A broad spectrum, one-step reverse-transcription PCR amplification of the neuraminidase gene from multiple subtypes of influenza A virus.
A broad spectrum, one-step reverse-transcription PCR amplification of the neuraminidase gene from multiple subtypes of influenza A virus.
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DOI：
10.1186/1743-422x-5-77
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发表时间：
2008-07-09
期刊：
影响因子：
4.8
通讯作者：
Barnard R
中科院分区：
文献类型：
--
作者：
Alvarez AC;Brunck ME;Boyd V;Lai R;Virtue E;Chen W;Bletchly C;Heine HG;Barnard R
甲型流感病毒的高致病性毒株在各种人类和动物宿主中的出现，具有广泛的地理分布，突出了快速识别和分型病毒对暴发管理和治疗的重要性。A型病毒可根据病毒包膜糖蛋白、血凝素和神经氨酸酶分为亚型。在这里，我们回顾了现有的特异性和扩增已公布的引物亚型神经氨酸酶基因，并描述了一个新的广谱引物对，可以检测所有9个亚型神经氨酸酶。对NCBI数据库中3，337个全长甲型流感神经氨酸酶片段的生物信息学分析揭示了先前未被引物靶向的半保守区域。从这些区域设计了两个带有M13标签的简并引物NA 8 F-M13和NA 10 R-M13，并用于产生253 bp的cDNA产物。使用来自代表9种神经氨酸酶亚型的超过32种不同培养甲型流感病毒株的RNA的降落方案，在31/32例（97%）病例中成功进行了一步RT-PCR检测。还分析了冷冻的盲态临床鼻咽抽吸物，大多数为N2亚型。将扩增的区域直接测序，然后用于数据库搜索以确认模板RNA的身份。产生的RT-PCR片段包括与奥司他韦耐药相关的突变位点之一H274 Y。我们的一步RT-PCR检测，然后测序是一种快速，准确，特异性的方法，用于检测和亚型不同的神经氨酸酶亚型从一系列的宿主物种和不同的地理位置。
The emergence of high pathogenicity strains of Influenza A virus in a variety of human and animal hosts, with wide geographic distribution, has highlighted the importance of rapid identification and subtyping of the virus for outbreak management and treatment. Type A virus can be classified into subtypes according to the viral envelope glycoproteins, hemagglutinin and neuraminidase. Here we review the existing specificity and amplification of published primers to subtype neuraminidase genes and describe a new broad spectrum primer pair that can detect all 9 neuraminidase subtypes. Bioinformatic analysis of 3,337 full-length influenza A neuraminidase segments in the NCBI database revealed semi-conserved regions not previously targeted by primers. Two degenerate primers with M13 tags, NA8F-M13 and NA10R-M13 were designed from these regions and used to generate a 253 bp cDNA product. One-step RT-PCR testing was successful in 31/32 (97%) cases using a touchdown protocol with RNA from over 32 different cultured influenza A virus strains representing the 9 neuraminidase subtypes. Frozen blinded clinical nasopharyngeal aspirates were also assayed and were mostly of subtype N2. The region amplified was direct sequenced and then used in database searches to confirm the identity of the template RNA. The RT-PCR fragment generated includes one of the mutation sites related to oseltamivir resistance, H274Y. Our one-step RT-PCR assay followed by sequencing is a rapid, accurate, and specific method for detection and subtyping of different neuraminidase subtypes from a range of host species and from different geographical locations.