Rapid Low Cost Molecular Detection of Influenza Virus Infections

描述（由申请人提供）：在发展中国家，急性呼吸道感染（ARI）每年导致约190万儿童死亡，其中20%在印度。幼儿的流感与高危成人的流感一样严重，而且常常未被发现。在亚热带地区，流感是儿童住院的一个重要原因，其发病率超过了温带地区报告的发病率。在印度因急性呼吸道感染住院的儿童中，有15%发现了流感。对发展中国家病毒感染的理解所面临的挑战之一是对超出现有实验室能力的诊断工具的需求。这方面的一个例子是分子诊断的应用，如逆转录聚合酶链反应（RTPCR）。虽然这些检测方法为病毒检测提供了优势，但它们的使用受到成本的限制。逆转录环介导等温扩增（LAMP）是一种特异性高、效率高、速度快、成本低的RNA扩增新技术。我们建议开发LAMP检测方法，用于印度流感病毒的研究。这将需要对人类和禽流感病毒进行检测并确定其类型和亚型。在Aim 1中，将使用人类流感病毒和人类和禽流感靶基因的体外转录rna建立LAMP测定方法。将对视觉检测系统和机器检测系统进行比较。还将开发流感的LAMP定量检测方法。在目标2中，LAMP检测将与培养和RTPCR检测流感病毒进行比较。将使用的样本来自德里现有的独特临床样本收集，包括从儿童病毒性急性呼吸道感染研究和国家流感病毒监测网络中前瞻性收集的样本。定量LAMP将用于检验病毒载量与儿童疾病严重程度相关的假设。本申请提出了开发新的研究技术，以解决流感流行病学和发病机制。此外，它还涉及一个高度优先的流感研究领域，即开发敏感、特异和快速的流感临床诊断测试。虽然这项建议的重点是印度的流感，但其结果应广泛适用，并有可能在全球使用。拟议的研究与公共卫生有关，因为流感病毒感染是发达国家和发展中国家儿童和成人疾病的重要原因。改进的病毒检测方法可以改善流感诊断，并支持抗流感药物和疫苗的研究。

DESCRIPTION (provided by applicant): Acute respiratory infections (ARI) result in ~ 1.9 million childhood deaths per year in developing countries, 20% of the deaths are in India. Influenza in young children is as severe as in high-risk adults and frequently goes unrecognized. In the subtropics, influenza is an important cause of hospitalization among children, with rates exceeding those reported for temperate regions. Influenza was found in 15% of the children hospitalized with ARI in India. Among the challenges to the understanding of viral infections in the developing world has been the requirement for diagnostic tools that exceed the available laboratory capacity. An example of this lies in the application of molecular diagnostics such as reverse transcription polymerase chain reaction (RTPCR). While these assays offer advantages for virus detection their use has been limited by cost. Reverse-transcription loop mediated isothermal amplification (LAMP) is a novel technique that can amplify RNA with high specificity, efficiency, rapidity and at a low cost. We propose to develop LAMP assays for use in the study of influenza virus in India. This will entail human and avian influenza virus detection and characterization as to type and subtype. In Aim 1 LAMP assays will be established using human influenza virus and in vitro transcribed RNAs from human and avian influenza target genes. Visual and machine detection systems will be compared. A quantitative LAMP assay for influenza will also be developed. In Aim 2 the LAMP assays will compared to culture and RTPCR for the detection of influenza virus. The samples to be used are from a unique collection of clinical samples available in Delhi, including prospectively collected samples from studies of viral ARI in children and a national influenza virus surveillance network. Quantitative LAMP will be used to test the hypothesis that viral loads correlate with disease severity in children. This application proposes to develop new research technology to address influenza epidemiology and pathogenesis. In addition, it addresses a high-priority influenza research area, the development of sensitive, specific, and rapid clinical diagnostic tests for influenza. Although the emphasis of this proposal is on influenza in India, the results should be broadly applicable, with the potential global use. NARRATIVE The proposed studies are relevant to public health because influenza virus infections are an important cause of disease among children and adults in both developed and developing countries. Improved virus detection methods can improve influenza diagnosis and support studies of antivirals and vaccines against influenza.