Low cost molecular assay for HIV

描述（由申请方提供）：我们建议开发一种快速、低成本、阈值检测方法，用于检测HIV疫苗接种试验参与者的HIV感染。我们建议开发的检测方法将使用我们专有的解旋酶依赖性扩增（HDA）平台以及专门设计用于进行分子检测的低成本设备，而不会用扩增产物污染实验室。HDA类似于聚合酶链式反应（PCR），因为它使用两个引物以指数方式扩增核酸。它与PCR的不同之处在于它是完全等温的（因此不需要昂贵的热循环仪）。我们建议使用侧流装置来检测扩增产物。此类装置已经证明了它们在分离核酸扩增产物中的实用性，并且广泛用于FDA装置数据库中列出的几种“中等复杂性”和CLIA豁免测试。扩增子的检测将通过使用与抗体偶联的乳胶颗粒来完成，所述抗体与用于检测HDA反应产物的探针结合。该检测试剂盒采用夹心形式检测掺入HDA引物之一和各检测探针中的半抗原（生物素、FITC和地高辛）。用于这些测定的侧向流动条具有两个捕获区，因此允许检测一种分析物（HIV）以及竞争性内部对照。未能在侧流装置窗口中给出条带的测定被评分为无效，而仅在对照线中具有条带的测定被评分为真阴性。在对照和测试线位置处具有条带的测定在阈值处被评分为阳性（通常为200个分析物拷贝）。在测试线中具有强条带但没有对照线的测定被评分为强阳性（通常超过5000个分析物拷贝）。初步研究表明，当使用序列特异性捕获方法从血浆样品中提取的核酸用作RT-HDA模板时，检测限为100拷贝/mL血浆。我们建议使用从5个高风险患者招募点获得的临床标本进一步测试HIV检测的特异性和灵敏度。试验将在BioHessel和范德比尔特大学进行。试剂盒将提供给Dr. Yi-Wei Tang（范德比尔特大学），以便其实验室评价检测试剂盒。此外，我们建议在南非国家传染病研究所（NICD）进行验证研究，以获得在南非销售试剂盒的许可。最后，我们还可能与Assuragen签订合同，为我们的竞争性内部控制设计装甲RNA的克隆。在II期结束时，我们将获得初步数据，以便向FDA提交IDE，启动临床研究，寻求在美国销售人体诊断用检测系统的上市前批准（PMA）。

DESCRIPTION (provided by applicant): We propose to develop a rapid, low cost, threshold assay for the detection of HIV infection of HIV vaccination trial participants. The assay we propose to develop will use our proprietary helicase dependent amplification (HDA) platform as well as a low cost device specifically designed to perform molecular tests without contaminating the laboratory with amplification products. HDA is similar to the polymerase chain reaction (PCR) in that it uses two primers to exponentially amplify nucleic acids. It is distinct from PCR in that it is entirely isothermal (and thus does not require costly thermocyclers). We propose to use a lateral flow device to detect amplification products. Such devices have already proven their utility in separating nucleic acid amplification products, and are widely used in several "moderate complexity" and CLIA-waived tests listed in the FDA device database. Detection of amplicons will be accomplished by using latex particles conjugated to antibodies that bind to the probes used to detect the HDA reaction products. The assay uses a sandwich format to detect haptens (biotin, FITC and Digoxigenin) incorporated into one of the HDA primers and into each of the detection probes. The lateral flow strip used for these assays has two capture zones, and thus allows for the detection of one analyte (HIV) as well as a competitive internal control. Assays that fail to give a band in the lateral flow device window are scored as invalid, while assays with a band in the control line alone are scored as true negative. Assays with bands at both the control and test line position are scored as positives at the threshold value (typically 200 copies of the analyte). Assays with a strong band in the test line but no control line are scored as strong positives (typically over 5000 copies of analyte). Preliminary studies suggest a limit of detection of 100 copies/mL plasma when nucleic acids extracted from plasma samples with a sequence-specific capture method are used as templates for RT-HDA. We propose to further test the specificity and sensitivity of the HIV test with clinical specimens obtained from 5 high-risk patient recruitment sites. Assays will be performed at BioHelix, and at Vanderbilt University. Kits will be supplied to Dr. Yi-Wei Tang (Vanderbilt University) so his laboratory can evaluate the assays. In addition, we propose to perform a validation study at the National Institute for Communicable Diseases (NICD) of South Africa to gain clearance to sell kits in South Africa. Finally, we may also contract with Assuragen to engineer a clone of Armored RNA for our competitive internal control. By the end of Phase II, we will have preliminary data for submission of an IDE to the FDA to kick-off a clinical study to seek pre market approval (PMA) for sale of the assay system for human diagnostics in the US.