Wright K E, Wilson G A, Novosad D, Dimock C, Tan D, Weber J M
Department of Microbiology and Immunology, Faculty of Medicine, University of Ottawa, Ontario, Canada.
J Clin Microbiol. 1995 May;33(5):1180-4. doi: 10.1128/jcm.33.5.1180-1184.1995.
Type A and B influenza viruses can cause a wide spectrum of illness, and these viruses are responsible for considerable mortality and morbidity. Rapid typing of isolates is desirable when amantadine treatment or prophylaxis of contacts of type A influenza virus carriers is considered, but the available rapid techniques lack sensitivity and standard diagnostic methods require expansion of virus in tissue culture or embryonated hens' eggs. We developed a series of oligonucleotide primers able to detect, type, and subtype type A influenza viruses in a single reverse transcription-PCR. RNA was isolated from clinical specimens, and cDNA was generated with random primers. PCR was carried out with a mixture of primers specific for influenza viruses of types B, A/H1 and A/H3, and subtyping of the neuraminidase was carried out on the same cDNA template under identical conditions. Amplified products were detected by ethidium bromide staining of amplified products after agarose gel electrophoresis. When it was used to test 98 clinical specimens, this method was comparable to standard culture techniques in the detection, typing, and subtyping of influenza viruses.
甲型和乙型流感病毒可引发广泛的疾病，这些病毒导致了相当高的死亡率和发病率。当考虑对甲型流感病毒携带者进行金刚烷胺治疗或对其接触者进行预防时，需要对分离株进行快速分型，但现有的快速技术缺乏敏感性，而标准诊断方法需要在组织培养或鸡胚中扩增病毒。我们开发了一系列寡核苷酸引物，能够通过单次逆转录-聚合酶链反应（RT-PCR）检测、分型和亚型鉴定甲型流感病毒。从临床标本中提取RNA，并用随机引物生成cDNA。使用针对乙型、A/H1和A/H3型流感病毒的引物混合物进行PCR，并在相同条件下对同一cDNA模板进行神经氨酸酶亚型鉴定。扩增产物通过琼脂糖凝胶电泳后用溴化乙锭染色进行检测。当用该方法检测98份临床标本时，在流感病毒的检测、分型和亚型鉴定方面，该方法与标准培养技术相当。
