Microchip to Detect Influenza Infection and Type in Nasopharyngeal Swabs

描述(申请人提供)：非常保守的估计认为，在1918年的流感疫情中，全世界总共有5000万人死亡。这些死亡中的大多数发生在24周内。减少快速传播的传染病造成的死亡人数绝对需要对感染者进行快速诊断、治疗和隔离。要取得成功，需要广泛使用简单、可靠和易于使用的分子诊断技术，以便在护理点最早检测到致病生物体。材料科学的最新进展使一系列具有功能的纳米和微米粒子成为可能。将这些颗粒作为复合材料加入到塑料微流控装置中，为从微升和亚微升样品体积中分离生物分子提供了新的机会。我们已经开发了一个塑料芯片实验室平台，用于样品制备、扩增和使用微流控通道检测人体样本中的病毒RNA。这些设备能够裂解病毒颗粒，并结合、浓缩和洗脱10个L或以下的流感感染哺乳动物样本的核酸。要测试的假设是，具有纳米级特征的塑料微流控诊断芯片可以在微升规模的人类鼻咽部样本中检测和识别甲型流感，其特异性和敏感性可与目前可用的护理点检测相媲美或更好。我们已经在试验台上演示了芯片的组件对感染流感的模拟样本起作用。我们的长期目标是开发一种便携式、相对便宜的分子诊断系统，可以用于各种医疗保健环境，以快速诊断和识别真实人类样本中的特定流感病毒株。此外，我们的目标是创造能够在低资源环境下使用的设备，这些设备由很少或根本没有硅或玻璃组件的聚合物材料制成。为了进一步推动这一努力，我们与波士顿大学医学中心的临床医生合作，开始对真实样本进行研究，并使用中等规模的患者组进行特异性和敏感性的初步研究。为了将这项技术应用于临床和现场，提出了以下目标：(1)优化芯片设计，以获得更快的响应时间和更好的性能。现在我们已经有了一个工作的原型系统，我们将检查每个单独的组件，以提高设备的效率。(2)优化检测方法(试剂和方法)，提高反应速度和可靠性。设备本身的工程需要与化验开发并驾齐驱。在微流控设备中，很少有像将台式分析缩小到小容量设置这样简单的事情。(3)使用人类鼻咽样本，确定微流控诊断在特异性和敏感性方面是否与最新的诊断方法相当或更好。样本将在北京医科大学急诊科从出现流感样症状的患者身上采集。基于芯片的检测将与病毒培养、血凝素抑制和台式RT-PCR检测进行比较。 要更有效地控制传染病的传播，就需要将预防工作和普遍提供廉价和准确的诊断方法结合起来。用于扩增和鉴定病毒核酸的探针可用于流感，但由于复杂的程序，病毒的分子检测并不广泛。需要专门设计测试方案(样品制备、分离、稀释、洗涤、阻断和检测)和设备，以便将这些技术从研究实验室转移到能够对公众健康产生更直接影响的领域。

DESCRIPTION (provided by applicant): Very conservative estimates hold that in total 50 million people died worldwide in the 1918 flu epidemic. Most of these deaths happened in a 24-week period. Reducing the death toll from fast spreading infectious diseases will absolutely require fast diagnosis, treatment and isolation of the infected. Success requires the wide availability of simple, robust, and easy to use molecular diagnostics for use at the point-of-care for the earliest detection of the causative organism. Recent advances in materials science have made available a wide array of functional nano and microscale particles. Incorporation of these particles as composites into plastic microfluidic devices provides new opportunities to separate biomolecules from microliter and sub-microliter sample volumes. We have developed a plastic lab-on-a-chip platform for sample preparation, amplification and detection of viral RNA in human samples using microfluidic channels. These devices are capable of lysing viral particles and binding, concentrating and eluting nucleic acids from influenza infected mammalian samples of 10 l or less. The hypothesis to be tested is that plastic microfluidic diagnostic chips with nanoscale features can detect and identify influenza A in microliter-scale human nasopharyngeal samples with a specificity and sensitivity comparable or better than presently available point-of-care testing. We have demonstrated at the bench that the components of the chip work with simulated samples infected with influenza. Our long term goal is to develop a portable, relatively inexpensive molecular diagnostic system that can be used in a variety of healthcare settings to quickly diagnose and identify specific flu strains in real human samples. In addition, we aim to create devices that can be used in low resource settings that are made of polymeric materials with few or no silicon or glass components. To further this effort, we have teamed with clinicians at the Boston University Medical Center to begin work with real samples and to perform an initial study of specificity and sensitivity using a moderately sized group of patients. In order to develop this technology for real-world clinical and field use the following aims are proposed: (1) Optimize the chip design to result in faster time-to-answer with better performance. Now that we have a working prototype system in hand, we will examine each of the individual components to improve the efficiency of the device. (2) Optimize the assay (reagents and protocol) for faster time-to-answer and reliability. Engineering of the device itself needs to proceed side by side with assay development. In microfluidic devices, it is rarely as simple as shrinking down a bench top assay into the small volume setting. (3) Determine whether the microfluidic diagnostic is comparable or better in specificity and sensitivity to state of the art diagnostic assays using human nasopharyngeal samples. Samples will be collected at BUMC Emergency Department from patients presenting with influenza-like symptoms. The chip based assay will be compared to viral culture, hemagglutinin inhibition and bench-top RT-PCR assays. More effective control of the spread of infectious diseases requires a combination of prevention efforts and the widespread availability of inexpensive and accurate diagnostics. Probes to amplify and identify viral nucleic acids are available for influenza, but molecular detection of the virus is not widely performed due to complicated protocols. Dedicated engineering of test protocols (sample preparation, separations, dilutions, washing, blocking, and detection) and devices is required to move these technologies out of the research laboratory and into the field where they can have a more immediate impact on public health.