Simple Inexpensive Assay for Five Common HIV Resistance Mutations

 描述(申请人提供)：在发展中国家，只有少数几种廉价的药物可以用于治疗艾滋病。其中有几个靶标是艾滋病毒逆转录酶(RT)。因此，当编码RT的基因发生突变时，患者使用这些药物的治疗往往会失败。因此，世界卫生组织最近报告说，在接受抗RT药物治疗12个月后，患者在RT出现以下突变时最常复发：(A)奈韦拉平K103N和Y181C；(B)替诺福韦和d4T，K65R；(C)3TC和FTC，M184V；以及(D)胸腺嘧啶核苷类似物，D67N。因此，无论是为了监测还是为了即时患者护理，NIAID和CDC都发布了一份SBIR商业技术转让邀请函，以开发一种在资源有限的环境中快速且廉价地检测患者样本中这五种突变的方法。此次征集寻求10,000个分子/毫升的检测水平作为基准规格。这里提出的工作将以更好的LOD(10-100分子/毫升)提供这种分析，利用在NIAID R01下开发的技术到FfAME，该技术将于2014年结束。因此，选择了一种STTRGRANT格式来提供一种具有以下特点的检测方法，这使得它变得简单和廉价：1.该检测方法将在一次检测中对包含与耐药性相关的突变的RT基因区域进行多路扩增，从而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)，这一性能规范成为可能。2.该方法将采用等温解旋酶依赖扩增(HDA)，而不是标准的聚合酶链式反应。这既避免了聚合酶链式反应仪器的成本，也避免了热循环的电力需求。多重HDA依赖于Benner实验室最近在NIAID R01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.为了确保高覆盖率，将使用覆盖目标位点周围约90%序列多样性的多个引物。SAMRS通过防止底物与底物的相互作用，使这种多种多样的底物成为可能，并允许其扩展，而无需重新设计多重构型。3.该检测方法将在SNP检测之前扩增目标XNA，利用FfAME-Firebird技术的套式聚合酶链式反应的极低噪声，该技术被称为“人工扩展遗传信息系统”(Aegis)。4.该方法将使用同样在这里开发的“正交信标”来检测扩增产物。它们还依靠宙斯盾技术来抑制背景噪音，允许肉眼检测到低至1010个扩音器。5.读数将使用固定信标，荧光由手持电池操作的LED产生，现场诊断，如果由手机摄像头捕获，则在远程评估中心进行诊断。6.如果观察到交叉反应，在第二阶段，将使用带有工程聚合酶的氨基可逆终止剂来减少交叉反应，这是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.