Point of care detection of HPV in saliva

扩展的护理点(POC)筛查和监控选项，可轻松检测疾病生物标志物 需要收集唾液等标本来降低衰弱的发病率和死亡率。 提高医疗系统的复原力，改善护理。创新科技精准医疗 麻省理工学院正在努力开发一种创新的诊断平台，可以 用于POC筛查和疾病生物标志物的监测。在我们最初的概念验证中，我们正在开发 唾液中高危HPV的POC检测。HPV是一种病毒感染，导致大多数口咽部 鳞癌(OPSCC)病例。最近的研究表明，人类乳头瘤病毒可以在 OPSCC患者的唾液。目前，OPSCC的初步筛查和诊断是在晚期进行的 以症状为基础的症状阶段，症状通常很轻微，可以模仿不太严重的情况，如感冒 或喉咙痛，延误诊断和治疗。HPV的DNA检测通常基于RT-PCR检测 从血清或唾液中收集并发送到中央实验室的病毒遗传物质。逆转录聚合酶链式反应 检测依赖于病毒遗传物质的扩增和检测。这些测试长期以来一直具有可操作性- 周转时间，而且成本很高。因此，目前还没有一种快速、准确、低成本的检测方法来进行高污染的常规监测。 风险群体(即年轻、性生活活跃、多性伴)和监测对治疗和复发的反应。 因此，在幸福感检查期间对唾液中的HPV进行非侵入性监测的POC测试可能具有重要的意义 影响和拯救生命。我们的目标是开发一种快速、易用的原型来检测人乳头瘤病毒。 唾液生化信号放大与CRISPR-CAS12电化学联用 读数和我们专有的坚固、准确和低成本的金叶电极。利用我们专有的金叶 电极结合CRISPR-Cas12法灵敏、特异地检测靶核 酸(NA)生物标记物，我们已经演示了用质粒DNA以及在 病人样本。我们对这项提议的总体目标是开发一种可重复使用的工艺来处理 研制了一种用于HPV16和HPV18型多重检测的电化学生物传感器样机 以及一个参考基因，以确保准确的测试结果。为了实现这一目标，我们将首先开发一种 我们的金叶电极用于检测HPV16、HPV18和最佳HPV16的工作流程 参考基因。然后，我们将首先用质粒DNA验证这个多重测试，然后用唾液验证 标本。我们预计，将易于制造的空间多路复用电极与CRISPR相结合- 用于检测特定核酸序列的Cas12a将使我们的第二阶段开发、调控 批准和商业化可扩展和制造的按需技术， 易于使用和低成本的需要点检测，并减轻HPV感染和OPSCC的负担。

Expanded point-of-care (POC) screening and monitoring options for detection of disease biomarkers from easily collectable specimens such as saliva are needed to reduce the incidence and mortality rates of debilitating diseases and enhance the resilience of the medical system, and improve care. InnoTech Precision Medicine with Massachusetts Institute of Technology is working to develop an innovative diagnostic platform that can be used for POC screening and monitoring of disease biomarkers. In our initial proof-of-concept, we are developing a POC test for detection of high-risk HPV in saliva. HPV is a viral infection that causes most Oropharyngeal squamous cell carcinoma (OPSCC) cases. Recent studies have demonstrated that HPV can be detected in saliva from patients with OPSCC. Currently, the initial screening and diagnosis of OPSCC is done at the late symptomatic stage based on symptoms that are often mild and can mimic less serious conditions such as cold or sore throat, delaying diagnosis and treatment. DNA tests for HPV are most often based on RT-PCR detection of the viral genetic material from serum or saliva that is collected and sent to central laboratories. RT-PCR-based tests rely on the amplification and detection of viral genetic material. These tests have long actionable turn- around-time, and high cost. Thus, there are no rapid, accurate and low-cost tests for routine monitoring of high- risk groups (i.e., young, sextually active, multiple partner) and monitoring of response to therapy and recurrence. Thus, a POC test for non-invasive monitoring of HPV in saliva during well-being exams can have a significant impact and save lives. Our objective is to develop a rapid, easy-to-use prototype for the detection of HPV in saliva based on the combination of biochemical signal amplification with CRISPR-Cas 12 electrochemical readout and our proprietary robust, accurate and low-cost gold-leaf electrodes. Utilizing our proprietary gold-leaf electrode along with a CRISPR-Cas12 assay for sensitive and specific electrochemical detection of target nucleic acid (NA) biomarkers, we have already demonstrated the detection of HPV 18 with plasmid DNA as well as in patient specimen. Our overall objective for this proposal is to develop a reproducible process for processing of saliva and develop a prototype electrochemical biosensor for the multiplex detection of HPV 16 and HPV 18 along with a reference gene to ensure accurate test results. To accomplish this goal, we will first develop a workflow for spatial multiplexing of our gold leaf electrodes for detection of HPV16, HPV 18 and an optimal reference gene. We will then verify this multiplexed test initially with plasmid DNA, and then with saliva specimens. We anticipate that combining easy-to-fabricate spatially multiplexed electrodes with CRISPR- Cas12a for the detection of a specific nucleic acid sequence will enable our Phase II development, regulatory approval and commercialization of a point-of-need technology that can be scaled and manufactured for accurate, easy-to-use and low-cost point-of-need testing and to reduce the burden of HPV infection and OPSCC.