Rapid and highly sensitive influenza detection with RNA FISH

使用 RNA FISH 快速、高灵敏度地检测流感

• 批准号: 9045376
• 负责人: Sydney Shaffer
• 金额: $ 3.03万
• 依托单位: UNIVERSITY OF PENNSYLVANIA
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-02-01 至 2018-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9045376
• 关键词: Address; Adenoviruses; Adverse effects; Antibiotics; Antibodies; Antiviral Agents; Antiviral Therapy; Biological Assay; Biological Models; Cell Culture System; Cell Culture Techniques; Cells; Cessation of life; Clinic; Clinical; Data; Detection; Development; Device Designs; Devices; Diagnosis; Diagnostic; Diagnostic tests; Discrimination; Drug resistance; Economics; Exhibits; Fluorescent in Situ Hybridization; Geometry; Gold; Health; Hospitalized Child; Hour; Human; Image; Infection; Influenza; Influenza A Virus, H1N1 Subtype; Influenza A virus; Influenza B Virus; Label; Laboratories; Lead; Left; Microfluidic Microchips; Modeling; Morbidity - disease rate; Nose; Oligonucleotides; Oseltamivir; Output; Patients; Pediatric Hospitals; Pharmaceutical Preparations; Philadelphia; Population; Protocols documentation; RNA; Recruitment Activity; Research Technics; Respiratory System; Respiratory tract structure; Reverse Transcriptase Polymerase Chain Reaction; Rhinovirus; Running; Sampling; Sensitivity and Specificity; Signal Transduction; Source; Specimen; Statistical Data Interpretation; Swab; Symptoms; System; Testing; Time; Translating; United States; Vaccination; Vaccine Production; Validation; Viral; Virus; anti-influenza; base; clinical care; comparative efficacy; design; diagnosis standard; drug resistant bacteria; influenzavirus; molecular diagnostics; mortality; novel diagnostics; point of care; point-of-care diagnostics; research clinical testing; research study; resistant strain; respiratory; viral detection; virology; zanamivir

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.

描述（由申请人提供）：流感每年感染5-20%的美国人口，导致39，000人死亡和871亿美元的经济产出损失。目前缓解这些影响的策略包括疫苗接种和抗病毒治疗。在后者中，最好的药物是奥司他韦和扎那米韦，它们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，这些药物的数量有限，价格昂贵，并且仅对特定的病毒亚型有效。此外，它们在早期给药时最有效，优选在症状的前12小时内。因此，为了指导临床治疗抗病毒药物的使用，临床医生需要准确和快速的流感诊断。目前可用的流感诊断要么是快速低灵敏度，要么是缓慢高灵敏度，并且存在对快速、灵敏和特异性流感诊断的未满足的临床需求。我们通过开发一种用于通过基于寡核苷酸的RNA荧光原位杂交（RNA FISH）超快速和高灵敏度检测流感的集成系统来解决这一需求。在我们的初步实验中，我们设计并测试了在流感感染的细胞培养模型中靶向流感病毒的RNA FISH探针，表明我们的探针通过明亮地标记受感染的细胞并使未感染的细胞未被检测到而显示出显著的背景信号。我们通过设计针对甲型流感H1N1、H3 N2和流感B的流感亚型特异性探针，进一步推动了该检测方法，发现这些探针组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。为了实现这种检测的诊断应用，我们接下来开发了一种封闭格式的微流体设备，以自动浓缩鼻拭子中的细胞，进行RNA FISH，并分析所得图像。该提案概述了将RNA FISH从研究技术转化为临床可行的流感诊断测试的下一步。在第一个目标中，我们重新配置了我们的微流控设备的几何形状，以便用一个芯片进行多种检测，从而使我们能够在一个标本上使用所有亚型特异性探针。接下来，我们将优化多重芯片，然后在这个平台上测试人类鼻腔标本。在目标二中，我们将验证基于RNA FISH的流感病毒检测来自病毒感染患者的临床鼻标本。我们将从费城儿童医院招募受试者，这些受试者以前曾在常规临床护理中进行过流感RT-PCR检测。有了阳性和阴性受试者的样本库，我们将制定一个运行微流控芯片的方案，并优化临床样本的检测。接下来，我们将确定该检测试剂盒用于流感检测和亚型区分的灵敏度和特异性。这些目标的成功完成将证明超快速RNA FISH是一种可行的和潜在的范式转变的床旁诊断。