Rapid DNA Assay System for Characterization of Infectious Disease Pathogens

描述(申请人提供)：我们建议使用高灵敏度的磁阵DNA检测系统，开发一种传染病病原体的快速检测方法。特别是，我们将利用MagArray平台开发一种针对大量HPV基因类型的分析方法。宫颈癌的病毒来源现已得到充分证实。最近的研究表明，在99.7%的宫颈癌中可以发现人乳头瘤病毒(HPV)DNA。由于不同的HPV亚型对癌症的发生具有不同的风险，因此能够区分亚型的检测方法可以指导临床治疗，并提供新的流行病学见解。目前的检测方法仅限于对100多种HPV类型中的30-35种进行基因分型，这通常是通过聚合酶链式反应完成的。使用从临床样本中提取的少量DNA对HPV进行检测、基因分型和广谱分析将是一项重大进展。在这里，我们建议采用磁阵列平台来检测广谱(所有类型)的HPV。磁阵平台的基础是使用磁性纳米颗粒来标记目标片段，而不是像目前的做法那样使用荧光分子。使用磁性纳米颗粒(通常直径15 nm)而不是荧光分子作为标签的优势是极大地提高了灵敏度。对于荧光标记，人们通常需要100,000个荧光标记的分子来获得足够的信噪比来进行可靠的识别。磁阵技术将作为一种灵敏、特定的诊断工具来检测多种HPV类型。在第一年，将对与宫颈癌有关的高危基因HPV-16和HPV-18进行可行性研究。在第二年，我们将用一个样本来确定至少20种HPV类型的敏感性和选择性，这些类型代表了大部分已知的类型，其中大多数与肛门和口腔癌密切相关。所有这些HPV类型都将在一次实验中被检测到。这将减少成本、打字所需的时间以及每种类型所需的DNA样本量。磁阵技术在人乳头瘤病毒相关肿瘤中的应用和发展将为其在其他感染性疾病和癌症研究及相关临床管理中的应用奠定基础。这项技术有可能被集成到一种紧凑的设备中，该设备简单易用，可以作为分子诊断应用的护理点系统。

DESCRIPTION (provided by applicant): We propose, using a high-sensitivity MagArray DNA detection system, to develop a rapid assay for infectious disease pathogens. In particular we will develop an assay for a large number of HPV genotypes using the MagArray platform. The viral origin of cervical cancer is now well established. Recent studies have shown that Human Papilloma Virus (HPV) DNA can be found in 99.7% of all cervical carcinomas. Because different HPV subtypes carry different risks for the development of cancer, a detection method that can distinguish subtypes could guide clinical care and offer new epidemiological insights. Current detection methods are limited to genotyping 30-35 of the more than 100 HPV types, and this is most often done by PCR. Detection, genotyping and analysis of broad spectrum of HPVs using a small amount of DNA that can be extracted from clinical samples would represent significant progress. Here we propose to adapt the MagArray platform to detect a broad spectrum (all types) of HPVs. The MagArray platform is based on using magnetic nanoparticles to tag the target fragments, rather than fluorescent molecules, as is the present practice. The advantage of using magnetic nanoparticles (typically 15 nm in diameter) as tags rather than fluorescent molecules is an enormous increase in sensitivity. With fluorescent labeling, one typically needs 100,000 fluorescently labeled molecules to obtain a signal/noise ratio adequate for reliable identification. MagArray technology will serve as a sensitive, specific diagnostic tool to detect multiple HPV types. In the first year feasibility studies will be performed for the high-risk genotypes HPV-16 and HPV-18 associated with cervical cancer. In the second year, we will determine the sensitivity and selectivity of at least 20 HPV types with a single sample, representing a large portion of the known types, most of which are strongly associated with anogenital and oral cancers. All these HPV types will be detected in a single experiment. This will reduce the cost, the time required for typing, and the amount of DNA sample required on a per-type basis. The application and development of MagArray technology to HPV-related cancer will serve as a basis for many applications in other infectious disease and cancer research and related clinical management. This technology has the potential to be integrated in a compact device that is simple and easy to use as a Point-of-Care system for molecular diagnostic applications.