Point of care diagnosis of HIV1 viral load using nano reagents and isothermal PCR

描述（由申请人提供）：我们的目标是开发一种具有高灵敏度、特异性、可靠性和可重复性的HIV-1病毒载量综合诊断测试，用于最低限度的基础设施设置。如果5%的目标人群能够及早获得治疗，这种用于围产期HIV-1诊断的检测每年可节省18万生命周期；如果100%的人群能够获得治疗，每年可节省多达250万生命周期。它还将有助于克服普遍获得治疗的主要挑战之一：缺乏对儿童艾滋病毒-1疾病的充分诊断和治疗（世卫组织）[1]。目前，定量HIV-1病毒载量的最灵敏、最可靠的检测方法依赖于核酸扩增和检测，但这些测试通常需要复杂的仪器和昂贵的试剂。另外，寡核苷酸包被的金纳米颗粒作为核酸检测中的靶向/报告剂具有很高的分析灵敏度[2,3]。为了达到所需的灵敏度，我们将开发一种HIV-1病毒载量测定方法，该方法将HIV-1 RNA的等温PCR扩增与靶向金纳米颗粒的使用和测试结果的比色定量相结合。我们将开发这种检测方法用于低资源环境，其基础设施要求最低，灵敏度和特异性可与发达国家可用的商业病毒载量检测方法相媲美。该提案的具体目标是：(1)开发一种廉价、敏感和特异性的诊断测试，以确定低资源环境中血清阳性患者的1型HIV病毒载量。该分析将结合：目标分离和等温扩增技术，从含有至少1000个病毒拷贝/ml的样品中产生至少104倍的扩增。然后将使用寡核苷酸靶向金纳米颗粒检测试验和定量读出来检测，以在POC中实现103至106 HIV-1病毒拷贝/ml的动态检测范围。(2)在临床标本中验证该方法测定HIV-1病毒载量的性能。我们将与耶鲁大学医学院具有HIV-1分子生物学专业知识的Richard Sutton博士合作，测试该检测方法从整个病毒颗粒和NIH/UNAIDS参考小组的M组分支中检测RNA的能力，并将该检测方法与RT-PCR进行比较。我们将与马拉维布兰太尔伊丽莎白女王中心医院的Elizabeth Molyneux博士合作，她在HIV-1的临床诊断方面具有专业知识，我们将开展一项初步研究，以确定这种新检测方法的敏感性和特异性。Molyneux博士团队的工作人员将前往休斯顿学习该检测方法，Richards-Kortum实验室的成员将前往马拉维与她的团队一起评估该检测方法在儿科临床样本中的应用，并与干血斑RT-PCR的金标准进行比较。

DESCRIPTION (provided by applicant): Our goal is to develop an integrated diagnostic test for HIV-1 viral load with high sensitivity, specificity, reliability, and reproducibility for use in minimal infrastructure settings. Such a test, applied in perinatal HIV-1 diagnosis, could save 180,000 DALYs each year if 5% of the targeted population has early access to therapy, and up to 2.5 million DALYs each year if 100% of the population has access to treatment [2]. It would also help to overcome one of the major challenges to universal access to therapy: the lack of adequate diagnosis and treatment of pediatric HIV-1 disease (WHO) [1]. Currently the most sensitive and reliable assays to quantify HIV-1 viral load rely on nucleic acid amplification and detection, but these tests often require sophisticated instrumentation and expensive reagents. Alternatively, the high analytical sensitivity of oligonucleotide-coated gold nanoparticles as targeting/reporting agents in nucleic acid tests has been demonstrated [2, 3]. To achieve the required sensitivity, we will develop an HIV-1 viral load assay which integrates isothermal PCR amplification of HIV-1 RNA with the use of targeted gold nanoparticles and colorimetric quantification of test results. We will develop this assay for use in low - resource settings, with minimal infrastructure requirements and a sensitivity and specificity comparable to that of commercial viral load assays available in the developed world. The specific aims of the proposal are to: (1) Develop an inexpensive, sensitive, and specific diagnostic test for determining type 1 HIV viral loads of seropositive patients in low-resource settings. The assay will combine: target isolation and isothermal amplification technologies to yield at least 104 fold amplification from samples containing a minimum of 1000 viral copies/ml. An oligonucleotide-targeted gold nanoparticle detection assay and a quantitative read-out will be then used to detect to achieve a dynamic detection range from 103 to 106 HIV-1 viral copies/ml at the POC. (2) Validate the performance of this assay for HIV-1 viral load determination in clinical specimens. In collaboration with Dr. Richard Sutton from Yale School of Medicine who has expertise in molecular biology of HIV-1, we will test the ability of the assay to detect RNA from whole viral particles, and from group M clades on an NIH/UNAIDS reference panel, comparing the assay to RT-PCR. In collaboration with Dr. Elizabeth Molyneux from Queen Elizabeth Central Hospital, Blantyre, Malawi who has expertise in clinical diagnosis of HIV-1, we will carry out a pilot study to determine the sensitivity and specificity of this new assay. Personnel from Dr. Molyneux's team will travel to Houston to learn the assay, and members of the Richards-Kortum lab will travel to Malawi to work with her group to evaluate the assay in pediatric clinical samples relative to the gold standard of dried blood spot RT-PCR. PUBLIC HEALTH RELEVANCE: Our goal is to develop an integrated diagnostic test for HIV viral load with high sensitivity, specificity, reliability, and reproducibility for use in minimal infrastructure settings. Viral load determination is needed to determine when to initiate therapy, monitor compliance, and most importantly, as an early indicator of therapeutic failure. Despite the encouraging trends in the scale-up of antiretroviral treatment in low- and middle-income countries reported by the WHO, reliable and accurate HIV load testing has yet to be introduced into the management of infected patients in low resource settings and remains one of the major challenges to universal access to therapy [1]. Such a diagnostic test, applied in perinatal diagnosis, could save 180,000 DALYs each year if 5% of the targeted population has early access to therapy, and up to 2.5 million DALYs if 100% of the population has access to treatment [2].