Townsend Michael B, Dawson Erica D, Mehlmann Martin, Smagala James A, Dankbar Daniela M, Moore Chad L, Smith Catherine B, Cox Nancy J, Kuchta Robert D, Rowlen Kathy L
Department of Chemistry and Biochemistry, UCB 215, University of Colorado, Boulder, CO 80309, USA.
J Clin Microbiol. 2006 Aug;44(8):2863-71. doi: 10.1128/JCM.00134-06.
Global surveillance of influenza is critical for improvements in disease management and is especially important for early detection, rapid intervention, and a possible reduction of the impact of an influenza pandemic. Enhanced surveillance requires rapid, robust, and inexpensive analytical techniques capable of providing a detailed analysis of influenza virus strains. Low-density oligonucleotide microarrays with highly multiplexed "signatures" for influenza viruses offer many of the desired characteristics. However, the high mutability of the influenza virus represents a design challenge. In order for an influenza virus microarray to be of utility, it must provide information for a wide range of viral strains and lineages. The design and characterization of an influenza microarray, the FluChip-55 microarray, for the relatively rapid identification of influenza A virus subtypes H1N1, H3N2, and H5N1 are described here. In this work, a small set of sequences was carefully selected to exhibit broad coverage for the influenza A and B viruses currently circulating in the human population as well as the avian A/H5N1 virus that has become enzootic in poultry in Southeast Asia and that has recently spread to Europe. A complete assay involving extraction and amplification of the viral RNA was developed and tested. In a blind study of 72 influenza virus isolates, RNA from a wide range of influenza A and B viruses was amplified, hybridized, labeled with a fluorophore, and imaged. The entire analysis time was less than 12 h. The combined results for two assays provided the absolutely correct types and subtypes for an average of 72% of the isolates, the correct type and partially correct subtype information for 13% of the isolates, the correct type only for 10% of the isolates, false-negative signals for 4% of the isolates, and false-positive signals for 1% of the isolates. In the overwhelming majority of cases in which incomplete subtyping was observed, the failure was due to the nucleic acid amplification step rather than limitations in the microarray.
全球流感监测对于改善疾病管理至关重要，对于早期发现、快速干预以及可能减轻流感大流行的影响尤为重要。加强监测需要快速、可靠且廉价的分析技术，能够对流感病毒株进行详细分析。具有针对流感病毒高度多重“特征”的低密度寡核苷酸微阵列具备许多所需特性。然而，流感病毒的高变异性带来了设计挑战。为使流感病毒微阵列有用，它必须能为广泛的病毒株和谱系提供信息。本文描述了一种用于相对快速鉴定甲型流感病毒H1N1、H3N2和H5N1亚型的流感微阵列——FluChip - 55微阵列的设计与特性。在这项工作中，精心挑选了一小部分序列，以广泛覆盖当前在人群中传播的甲型和乙型流感病毒，以及在东南亚家禽中已成为地方性流行且最近传播到欧洲的禽甲型H5N1病毒。开发并测试了一种完整的检测方法，包括病毒RNA的提取和扩增。在对72株流感病毒分离株的盲测中，对来自多种甲型和乙型流感病毒的RNA进行了扩增、杂交、用荧光团标记并成像。整个分析时间不到12小时。两种检测方法的综合结果显示，平均72%的分离株得到了完全正确的类型和亚型，13%的分离株得到了正确的类型和部分正确的亚型信息，10%的分离株仅得到了正确的类型，4%的分离株出现假阴性信号，1%的分离株出现假阳性信号。在绝大多数观察到亚型分型不完全的情况下，失败是由于核酸扩增步骤而非微阵列的局限性。
