NASBA快速检测禽流感H5亚型病毒

采用建立的依赖核酸序列的扩增 (Nucleic acid sequence-based amplification, NASBA) 对禽流感病毒3株H5亚型、1株H1、H3、H6亚型、3株禽流感H9亚型、5株不同宿主来源的新城疫病毒、鸭肝炎病毒、鸭瘟病毒、SPF鸡胚尿囊液及禽流感 (H9) 疫苗、新城疫疫苗、传染性法氏囊病疫苗、传染性支气管炎疫苗进行检测,结果NASBA (H5试剂) 仅检测到禽流感病毒H5亚型, 表明方法的特异性强。采用已知禽流感病毒A/Chicken/HK/1000/97 (H5N1) 的鸡胚尿囊液 (ELD5010-7.5/mL), 经10倍连续稀释,将经典的鸡胚病原分离法和NASBA进行比较,二种方法的灵敏度相当。用A/Chicken/HK/1000/97 (H5N1) 病毒人工感染SPF鸡、商品鸡,采用NASBA和病原分离法同时对人工感染鸡的粪拭子、血液进行了动态检测;采集感染死亡鸡的组织脏器,共检测了101个组织脏器,两种方法的符合率为90% (87/97)。

Nucleic acid sequence-based amplification with electrochemiluminescent detection (NASBA/ ECL) of avian influenza virus subtype H5 was developed,comparing with virus isolation in embryonated chicken eggs. The NASBA technique described here can detect three isolates of infuenza A subtype H5 and do not detect infuenza A subtype H1,3,6,9, other avian viruses and allantoic fluid of SPF chicken, showing rapidness and high specificity for identificating infuenza A subtype H5 viruses. Dilution of a known virus was used to determine the limit of sensitivity for both NASBA and classic virus isolation. The NASBA/ECL method was equivalent in sensitivity to virus isolation in eggs,with limit of 10+{-1.5} ELD-{50} dose. Virus was isolated from anal swabs, blood and tissues of chickens artificially infected with highly pathogenic avian influenza A/Chicken/Hong Kong/1000/97 (H5N1). NASBA/ ECL could detect viral nucleic acid in anal swabs from the day following artificial infection until death. Conversely, Nucleic acid molecule could only be detected in blood by the NASBA/ ECL and virus isolation immediately prior to death. Thus , blood and/ or anal swabs are a suitable source of material for the detection of avian influenza in dead birds, but anal swabs are more suitable for detection of viral genetic material in live birds. When samples fro m chickens artificially infected with avian influenza virus were analysed by both egg culture and NASBA/ ECL met hods in parallel , the result s agreed in 87/ 97 (90%) .