Molecular tests for malaria that can be performed with unprocessed samples

描述（由申请人提供）：该SBIR-AT-NIAID第一期申请解决了资源有限环境下疟疾诊断的问题。BioHelix开发了一种等温核酸扩增化学，称为解旋酶依赖性扩增（HDA），可耐受全血中存在的DNA扩增抑制剂。本课题将重点研究该技术在无需核酸提取的疟疾无性寄生虫检测中的应用。根据我们的初步结果，物种特异性分子检测（~100个核酸拷贝/5L血液）的预期灵敏度优于目前无性期快速检测（200个拷贝/5L血液）。我们这个为期两年的项目的具体目标是：1。开发一种HDA检测方法来检测恶性疟原虫、间日疟原虫、卵形疟原虫和疟疾疟原虫。2. 开发竞争性内部控制（CIC）的物种特异性分析，以及2-plex分析，以检测CIC与全血中的目标。3. 为提议的检测试剂盒开发制造工艺，使该技术能够在现场使用。4. 相对于金标准测试，评估分析的性能。5. 获得该检测的CE标志。6. 向FDA提交预ide，作为寻求FDA批准测试的初步步骤。

DESCRIPTION (provided by applicant): This SBIR-AT-NIAID Phase I application addresses the problem of malaria diagnostics in resource- limited settings. BioHelix, has developed an isothermal nucleic acid amplification chemistry called helicase dependent amplification (HDA) that is tolerant of the DNA amplification inhibitors present in whole blood. This proposal will focus on the application of this technology to the detection of malarial asexual parasites without nucleic acid extraction. Based on our preliminary results, the expected sensitivity of the species-specific molecular test (~100 nucleic acid copies/5L blood) better than that possible with current rapid tests for asexual stages (200 copies of parasite/5L blood). Our specific objectives for this 2-year-long project are: 1. Develop an HDA assay to detect P. falciparum, P. vivax, P. ovale, and P. malariae. 2. Develop competitive internal controls (CIC) for the species-specific assay, as well as a 2-plex assay to detect the CIC with the targets in whole blood. 3. Develop manufacturing processes for the proposed assay kit that will enable field-use of the technology. 4. Evaluate the performance of the assay relative to the gold-standard tests. 5. Obtain CE marking for the assay. 6. Submit a pre-IDE to FDA as a preliminary step to seeking FDA clearance for the test. PUBLIC HEALTH RELEVANCE: As malaria is the leading cause death in endemic regions, the current treatment approach is not based on laboratory, or microscopy testing because establishing quality-assured microscopy in rural and resource- poor settings is difficult. The liberal use of anti-malarial drugs that results from such practices presents a problem in that resistance to first line drug treatments is wide-spread. This SBIR-AT-NIAID proposal focuses on the development of a species-specific for the detection of Plasmodium in blood. More sensitive, species- specific, and stage-specific diagnostic tests will help focus drug treatment, and may slow the spread of anti- malarial drug resistance in the pathogen population. Based on our preliminary results, the expected sensitivity of the species-specific DNA test (~100 nucleic acid copies/5L blood) will be better than that possible with current rapid tests for asexual stages (200 copies of parasite/5L blood). A more sensitive test that can be performed in the field near patients would greatly enhance malaria fighting activities. We anticipate tests in the range of $7-$10 for the HDA tests should be possible, if total test volumes reach 200,000.