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

项目总结/摘要 季节性流感的全球年发病率在成人中高达10%，在儿童中高达30%。 大约有300万到500万的感染会导致严重的疾病， 案件是致命的。抗病毒药物可以减轻严重程度，并降低死亡率时，48小时内给予的 出现症状。因此，敏感和特异性的床旁（POC）诊断工具，特别是那些 可以在家里使用，在加速适当的治疗和减轻病毒传播方面是非常宝贵的。而 TaqMan实时逆转录聚合酶链反应（RT-PCR）和病毒培养继续在临床上应用。 诊断金标准，几种快速POC流感诊断技术已经 商业化。虽然结果在15分钟内可用，但大多数这些测试的敏感性较差（40 - 70%）。 与黄金标准相比）。基于等温核酸扩增的快速检测（Alere i influenza A 和B试验）最近也变得可用，虽然它们非常特异和敏感，但它们可以 对于广泛的POC使用来说太贵了-每次测试70至85美元，并且需要Alere i仪器本身 费用约为8500美元。为了降低检测成本，使核酸诊断更广泛地用于... 家庭测试我们建议开发一种一锅法，强大的，廉价的POC测试，可以区分甲型流感 和B病毒。该技术的关键是通过环介导等温扩增进行病毒RNA扩增 （LAMP）和将所得核酸扩增子转化为葡萄糖以通过广泛可用的， 非处方血糖仪总的来说，在Paratus Diagnostics和Ellington实验室的合作中， 在德克萨斯大学奥斯汀分校，我们将开发一种Paratus耗材盒（PCC）， 从患者样本中提取、扩增和转导病原体遗传特征的一体化设备， 血糖仪的读数，预计成本约为5美元/设备。特别是，我们将开发反向 对甲型和B型流感病毒基质基因具有特异性的转录（RT）-LAMP试验（目的1，目标1），以及 通过我们新的、正在申请专利的序列特异性寡核苷酸链将扩增子转化为葡萄糖 流离失所（OSD）记者。(Aim 1，目标2）。我们将在PCC（Aim）上自动进行分子测定 2、目标3）和使用生物样品完善适合现场需要的样品提取程序 只有直观的用户输入（目标2，目标4）。PCC和分子测定的诊断效用将是 通过将其稳健性及其阳性和阴性预测值与 当用盲态替代临床样品进行挑战时，TaqMan实时RT-PCR（目的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品， 最终进行实地试验。

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.