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Universal Primer Set for Amplification and Sequencing of HA0 Cleavage Sites of All Influenza A Viruses▿ †

机译:通用引物组,用于扩增和测序所有甲型流感病毒的HA0切割位点▿†

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摘要

Sequence analysis of the endoproteolytic cleavage site within the hemagglutinin (HA) precursor protein HA0 is fundamental for studies of the molecular biology of influenza A viruses, in particular, for molecular pathotyping of subtype H5 and H7 isolates. A current problem for routine diagnostics is the emergence of new strains of the H5 or H7 subtype or even other subtypes which escape detection by commonly used reverse transcription-PCR (RT-PCR) protocols. Here, the first pan-HA (PanHA) RT-PCR assay targeting the HA0 cleavage site of influenza A viruses of all 16 HA subtypes is reported. The assay was assessed in comparison to H5 and H7 subtype-specific RT-PCRs for the HA0 cleavage site and a real-time RT-PCR detecting the M gene. A panel of 92 influenza A viruses was used for validation. Sequence data for influenza A viruses from 32 allantoic fluid samples and 11 diagnostic swab samples of all 16 HA subtypes were generated by direct sequencing of the PanHA RT-PCR products. The results demonstrate that the new PanHA RT-PCR assay—followed by cycle sequencing—can complement existing methods and strengthen the reliability of influenza A virus diagnostics, allowing both molecular pathotyping (H5 and H7) and subtyping (non-H5 or -H7) within a single approach.
机译:血凝素(HA)前体蛋白HA0中的蛋白水解酶切位点的序列分析对于研究甲型流感病毒的分子生物学,特别是对于H5和H7亚型分离株的分子病理分型至关重要。常规诊断的当前问题是出现了H5或H7亚型甚至其他亚型的新菌株,这些菌株无法通过常用的逆转录PCR(RT-PCR)方案进行检测。在此,报道了针对所有16种HA亚型的甲型流感病毒的HA0切割位点的首次泛HA(PanHA)RT-PCR分析。与针对H0切割位点的H5和H7亚型特异性RT-PCR和检测M基因的实时RT-PCR相比,评估了该测定法。使用一组92种甲型流感病毒进行验证。通过对PanHA RT-PCR产物进行直接测序,从32种尿囊液样本和11种诊断性拭子样本中获得了所有16种HA亚型的甲型流感病毒的序列数据。结果表明,新的PanHA RT-PCR测定法(随后进行循环测序)可以补充现有方法并增强A型流感病毒诊断的可靠性,允许进行分子病理分型(H5和H7)和亚型分型(非H5或-H7)在单一方法中。

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