Strain Specific Detection of Influenza at the Point-of-Care

描述（由申请人提供）：在护理点进行流感病毒株特异性检测流感病毒在世界范围内造成严重的发病率和死亡率；世界卫生组织（WHO）估计每年有25万~ 50万人死亡。特别是，一种高致病性毒株——禽流感病毒H5N1（15,56）——可能造成大流行的威胁，因为它可以迅速变异以获得在人与人之间传播的能力（23,49）。从患者样本（如鼻拭子或粘液）中检测甲型流感的最低人类感染剂量（约100个病毒颗粒）是一项重大的分析挑战。病毒培养检测灵敏、特异；然而，这种方法是缓慢的（3-10天），不能在POC进行。基于表面标记的检测（如ELISA）提供了一种简单而快速的检测方法，然而，它的检测限很差（~ 105个病毒颗粒/ml）（9,21,59）。目前，聚合酶链反应（PCR）在更短的周转时间（2-4小时）内提供了最高的灵敏度（29,61,67）。然而，由于POC操作困难，目前患者样本在中心实验室进行分析，通常需要2-3天。目前在该领域，还没有能够直接从患者样本中进行高灵敏度和特异性流感检测的自动化技术解决方案，世卫组织特别强调了填补这一重要技术空白的迫切需要（75）。为了解决这一重要问题，这组具有互补技能（流感病毒学，适体生物化学和微流控装置工程）的三位PI建议开发一个强大的，现场便携式的微流控平台，用于POC的流感检测。这一努力在许多方面都是独一无二的；首先，为了避免外壳蛋白快速突变带来的问题，我们提出了一种新的DNA适体试剂，它可以特异性地结合病毒的核蛋白复合体。目前，该核蛋白复合物的适配体尚未报道，由于该核蛋白是保守的，因此它将作为标记甲型流感的通用标签。其次，将适配体试剂偶联到磁珠上，使用我们独特的微磁技术从鼻拭子样品中纯化核蛋白复合物。第三，利用我们在小型遗传分析系统方面的专业知识，我们将开发出能够在单个单片设备中集成微磁样品纯化，2)逆转录- pcr扩增和3)序列特异性电化学检测的IMED芯片。该项目的成功完成将实现定量的“从样本到答案”能力——系统的输入将是未经处理的患者样本，输出将是电化学信号，该信号将与样本中特定流感RNA序列的拷贝数成正比。这种新型亲和试剂与高度集成的一次性设备的强大组合将使开发急需的有效POC诊断系统成为可能。由于甲型流感病毒的包膜在不同亚型之间存在差异并不断进化，因此迫切需要一种现场便携式遗传检测平台，能够以高灵敏度和特异性从未处理的样品中快速识别病毒病原体的致病性菌株。为了找到有效的解决方案，三个具有互补技能的PI（流感病毒学，适体生物化学和微流控设备工程）建议开发一种有效的流感即时诊断系统：1)生成特异性的，高亲和力的DNA适体来标记甲型流感的保守区域；2)开发一种高度集成的微流控系统，能够进行磁性样品制备。单芯片RT-PCR扩增及序列特异性电化学检测。通过将这些功能集成到单片芯片中，该项目的成功将产生一种具有前所未有功能的新型POC分析系统，这将对甲型流感检测以及食品安全，环境监测和国土安全等许多其他应用产生重大影响。1

DESCRIPTION (provided by applicant): Strain Specific Detection of Influenza at the Point-of-Care 1. PROJECT SUMMARY Influenza viruses cause significant morbidity and mortality worldwide; World Health Organization (WHO) estimates 250,000 ~ 500,000 yearly deaths. In particular, a highly pathogenic strain - avian influenza virus H5N1 (15, 56) - poses a threat of a possible pandemic because it can rapidly mutate to acquire the ability to transmit among humans (23, 49). The detection of minimal human infectious dose (~100 viral particles) (65) of Influenza A from patient samples (e.g. nasal swabs or mucus) is a significant analytical challenge. Detection by viral culture is sensitive and specific; however, this method is slow (3-10 days) and cannot be performed at POC. Surface marker-based detection (e.g. ELISA) provide a simple and rapid testing, however, it suffers from a poor limit of detection (~ 105 viral particles/ml) (9, 21, 59). Currently, polymerase chain reaction (PCR) offers the highest sensitivity in a much shorter turn-around time (2-4 hours) (29, 61, 67). Nevertheless, due to the difficulties in POC operation, patient samples are currently analyzed at central laboratories which typically requires ~2-3 days. Currently in the field, there is no automated technology solution that can perform specific influenza detection directly from patient samples with high sensitivity and specificity, and the WHO has specifically emphasized the critical need to fill this important technological gap (75). Towards a solution to this important problem, this group of three PI's with complementary skill sets (influenza virology, aptamer biochemistry and microfluidic device engineering) propose to develop a powerful, field- portable, microfluidic platform for Influenza detection at POC. This effort is truly unique in many facets; first, to circumvent the problem posed by rapid mutation of the coat protein, we propose to generate novel DNA aptamer reagents which will specifically bind to the nucleoprotein complex of the virus. Currently, there are no reported aptamers for the nucleoprotein complex, and due to the fact that the nucleoprotein is conserved, it will serve as a universal tag to label Influenza A. Secondly, the aptamer reagent will be conjugated to magnetic beads to purify the nucleoprotein complex from nasal swab samples using our unique micromagnetics technology. Thirdly, by leveraging our expertise in miniaturized genetic analysis systems, we will develop the IMED chip capable of 1) integrated micromagnetic sample purification, 2) reverse transcription-PCR amplification and 3) sequence-specific electrochemical detection in a single monolithic device. The successful completion of this project will allow a quantitative "sample-to-answer" capability - the input into the system will be unprocessed patient samples, and the output will be an electrochemical signal that will be directly proportional to the number of copies of specific influenza RNA sequences in the sample. Such powerful combination of novel affinity reagents with highly integrated disposable devices will enable the development of critically needed effective POC diagnostics systems. PUBLIC HEALTH RELEVANCE: Strain Specific Detection of Influenza at the Point-of-Care Due to the fact that the envelope of influenza A virus differs among subtypes and evolves continuously, there is an urgent need for a field-portable genetic detection platform that can rapidly identify pathogenic strain of viral pathogens from unprocessed samples with high sensitivity and specificity. Towards an effective solution, the three PI's with complementary skill sets (influenza virology, aptamer biochemistry and microfluidic device engineering) propose to develop an effective point-of-care diagnostic system for influenza by 1) generating specific, high affinity DNA aptamers to tag the conserved regions of Influenza A, and 2) developing a highly integrated microfluidic system capable of magnetic sample preparation, RT-PCR amplification and sequence specific electrochemical detection in a single chip. By integrating the functions in a monolithic chip, the success of this project will yield a novel POC analysis system with unprecedented capabilities which will have a significant impact for Influenza A detection as well as many other applications in food safety, environmental monitoring and homeland security. 1