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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 Premier 5.0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440 bp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30 pg的DHVⅠ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-ELISA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 obtained DHV Ⅰ RNA polymerase gene sequence, Primer Premier 5.0 software was used to design a pair of primers and a RT-PCR method for detecting DHVⅠ was established. The method can be amplified from DHV Ⅰ to 440 bp band, while the normal duck embryo allantoic fluid, healthy duck liver, duck plague virus, Muscovy duck parvovirus, goose parvovirus, Gosling new virus enteritis virus, Influenza virus (H5N2 subtype), duck viral swine head virus, Pasteurella multocida (5: A) amplified results were negative. The method can detect 30 pg of DHV I nucleic acid template as low as possible. RT-PCR detection of DHV Ⅰ virulent CHV-1 strain of artificial death of infected duck test results and virus isolation and Dot-ELISA test results were 100% positive rate, the spleen, lung, brain disease detection rate Significantly (P <0.01) was higher than virus isolation and Dot-ELISA. RT-PCR, virus isolation and Dot-ELISA detected 100% (19/19) cases of clinically detected liver disease which was diagnosed as duck hepatitis by virus isolation and collected at -20 ℃ from 1987 to 2005, respectively , 36.84% (7/19) and 57.98% (11/19) respectively. The detection rates of 48 liver disease materials collected from 2000 to 2005 and stored at -20 ℃ in duck populations from 18 provinces in China were 100% (48/48), 56.25% (27/48) and 75.00% (36/48). The results showed that the established RT-PCR method has good specificity and sensitivity, which can be used for the isolation and identification of DHVⅠ, the detection of clinical materials and the molecular epidemiological investigation.