A reverse transcription-PCR for subtyping of the neuraminidase of avian influenza viruses
A reverse transcription-PCR for subtyping of the neuraminidase of avian influenza viruses
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DOI：
10.1016/j.jviromet.2008.10.001
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发表时间：
2009-02-01
影响因子：
3.1
通讯作者：
Liu, Xiu-Fan
中科院分区：
文献类型：
--
作者：
Qiu, Bao-Feng;Liu, Wu-Jie;Liu, Xiu-Fan
到目前为止，已经鉴定了九种禽流感病毒的神经氨酸酶（NA）亚型。为了快速区分禽流感病毒的核酸，建立了逆转录PCR（RT-PCR）方法。根据GenBank中509个NA全序列设计了9对NA特异性引物进行RT-PCR扩增。设计引物以扩增部分NA基因，并且每对引物对于单个NA亚型（N1-N9）是独特的。通过在一组单独的管中同时进行9次RT-PCR，通过随后的琼脂糖凝胶电泳和溴化乙锭染色来确定NA的亚型，因为9次RT-PCR中只有一次会给出每种病毒株的预期大小的产物。与已建立的101株禽流感病毒参考株或分离株序列分析方法比较，RT-PCR方法对禽流感病毒亚型分型的敏感性为97.3%，特异性为91.1%。这些结果表明，下文所述的RT-PCR方法为常规NA亚型分型方法提供了一种特异性和敏感性的替代方法。(C)2008 Elsevier B. V.保留所有权利。
To date, nine neuraminidase (NA) subtypes of avian influenza viruses have been identified. In order to differentiate the NA of avian influenza viruses rapidly, a reverse transcription PCR (RT-PCR) was developed. Nine pairs of NA-specific primers for the RT-PCR were designed based on the analysis of 509 complete NA sequences in GenBank. The primers were designed to amplify partial NA genes and each pair is unique to a single NA subtype (N1-N9). By nine RT-PCRs simultaneously in a set of separate tubes, the subtype of NA was determined by subsequent agarose gel electrophoresis and ethidium bromide staining, since only one of the nine RT-PCRs would give a product of expected size for each virus strain. In comparison with the established method of sequence analysis of 101 reference strains or isolates of avian influenza viruses, the RT-PCR method had a sensitivity of 97.3% and a specificity of 91.1% in subtyping avian influenza viruses. These results indicate that the RT-PCR method described below provides a specific and sensitive alternative to conventional NA-subtyping methods. (C) 2008 Elsevier B.V. All rights reserved.