A sample-to-answer diagnostic cartridge that identifies influenza using a glucometer

使用血糖仪识别流感的样本到答案诊断盒

• 批准号: 9200472
• 负责人: John Carrano
• 金额: $ 13.77万
• 依托单位: PARATUS DIAGNOSTICS, LLC
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-07-04 至 2017-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9200472
• 关键词: Adoption; Adult; Antiviral Agents; Bedside Testings; Biological; Biological Assay; Blinded; Child; Clinical; Communicable Diseases; Cytolysis; Data; Detection; Devices; Diagnostic; Diagnostic tests; Disease; Elderly; Evaluation; Future; Genes; Glucose; Goals; Gold; Hand; Head; Heating; Home environment; Human; Incidence; Infection; Influenza; Influenza A Virus, H1N1 Subtype; Influenza A Virus, H3N2 Subtype; Influenza A virus; Influenza B Virus; Invertase; Label; Legal patent; Light; Mediating; Methods; Molecular; National Institute of Allergy and Infectious Disease; Nose; Nucleic Acid Amplification Tests; Nucleic Acids; Oligonucleotide Primers; Oligonucleotides; Patients; Performance; Pharyngeal structure; Positioning Attribute; Predictive Value; Preparation; Procedures; Public Health; RNA; RNA amplification; Reaction; Reaction Time; Reading; Reporter; Resources; Reverse Transcriptase Polymerase Chain Reaction; Reverse Transcription; Sampling; Sensitivity and Specificity; Severities; Signal Transduction; Specificity; Symptoms; Technology; Testing; Texas; Time; Universities; Validation; Viral; Virion; Virus; abstracting; assay development; austin; base; cost; design; effective therapy; flu; genetic signature; glucose monitor; influenzavirus; instrument; mortality; novel; pandemic disease; pathogen; point of care; point-of-care diagnostics; respiratory; seasonal influenza; tool; viral RNA

Project Summary/Abstract Seasonal influenza has an annual global incidence of up to 10% in adults and up to 30% in children. About 3 to 5 million infections result in severe disease while somewhere between a quarter and a half million cases are fatal. Antiviral drugs can lessen the severity and reduce mortality when administered within 48 h of onset of symptoms. Hence, sensitive and specific point-of-care (POC) diagnostic tools, especially those that can be used at home, can be invaluable in expediting appropriate therapy and mitigating viral spread. While TaqMan real-time reverse transcriptase polymerase chain reaction (RT-PCR) and viral culture continue to be the diagnostic gold standards, several rapid POC influenza diagnostic technologies have been commercialized. Although results are available within 15 min, most of these tests have poor sensitivity (40-70% compared to gold standards). Rapid tests based on isothermal nucleic acid amplification (Alere i influenza A and B test) have also recently become available, and while they are very specific and sensitive, they can be too expensive for widespread POC use - $70 to $85 per test, and requiring the Alere i instrument that itself costs about $8500. To reduce testing cost and make nucleic acid diagnostics more widely available for at- home testing we propose to develop a one-pot, robust, and cheap POC test that can distinguish influenza A and B viruses. Key to this technology is viral RNA amplification by loop-mediated isothermal amplification (LAMP) and conversion of the resulting nucleic acid amplicons into glucose for readout via widely available, over-the-counter glucometers. Overall, in a partnership between Paratus Diagnostics and the Ellington lab at the University of Texas at Austin, we will develop a Paratus Consumable Cartridge (PCC) that will serve as an all-in-one device that extracts, amplifies, and transduces pathogen genetic signatures from patient samples to glucometers for readout, and is projected to cost about $5/device. In particular, we will develop reverse transcription (RT)-LAMP assays specific for the influenza A and B virus matrix genes (Aim 1, Objective 1) and transduce the amplicons into glucose via our novel, patent-pending sequence-specific oligonucleotide strand displacement (OSD) reporters. (Aim 1, Objective 2). We will automate the molecular assay on the PCC (Aim 2, Objective 3) and using biological samples perfect field-appropriate sample extraction procedures requiring only intuitive user input (Aim 2, Objective 4). Diagnostic utility of the PCC and the molecular assay will be demonstrated by comparing its robustness and its positive and negative predictive values with those of TaqMan real-time RT-PCR when challenged with blinded surrogate clinical samples (Aim 2, Objective 5). These data will position the PCC-based POC diagnostic for further validation with clinical samples and eventually field trials.

项目摘要/摘要 季节性流感每年在全球成人中的发病率高达10%，在儿童中高达30%。 大约300万到500万的感染导致严重的疾病，而大约25万到50万的感染导致严重的疾病 病例是致命的。在发病后48小时内服用抗病毒药物可减轻病情，降低死亡率。 出现症状。因此，敏感和特定的护理点(POC)诊断工具，特别是那些 可在家中使用，在加快适当治疗和减缓病毒传播方面可能是无价的。而当 TaqMan实时逆转录聚合酶链式反应(RT-PCR)和病毒培养 诊断金标准、几种POC流感快速诊断技术已被 商业化了。虽然在15分钟内就可以得到结果，但大多数这些测试的灵敏度都很低(40%-70% 与黄金标准相比)。基于恒温核酸扩增的快速检测(ALERE I流感A 和B测试)最近也出现了，虽然它们非常具体和敏感，但它们可以 对于广泛使用的PoC来说太贵了-每次测试70到85美元，并且需要Alere I仪器本身 价格约为8500美元。为了降低检测成本，并使核酸诊断更广泛地应用于- 家居检测我们建议开发一种一锅、坚固和廉价的POC检测方法，可以区分甲型流感 和B型病毒。这项技术的关键是通过环介导的等温扩增来扩增病毒RNA (LAMP)并将所得到的核酸扩增物转化为葡萄糖以通过广泛可得的方式读出， 非处方血糖仪。总体而言，在Paratus诊断公司和艾灵顿实验室之间的合作中， 得克萨斯大学奥斯汀分校，我们将开发一种Paratus消耗品墨盒(PCC)，它将用作 从患者样本中提取、放大和传递病原体遗传特征的一体机 用于读数的血糖仪，预计每台设备的成本约为5美元。特别是，我们将发展反向 转录(RT)-LAMP检测甲型和乙型流感病毒基质基因的特异性(目标1，目标1)和 通过我们正在申请专利的新型序列特异性寡核苷酸链将扩增产物转化为葡萄糖 流离失所(OSD)记者。(目标1、目标2)。我们将在PCC(AIM)上自动进行分子分析 2，目标3)和使用生物样本完善的适合现场的样本提取程序，要求 只有直观的用户输入(目标2、目标4)。PCC和分子分析的诊断效用将是 通过将其稳健性及其正负预测值与 TaqMan实时逆转录聚合酶链式反应(TaqMan Real Time RT-PCR)，当挑战盲法替代临床样本时(目标2，目标5)。 这些数据将定位基于PCC的POC诊断，以进一步验证临床样本和 最终进行了实地试验。