 孟秀祥, 冯金朝, 周宜君, 等. 2006. 野捕和圈养繁殖雄性马麝行为格局的比较[J]. 应用生态学报, 17(11): 2084-2087. (Meng X X, Feng J Z, Zhou Y J, et al.2006. Behavioral patterns of wild-caught and captive-bred male musk deer[J]. Chinese Journal of Applied Ecology, 17(11): 2084-2087.) [11] 王芳, 李超美, 杨龙圣, 等. 2007. 兔出血症病毒RTPCR检测方法的建立及应用[J]. 农业生物技术学报, 15(3): 409-413. (Wang F, Li C M, Yang L S, et al.2007. Establishment and application of RTPCR test for Rabbit hemorrhagic disease virus[J]. Journal of Agricultural Biotechnology, 15(3): 409-413.) [12] 吴家炎, 王伟. 2006. 中国麝类[M]. 北京: 中国林业出版社, pp. 175-185. (Wu J Y, Wang W.2006. The musk deer in China[M]. Beijing: China Forestry Publishing House, pp. 175-185.) [13] 杨泽晓, 赵希仑, 李岩, 等. 2016. 兔出血症病毒与兔出血症病毒2型复合RT-PCR检测方法的初步研究[J]. 中国兽医科学, 46(06): 710-716. (Yang Z X, Zhao X L, Li Y, et al.2016. Development of a complex RT-PCR for the detection and differentiation of Rabbit hemorrhagic disease virus and Rabbit hemorrhagic disease virus 2[J]. Chinese Veterinary Science, 46(06): 710-716.) [14] 朱裕华, 胡安康, 陈全刚, 等. 2016. 兔病毒血症RT-PCR检测方法的建立及初步应用[J]. 上海畜牧兽医通讯, (06): 11-13. (Zhu Y H, Hu A K, Chen Q G, et al. 2016. Establishment and preliminary application of RTPCR test for Rabbit hemorrhagic disease virus[J]. Shanghai Journal of Animal Husbandry and Veterinary Medicine, (06): 11-13.) [15] Geng S, Ma S.2000. Decline of musk deer in China and prospects for management[J]. Environmental Conservation, 26(7): 323-325.
