Ⅰ型鸭病毒性肝炎病毒RT-PCR检测方法的建立  

Development of a RT-PCR assay to detect duck hepatitis virus typeⅠ

作　　者：程安春[1] 汪铭书[1] 信洪一[1] 陈海军[1] 杨苗[1] 郭宇飞[1] 朱德康[1] 贾仁勇[2] 袁桂萍[1] 陈孝跃[1] 

机构地区：[1]四川农业大学动物科技学院禽病防治研究中心,四川雅安625014 [2]动物疫病与人类健康四川省重点实验室,四川雅安625014

出　　处：《中国兽医科学》2007年第1期38-42,共5页Chinese Veterinary Science

基　　金：国家科技攻关重大项目(2004BA901A03);国家农业科技成果转化资金项目(2006GB2F000249);教育部"新世纪优秀人才支持计划"项目(NCET-04-0906);四川省基础研究重大项目(05JY029-109);四川省杰出青年学科带头人基金后续资助项目(03ZQ026-028);四川省重点建设学科项目(SZD0418)

摘　　要：根据获得的Ⅰ型鸭病毒性肝炎病毒（DHVⅠ）RNA聚合酶基因序列，应用Primer Premier5．0软件设计了1对引物，建立了检测DHVⅠ的RT-PCR方法。该法能从DHVⅠ中扩增到440bp的条带，而对正常鸭胚尿囊液、健康鸭肝、鸭瘟病毒、番鸭细小病毒、鹅细小病毒、雏鹅新型病毒性肠炎病毒、禽流感病毒（H5N2亚型）、鸭病毒性肿头出血症病毒、鸭源多杀性巴氏杆菌（5：A）的扩增结果均为阴性。该法最低可以检测到30pg的DHV工核酸模板。RT-PCR对DHVⅠ强毒CHv-1株人工感染发病死亡鸭肝的检测结果与病毒分离和Dot—ELISA检测结果的阳性检出率均为100％，对脾、肺、脑病料的检出率显著（P≤0．01）高于病毒分离和Dot-ELISA。RT-PCR、病毒分离和Dot—ELSA对1987～2005年采集且保存于-20℃的经病毒分离已确诊为鸭肝炎的临床送检肝病料的检出率分别为100％（19／19）、36．84％（7／19）和57．98％（11／19），对2000-2005年采集且于-20℃保存的来源于中国18个省份发病鸭群的48份肝病料的检出率分别为100％（48／48）、56．25％（27／48）和75．00％（36／48）。结果表明，建立的RT—PCR方法具有良好的特异性和敏感性，可用于DHVⅠ的分离鉴定、临床病料的检测和分子流行病学调查。According to the sequence of RNA polymerase encoding region in genome of serotype I duck hepatitis virus （DHV Ⅰ ）, primers were designed with help of Primer Premier 5.0 design software , and a RT PCR assay for the detection of DHV Ⅰ was developed. A specific 440 bp fragment was amplified from RNA templates of DHV Ⅰ virulence CHv-1 strain, but no bands were amplified with templates extracted respectively from normal duck embryo allantoic fluid, health duck liver tissue, duck plague virus （DPV）, Muscovy parvovirus（MPV）,gosling parvovirus（GPV）, gosling new type viral enteritis virus （NGVEV）, avian influence virus （AIV/H5N2 subtype）, duck swollenhead hemorrhagic disease virus （DSHDV）, Pasteurella multocida （PM） （5 ： A） from dead duck. The sensitivity of the RT-PCR assay was 30 pg. All results were 100 percent positive when the livers of dead ducks which were experimentally infected with DHV Ⅰ virulence CHv-1 strain were detected by the RT PCR, virus isolation and Dot ELISA. The positive rates by RT-PCR were significantly higher（P≤0.01）than those by the virus isolation and the Dot-ELISA when samples were from spleen, lung and brain. The positive rates were 100%（19/19）,36. 84% （7/19） and 57.98%（11/19）when clinical liver samples collected from 1987 to 2005 and stored at -20℃, which had been diagnosed as DHV Ⅰ by the virus isolation, were detected by the RT PCR, the virus isolation and the Dot-ELISA, respectively. The positive rates were 100% （48/48）, 56.25 % （27/48） and 75. 00%（36/48） when liver samples of clinical cases from 18 provinces of China collected from 2000 to 2005 and stored at -20 ℃were detected by the RT-PCR, the virus isolation and the Dot-ELISA, respectively. Results showed that the RT-PCR is specific and sensitive, and can be used as a method to identify DHV Ⅰisolates, detect samples of clinical cases and investigate molecular epidemiology of DVH Ⅰ.

关 键 词：Ⅰ型鸭病毒性肝炎病毒 RT—PCR检测 应用 

分 类 号：S852.659.6[农业科学—基础兽医学]