Universal primer set for the full-length amplification of all influenza A viruses
Universal primer set for the full-length amplification of all influenza A viruses
复制标题
DOI：
10.1007/s007050170002
复制
发表时间：
2001-01-01
影响因子：
2.7
通讯作者：
Perez, DR
中科院分区：
文献类型：
--
作者：
Hoffmann, E;Stech, J;Perez, DR
为了系统地鉴定和分析甲型流感病毒的15种HA和9种NA亚型，我们需要可靠、简单的方法，不仅要表征部分序列，还要分析整个甲型流感基因组。我们设计的引物是基于基因组病毒RNA的15和21个末端片段特异性核苷酸在所有甲型流感病毒之间是保守的，并且每个片段都是独特的。为每个片段设计的引物在其3 '端含有流感病毒特异性核苷酸，在5'端含有非流感病毒核苷酸。使用这组引物，我们能够扩增N1、N2、N4、N5和N8亚型的所有8个片段。对于N3、N6、N7和N9亚型，神经氨酸酶基因的区段特异性序列是不同的。因此，我们优化了引物设计，以允许这些神经氨酸酶基因的扩增。所得引物组适用于所有甲型流感病毒，以产生全长cDNA，对病毒进行亚型分析，对其DNA进行测序，并构建用于反向遗传学系统的表达质粒。
To systematically identify and analyze the 15 HA and 9 NA subtypes of influenza A virus, we need reliable, simple methods that not only characterize partial sequences but analyze the entire influenza A genome. We designed primers based on the fact that the 15 and 21 terminal segment specific nucleotides of the genomic viral RNA are conserved between all influenza A viruses and unique for each segment. The primers designed for each segment contain influenza virus specific nucleotides at their 3'-end and non-influenza virus nucleotides at the 5'-end. With this set of primers, we were able to amplify all eight segments of N1, N2, N4, N5, and N8 subtypes. For N3, N6, N7, and N9 subtypes, the segment specific sequences of the neuraminidase genes are different. Therefore, we optimized the primer design to allow the amplification of those neuraminidase genes as well. The resultant primer set is suitable for all influenza A viruses to generate full-length cDNAs, to subtype viruses, to sequence their DNA, and to construct expression plasmids for reverse genetics systems.