Rapid and highly sensitive influenza detection with RNA FISH

描述(申请人提供)：流感每年感染5-20%的美国人口，导致39,000人死亡，经济产出损失871亿美元。目前减轻这些影响的策略包括接种疫苗和抗病毒疗法。在后者中，可用的最佳药物是奥司他韦和扎那米韦，这两种药物可以将流感感染的持续时间缩短30%。这些抗病毒药物的副作用相对较少，因此它们的处方适用于确诊的流感感染病例。不幸的是，这些药物数量有限，价格昂贵，而且只对特定的病毒亚型有效。此外，如果及早给药，最好是在症状出现的前12小时内，它们是最有效的。因此，为了指导抗病毒药物的临床护理，临床医生需要对流感进行准确和快速的诊断。目前可用的流感诊断要么是低灵敏度的快速诊断，要么是高灵敏度的慢诊断，而对快速、敏感和特异的流感诊断的临床需求还没有得到满足。我们通过开发一种通过基于寡核苷酸的RNA荧光原位杂交(RNA FISH)来超快速和高灵敏地检测流感的集成系统来满足这一需求。在我们的初步实验中，我们在流感感染的细胞培养模型中设计并测试了针对流感病毒的RNA鱼类探针，表明我们的探针通过明亮地标记受感染的细胞并未检测到受感染的细胞而显示出与背景的戏剧性信号。我们通过设计针对甲型H1N1流感、H3N1流感和B型流感的流感亚型特异性探针进一步推动了检测，发现这些探针集具有区分亚型的能力，在感染人类毒株的细胞培养模型系统中具有几乎完美的区分能力。为了使这项检测的诊断应用成为可能，我们接下来开发了一种封闭格式的微流控设备，可以自动浓缩鼻拭子中的细胞，执行RNA FISH，并分析结果图像。该提案概述了将RNA FISH从一种研究技术转化为临床可行的流感诊断测试的下一步步骤。在第一个目标中，我们重新配置我们的微流控设备的几何结构，以便在一个芯片上执行多个分析，从而允许我们在一个样本上使用我们所有的亚型特定探针。接下来，我们将对多路复用芯片进行优化，然后在这个平台上对人鼻标本进行测试。在第二个目标中，我们将验证基于RNA FISH的流感病毒感染患者的临床鼻样检测。我们将从费城儿童医院招募受试者，这些受试者之前曾作为常规临床护理的一部分进行过流感RT-PCR检测。有了阳性和阴性受试者，我们将制定运行微流控芯片的程序，并对临床样本进行优化分析。接下来，我们将确定该检测方法用于流感检测和亚型识别的敏感性和特异性。这些目标的成功实现将证明超快速RNA FISH是一种可行的、潜在的范式转换的医疗保健点诊断方法。

DESCRIPTION (provided by applicant): Influenza annually infects 5-20% of the US population leading to 39,000 deaths and $87.1 billion lost in economic output. Current strategies for mitigating these effects include vaccination and antiviral therapies. Of the latter, the best available medications are oseltamivir and zanamivir, which can reduce the duration of influenza infections by 30%. These antivirals have relatively few side effects, and thereby their prescription is indicated in confirmed cases of influenza infection. Unfortunately, these medications are available in limited quantities, are costly, and are only effective against particular viral subtypes. Furthermore, they are most effective when administered early, preferably within the first 12 hours of symptoms. Thus, in order to guide clinical care with regards to antiviral usage, clinicians need accurate and rapid diagnostics for influenza. Currently available influenza diagnostic are either fast with low sensitivity or slow with high sensitivity, nd there is an unmet clinical need for fast, sensitive, and specific influenza diagnostics. We address this need by developing an integrated system for ultra-rapid and highly sensitive detection of influenza via oligonucleotide- based RNA fluorescent in situ hybridization (RNA FISH). In our preliminary experiments, we designed and tested RNA FISH probes targeting the influenza virus in a cell culture model of influenza infection, showing that our probes exhibit dramatic signal to background by brightly labeling infected cells and leaving uninfected cells undetected. We pushed the assay further by designing influenza subtype-specific probes to target influenza A H1N1, H3N2 and influenza B, finding that these probe sets are of distinguishing subtypes with virtually perfect discriminative ability in cell culture models system infected with the human strains. To enable diagnostic applications of this assay, we next developed a closed-format microfluidic device to automatically concentrate cells from a nasal swab, perform RNA FISH, and analyze the resulting images. This proposal outlines the next steps in translating RNA FISH from a research technique into a clinically viable diagnostic test for influenza. In the first aim, we reconfigure the geometry of our microfluidic device to perform multiple assays with one chip, thereby allowing us to use all of our subtype specific probes on one specimen. Next, we will optimize the multiplex chip and then test human nasal specimens on this platform. In aim two, we will validate RNA FISH based influenza detection on clinical nasal specimens from patients infected with the virus. We will recruit subjects from the Children's Hospital of Philadelphia who previously had influenza RT-PCR tests performed as part of their routine clinical care. With a pool of positive and negative subjects, we will formalie a protocol for running the microfluidic chip and optimize the assay on clinical samples. Next, we will establish the sensitivity and specificity of the assay for influenza detection and subtype discrimination. Successful completion of these aims will prove that ultra-rapid RNA FISH is a viable and potentially paradigm shifting point-of-care diagnostic.