Simple Inexpensive Assay for Five Common HIV Resistance Mutations

 描述（由申请人提供）：只有少数廉价的药物可以在发展中国家用于治疗艾滋病。其中一些靶向HIV逆转录酶（RT）。因此，当编码RT的基因发生突变时，用这些药物进行的患者治疗常常失败。因此，WHO最近报道，在用抗RT药物治疗12个月后，当RT中出现以下突变时，患者最常复发：（a）奈韦拉平，K103 N和Y181 C;（B）替诺福韦和d4 T，K65 R;（c）3 TC和FTC，M184 V;和（d）胸苷类似物，D 67 N。因此，为了监测和立即的患者护理，NIAID和CDC发布了一项SBIR征求商业技术转让，以开发一种可以在资源有限的环境中快速，廉价地检测患者样本中这五种突变的检测方法。该招标寻求的基准质量标准为10，000分子/mL的检测水平（LOD）。本文提出的工作将提供具有更好的LOD（10-100分子/mL）的该测定，利用在2014年结束的FfAME的NIAID R 01下开发的技术。因此，选择了STTR资助形式来提供具有以下特征的测定，这些特征使其容易且便宜：1.该检测试剂盒将在一次检测中对含有耐药突变的RT基因区域进行多重扩增，避免了对每个等位基因进行五次单独检测的成本。由于FfAME-Firebird自回避分子识别系统（SAMRS），该性能规范是可能的。2.该检测将利用等温解旋酶依赖性扩增（HDA），而不是标准PCR。这避免了PCR仪器的成本和热循环的功率需求。多重HDA依赖于Benner实验室最近在NIAID R 01资助下开发的技术，包括SAMRS-逆转录酶HDA，其检测水平（LOD）为10 ~ 100个分子。3.为确保高覆盖率，将使用覆盖靶位点周围约90%序列多样性的多个引物。SAMRS通过防止引物-引物相互作用，使得这种引物多样性成为可能，并允许它们被扩增，而不需要重新设计多重引物。3.该检测试剂盒将在SNP检测之前扩增目标xNA，利用称为“人工扩增遗传信息系统”（AEGIS）的FfAME-Firebird技术的巢式PCR的极低噪音。4.该检测试剂盒将使用“正交信标”检测扩增子，也是在此开发的。这些也依赖于AEGIS技术来抑制背景噪声，允许通过眼睛检测少至1010个扩增子。5.读出将使用固定的信标，由手持电池供电的LED产生荧光，现场进行诊断，或者如果通过手机摄像头捕获，则在远程评估中心进行诊断。6.如果观察到交叉反应性，在第2阶段，它将使用氨氧基可逆终止剂与工程聚合酶一起减少，这是来自Benner实验室的另一项创新。

 DESCRIPTION (provided by applicant): Only a few inexpensive drugs can be used in the developing world for the treatment of AIDS. Several of these target the HIV reverse transcriptase (RT). Accordingly, patient therapy with these drugs often fails when the gene encoding RT undergoes mutation. Thus, the WHO recently reported that after 12 months of treatment with anti-RT drugs, patients most often relapsed when the following mutations arose in RT: (a) for nevirapine, K103N and Y181C; (b) for tenofovir and d4T, K65R; (c) for 3TC and FTC, M184V; and (d) for thymidine analogs, D67N. Therefore, both for surveillance and for immediate patient care, the NIAID and the CDC issued an SBIR solicitation for commercial technology transfer to develop an assay that could quickly and inexpensively detect these five mutations in patient samples in a resource-limited environment. The solicitation sought, as a benchmark specification, a level of detection (LOD) of 10,000 molecules/mL. The work proposed here will deliver this assay with a much better LOD (10-100 molecules/mL), exploiting technology developed under an NIAID R01 to the FfAME that ends in 2014. Thus, an STTR grant format has been chosen to deliver an assay with the following features that make it easy and inexpensive: 1. The assay will do multiplexed amplification for regions of the RT gene that contain resistance-conferring mutations in one assay, avoiding the cost of five separate assays for each of the alleles. This performance specification is possible because of FfAME-Firebird self-avoiding molecular recognition systems (SAMRS). 2. The assay will exploit the isothermal helicase-dependent amplification (HDA), not standard PCR. This avoids both the cost of a PCR instrument and the power demands of thermal cycling. Multiplexed HDA relies on technology recently developed in the Benner laboratory under the NIAID R01 grant, including SAMRS-reverse transcriptase HDA, which has a level of detection (LOD) of 10 ~ 100 molecules. 3. To ensure high coverage, multiple primers covering ~90% of the sequence diversity surrounding the target site will be used. SAMRS, by preventing primer-primer interactions, makes this multiplicity of primers possible, and allows them to be expanded, without needing to redesign the multiplex. 3. The assay will amplify target xNA before SNP detection, exploiting the very low noise of nested PCR using the FfAME-Firebird technology known as "artificially expanded genetic information systems" (AEGIS). 4. The assay will detect amplicons using "orthogonal beacons", also developed here. These also rely on AEGIS technology to suppress background noise, allowing detection by eye of as few as 1010 amplicons. 5. Readout will use immobilized beacons, with fluorescence generated by a hand-held battery-operated LED, with diagnosis made on the spot or, if captured by a cell phone camera, at a remote evaluation center. 6. Should cross-reactivity be observed, in Phase 2, it will be reduced using aminoxy reversible terminators with engineered polymerases, another innovation coming from the Benner laboratory.