Cartridge-Format Branched DNA System for HIV Viral Load Measurement in Low-Resour

描述（由申请人提供）：在低资源环境中测量HIV病毒载量是一个持续需要的领域。两种技术的结合为改善服务不足的患者群体获得艾滋病毒载量测量带来了巨大希望。第一种是分支DNA（bDNA），这是一种黄金标准的核酸化学方法，它用多达10，000个信号分子标记病毒RNA的每条链，从而能够在1 mL血浆样本中检测到少至75个病毒RNA拷贝，并且由于样本污染导致的检测失败风险很低。第二种是SCA微流体技术，用于高性能的检测盒上样品/试剂处理，检测盒和仪器之间具有静态接口。该第II阶段项目的起点是成功的第I阶段工作，以缩短bDNA检测的持续时间，同时使检测适应于封闭式检测盒形式。新开发的盒式bDNA检测试剂盒（EO-NAT HIV Assay）包括重新设计的捕获探针和信号化学，同时保留了原始检测试剂盒的多层探针配置和高特异性合成碱基。bDNA检测试剂盒中的初级杂交步骤在带有嵌入式SCA微型泵的封闭式检测盒中运行，速度提高了8倍：在常规参考实验室仪器上运行初级杂交步骤需要16小时，而在EO-NAT HIV嵌入式检测试剂盒中，该步骤仅需120分钟。总的测试周转时间已减少到不到四个小时，这一时间有利于治疗艾滋病毒/艾滋病患者获得有限的护理，对他们来说，实现良好的临床结果往往取决于在同一次访问中进行病毒载量测试和制定管理计划。II期项目工作将两个额外的技术要素纳入EO-NAT HIV检测试剂盒。杂交的bDNA复合物的检测是通过具有亚纳摩尔浓度的信号分子和多路复用的电化学发光的特殊形式。样品制备，包括裂解和预浓缩，是通过嵌入集成的光聚合整料。在第二阶段的第二年，生产了集成所有四种技术元素的检测盒-检测盒优化的bDNA化学、SCA微流体技术、mECL检测和提取整体。这些测试卡片和原型EO- NAT仪器在临床标本上进行测试，以验证系统的临床灵敏度和特异性。在第二阶段的最后阶段，符合ISO 13485标准的试点生产将加速进行，为向监管机构提交文件做准备。本项目的中心假设是，在EO-NAT仪器上运行的EO-NAT HIV Assay可在20 μ L样本中检出少至50个拷贝的HIV RNA，灵敏度为98%（n=100），并且在检测阴性样本（n=100）时特异性大于90%。公共卫生相关性：血浆病毒载量，通过复杂的血液测试确定，是艾滋病毒/艾滋病患者病情如何以及他或她对治疗反应如何的指标。定期测量病毒载量对于照顾艾滋病毒/艾滋病患者很重要，但这些测试目前并不适用于所有人。该项目开发了一种新的系统，使病毒载量测量更便宜，更容易为远离先进医疗设施的医生和患者提供。

DESCRIPTION (provided by applicant): HIV viral load measurement in low-resource settings is an area of persistent need. A combination of two technologies holds great promise for improving access to HIV viral load measurement among underserved patient populations. The first is branched DNA (bDNA), a gold-standard nucleic acid chemistry which labels each strand of viral RNA with as many as 10,000 signaling molecules to enable detection of as few as 75 copies of viral RNA in a 1 mL plasma sample with low risk of test failure from sample contamination. The second is SCA microfluidic technology for high-performance on-cartridge sample/reagent processing with a static interface between cartridge and instrument. The starting point for this Phase II project is the successful Phase I work to shorten the bDNA assay's duration while simultaneously adapting the assay to an enclosed-cartridge format. The newly developed cartridge- format bDNA assay, the EO-NAT HIV Assay, includes re-engineered capture probes and signaling chemistry while retaining the original assay's multi-tiered probe configuration and high-specificity synthetic bases. Running in enclosed cartridges with cartridge-integrated SCA micropumps, the primary hybridization step in the bDNA assay has been speeded up eight-fold: whereas the primary hybridization step takes 16 hours running on conventional reference laboratory instruments, in the EO-NAT HIV cartridge-format assay this step requires only 120 minutes. Total test turnaround time has been reduced to less than four hours, a duration which facilitates treatment of HIV/AIDS patients with limited access to care, for whom achieving favorable clinical outcomes often depends on carrying out viral load testing and developing a management plan in the same visit. The Phase II project work incorporates two additional technological elements into the EO-NAT HIV assay. Detection of hybridized bDNA complexes is by a specialized form of electrochemiluminescence with sub- nanomolar concentrations of signaling molecules and multiplexing. Sample preparation, including lysing and preconcentration, is by cartridge-integrated photopolymerized monoliths. During the second year of Phase II, cartridges integrating all four technological elements-cartridge-optimized bDNA chemistry, SCA microfluidic technology, mECL detection, and extraction monoliths-are produced. These cartridges and prototype EO- NAT instruments are tested on clinical specimens to validate the clinical sensitivity and specificity of the system. In the final portion of Phase II, pilot ISO 13485-compliant manufacturing is ramped up in preparation for filings with regulatory agencies. The central hypothesis of this project is that the EO-NAT HIV Assay running on EO-NAT instruments detects as few as 50 copies of HIV RNA in 20 microliter samples with 98% sensitivity (n=100) and exhibits specificity greater than 90% in testing of negative samples (n=100). PUBLIC HEALTH RELEVANCE: Plasma viral load, determined by complex blood tests, is an indication of how sick an HIV/AIDS patient is and how well he or she is responding to treatment. Regular viral load measurement is important in caring for HIV/AIDS patients, but these tests are currently not available for everyone. This project develops a new system which makes viral load measurement less expensive and more readily available to doctors and patients who live far from sophisticated medical facilities.