采用建立的依赖核酸序列的扩增(Nucleicacidsequencebasedamplification,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