Rapid molecular subtyping by reverse transcription polymerase chain reaction of the neuraminidase gene of avian influenza A viruses
Rapid molecular subtyping by reverse transcription polymerase chain reaction of the neuraminidase gene of avian influenza A viruses
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DOI：
10.1016/j.vetmic.2008.09.077
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发表时间：
2009-03-30
影响因子：
3.3
通讯作者：
Harder, T.
中科院分区：
文献类型：
--
作者：
Fereidouni, S. R.;Starick, E.;Harder, T.
准确鉴定甲型流感病毒的血凝素（HA）和神经氨酸酶（NA）亚型是针对禽流感病毒（AIV）的监测计划的一个组成部分。使用高灵敏度的分子筛选方法，如泛流感特异性实时RT-PCR（rRT-PCR）产生越来越多的样品，这些样品对AIV RNA呈阳性，但病毒分离呈阴性，因此需要分子分型，而不是血清学分型。我们开发了所有已知的九种AIV NA亚型的特异性RT-PCR检测方法。使用来自不同动物种属的43株参考分离株进行的验证显示了灵敏度和特异性方面的良好性能特征。基于参考分离株的连续10倍稀释系列，M基因特异性rRT-PCR中C-t 32的基准值变得明显，低于该基准值，所有9种NA亚型均可通过亚型特异性RT-PCR轻易检出。对于N1、N2、N4和N6亚型，检测范围扩展至C-t值高达35的稀释液。通过对119份经M基因特异性rRT-PCR检测为AIV阳性的野生鸟类标本进行分析，评价了整套常规NA特异性RT-PCR方法的诊断适用性。通过对从该组产生的41个田间分离株的NA扩增子进行测序和NA抑制测定来确认诊断灵敏度和特异性。提出了一种适用于rRT-PCR阳性禽流感病毒监测样本的通用分子HA/NA亚型分型算法，该算法可对经典的A型流感病毒血清学亚型分型方法进行补充。(C)2008 Elsevier B. V.保留所有权利。
Accurate identification of hemagglutinin (HA) and neuraminidase (NA) subtypes of influenza A viruses is an integral part of monitoring programs targeting avian influenza viruses (AIV). Use of highly sensitive molecular screening methods such as pan influenza-specific real-time RT-PCR (rRT-PCR) yields an increasing number of samples which are positive for AIV RNA but negative by virus isolation and, therefore, require molecular, instead of serological, subtyping. We developed specific RT-PCR assays for all known nine AIV NA subtypes. Validation using 43 reference isolates from different animal species revealed good performance characteristics regarding sensitivity and specificity. On basis of serial tenfold dilution series of reference isolates a benchmark value of C-t 32 in an M gene-specific rRT-PCR became evident below which all nine NA subtypes were readily detectable by the subtype-specific RT-PCRs. For subtypes N1, N2, N4 and N6 detection was extended to dilutions with C-t values of up to 35. Diagnostic applicability of the whole set of conventional NA-specific RT-PCRs was evaluated by analysis of 119 different diagnostic samples from wild birds which proved to be positive for AIV by M gene-specific rRT-PCR. Diagnostic sensitivity and specificity was confirmed by sequencing NA amplicons from 41 field isolates generated from this set and by NA inhibition assays. A universal molecular HA/NA subtyping algorithm for rRT-PCR positive avian influenza virus monitoring samples is proposed which may complement classical serological subtyping of influenza A virus isolates. (C) 2008 Elsevier B.V. All rights reserved.