Molecular tests for malaria that can be performed with unprocessed samples

描述（由申请人提供）：该SBIR-AT-NIAID第I阶段应用解决了资源有限环境中的疟疾诊断问题。BioHancing公司开发了一种称为解旋酶依赖性扩增（HDA）的等温核酸扩增化学，它能耐受全血中存在的DNA扩增抑制剂。该提案将重点关注该技术在不提取核酸的情况下检测疟疾无性寄生虫的应用。根据我们的初步结果，种属特异性分子检测的预期灵敏度（约100个核酸拷贝/5L血液）优于目前无性阶段快速检测的可能灵敏度（200个寄生虫拷贝/5L血液）。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，用于检测恶性疟原虫、间日疟原虫、卵形疟原虫和三日疟原虫。 2.开发用于种属特异性测定的竞争性内部对照（CIC），以及用于检测全血中具有靶标的CIC的2重测定。 3.为拟议的检测试剂盒制定生产工艺，以便能够在现场使用该技术。 4.评价相对于金标准检测的检测性能。 5.获得检测试剂盒的CE认证。 6.向FDA提交预IDE，作为寻求FDA批准该试验的初步步骤。 公共卫生相关性：由于疟疾是流行地区的主要死亡原因，目前的治疗方法不是基于实验室或显微镜检测，因为在农村和资源贫乏的环境中建立有质量保证的显微镜是困难的。这种做法导致的抗疟疾药物的自由使用带来了一个问题，即对一线药物治疗的耐药性广泛传播。该SBIR-AT-NIAID提案的重点是开发一种用于检测血液中疟原虫的物种特异性。更敏感的、物种特异性的和阶段特异性的诊断测试将有助于集中药物治疗，并可能减缓病原体群体中抗疟疾药物耐药性的传播。根据我们的初步结果，种属特异性DNA检测的预期灵敏度（约100个核酸拷贝/5L血液）将优于目前无性阶段快速检测的可能灵敏度（200个寄生虫拷贝/5L血液）。一种可以在病人附近进行的更敏感的测试将大大加强疟疾防治活动。我们预计，如果总测试量达到200，000，HDA测试的测试价格在7 - 10美元之间应该是可能的。

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.