Use of the DNA flow-thru chip, a three-dimensional biochip, for typing and subtyping of influenza viruses
Use of the DNA flow-thru chip, a three-dimensional biochip, for typing and subtyping of influenza viruses
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DOI：
10.1128/jcm.42.5.2173-2185.2004
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发表时间：
2004-05-01
影响因子：
9.4
通讯作者：
Steel, A
中科院分区：
文献类型：
--
作者：
Kessler, N;Ferraris, O;Steel, A
基于外部血凝素和神经氨酸酶糖蛋白的抗原性差异进一步分型的甲型流感病毒和B型流感病毒是呼吸道疾病的主要病毒病因，并可引起广泛的疾病。每年这些病毒都是反复流行的原因，通常与遗传变异有关。需要有敏感和快速的诊断技术，以改进感染诊断和监测系统的质量。一种新的三维生物芯片平台（Flow-Thru Chip; MetriGenix）用于开发一种快速可靠的流感病毒分型和亚型分子方法。选择固定在硅晶片微通道中的寡核苷酸探针以识别甲型流感病毒基质蛋白基因的多个片段;流感B病毒NS基因; H1、H3和H5血凝素基因;以及N1和N2神经氨酸酶基因。将来自多重或随机逆转录-PCR的生物素化扩增子与流感病毒芯片上的阵列寡核苷酸杂交，然后用辣根过氧化物酶-链霉亲和素染色，并通过使用荧光底物成像。从将样品移液到芯片盒中的时间到分析结果的时间，芯片分析程序在不到5小时内进行。随机PCR在产物与流感病毒芯片杂交的特异性方面表现出比多重PCR更高的性能水平。对甲型流感病毒（H1N1、H3 N2、H1 N2和H5 N1）和B流感病毒的分析表明，这种基于微阵列的方法能够通过使用随机PCR产物快速、明确地鉴定所有类型和亚型的病毒。为每个选定基因设计的探针的冗余度产生了病毒亚型的额外置信标准，已知这些病毒的某些组分存在抗原变异。
Influenza A viruses, which are further subtyped on the basis of antigenic differences in external hemagglutinin and neuraminidase glycoproteins, and influenza B viruses are prominent among the viral causes of respiratory diseases and can cause a wide spectrum of illness. Each year these viruses are responsible for recurrent epidemics, frequently in association with genetic variation. There is a requirement for sensitive and rapid diagnostic techniques in order to improve both the diagnosis of infections and the quality of surveillance systems. A new three-dimensional biochip platform (Flow-Thru Chip; MetriGenix) was used to develop a rapid and reliable molecular method for the typing and subtyping of influenza viruses. Oligonucleotide probes immobilized in microchannels of a silicon wafer were selected to recognize multiple fragments of the influenza A virus matrix protein gene; the influenza B virus NS gene; the H1, H3, and H5 hemagglutinin genes; and the N1 and N2 neuraminidase genes. Biotinylated amplicons resulting from either multiplex or random reverse transcription-PCR were hybridized to arrayed oligonucleotides on the influenza virus chip before they were stained with horseradish peroxidase-streptavidin and were imaged by use of a chemiluminescent substrate. The chip analysis procedure, from the time of pipetting of the sample into the chip cartridge to the time of analysis of the results, was performed in less than 5 h. The random PCR exhibited a higher level of performance than the multiplex PCR in terms of the specificity of product hybridization to the influenza virus chip. Analysis of influenza A viruses (H1N1, H3N2, H1N2, and H5N1) and influenza B viruses showed that this microarray-based method is capable of the rapid and unambiguous identification of all types and subtypes of viruses by use of random PCR products. The redundancy of the probes designed for each gene selected yielded an additional criterion of confidence for the subtyping of viruses which are known for antigenic variations in some of their components.