Improved Chlamydia Test Using Aptamers on a Portable POC Microfluidic Platform

描述(由申请人提供)：该计划的基本目标是开发一种基于适配子的高保真的生殖器衣原体感染检测方法，允许快速和定量地确定不同血清类型的沙眼衣原体感染水平。最终目标是将这种测试集成到一种廉价的便携式微流控设备中，可以在任何医生的办公室使用。该设备将提供有关一些未得到充分服务的人群的重要健康方面的重要信息。关键成分是一系列血清型特异性DNA适配子，它们对检测沙眼衣原体表现出非常高的灵敏度和特异度(99,999%)。DNA适配子作为抗体的增强替代品，并与微流控版本的夹心ELISA中的目标微生物发生特异性反应，该微流控版本的夹心ELISA是由团队成员之一Claros Diagnostics(剑桥大学马萨诸塞州)最近开发的便携式手持设备实现的。提出了一种两阶段方案。第一阶段最终在约翰·霍普金斯大学(马里兰州巴尔的摩)国际性病、呼吸和生物研究实验室对沙眼衣原体适配子试验进行验证。该计划的第二阶段将集中于将检测、生物化学和仪器网格结合在一个新的护理点(医生办公室)沙眼衣原体检测中。提案团队积累的初步数据涵盖了约翰·霍普金斯大学夏洛特·A·盖多斯教授小组带来的沙眼衣原体感染的临床和社会方面的丰富经验和深厚知识。Claros Diagnostics的微流体检测平台作为一个小型设备实现，能够处理微型生物样本，并允许快速、可重复地处理微升液体体积。这项新的适配子选择技术已经由Infositex公司开发，并已成功地应用于生产高质量的DNA适配子，用于美国国防部的应用，如炭疽毒素和传染性普恩的检测。 公共卫生相关性：目前，生殖器衣原体病最敏感的诊断方法是实时定量聚合酶链式反应qRT-PCR。根据最近的报道，沙眼衣原体的主要外膜蛋白(OMP-1)基因和多态蛋白H(PMP-H)基因的L血清特异性区域可用于检测沙眼衣原体。每一次实时聚合酶链式反应的检测极限是每次反应50个基因组拷贝(Schaeffer，2008)。然而，作者承认，用这种方法区分一些沙眼衣原体血清变种是困难的。如果大规模实施，对于服务不足的人群来说，沙眼衣原体RT-PCR诊断的成本和诊断时间可能会变得令人望而却步。因此，对患者来源的样本进行快速和负担得起的护理点分析可以彻底改变生殖器衣原体感染的诊断领域。我们的技术将为医生提供廉价、简单的分析和明确的读数，建议或排除在医生办公室进行进一步治疗的必要性。总而言之，这里提出的检测-设备组合将接触到服务不足的人群，当能够进行生殖器衣原体感染的护理点诊断时，他们可以得到更充分的保护。

DESCRIPTION (provided by applicant): The fundamental goal of this program is to develop a point of care high fidelity, aptamer based assay for genital Chlamydia infections that allows fast and quantitative determination of levels of infection with different sero-types of Chlamydia trachomatis. The ultimate goal is to have this assay be integrated into an inexpensive portable microfluidics device that can be used in any physician's office. This device will provide vital information on important health aspects of some underserved populations. The key element is a family of serotype specific DNA aptamers that exhibit very high sensitivity and specificity (99,999 %) of detection of C. trachomatis. The DNA aptamers act as an enhanced replacement for the antibodies and specifically react with the target microorganisms in a microfluidic version of sandwich ELISA realized in a portable hand held device recently developed by one of the team members, Claros Diagnostics (Cambridge MA). A two phase program is proposed. The Phase I culminates in the validation of the aptamer assay for Chlamydia trachomatis in the International STD, Respiratory, and Biothreat Research Laboratory of Johns Hopkins University (Baltimore, MD). The Phase II of the program will focus on bringing together the detection biochemistry and instrumental grid in a new point of care (physician's office) assay for Chlamydia trachomatis. The preliminary data accumulated by the proposal team encompass extensive experience and profound knowledge of clinical and societal aspects of C. trachomatis infections brought by the group of Professor Charlotte A. Gaydos, Johns Hopkins University. Claros Diagnostics' microfluidics detection platform realized as a small device is capable of dealing with microscale bio-samples and allows rapid reproducible handling of microliter liquid volumes. The novel aptamer selection technology has been developed by Infoscitex Corporation and has been successfully applied to produce high quality DNA aptamers for the US Department of Defense applications, such as detection of anthrax toxin and infectious prions. PUBLIC HEALTH RELEVANCE: Currently, the most sensitive diagnosis of genital chlamydiosis employs quantitative real-time polymerase chain reaction qRT-PCR. As recently reported, the major outer membrane protein (omp-1) -gene and a L- serovar-specific region of the polymorphic protein H (pmp-H) -gene could be used to detect C. trachomatis. The detection limit of each real-time PCR was 50 genome copies per reaction (Schaeffer, 2008). However, difficulties in distinguishing some of the C. trachomatis sero-variants by this method were acknowledged by the authors. If implemented on a large scale, the costs of RT-PCR diagnosis for C. trachomatis and time to diagnosis may, unfortunately, become prohibitive for the underserved populations. Therefore, a rapid and affordable point of care analysis of patient-derived specimens can revolutionize the field of diagnosis of genital chlamydial infections. Our technology will provide physicians with an inexpensive simple assay and unambiguous readout that will suggest, or rule out, the necessity of further treatment right in the physician's office. In summary, the assay-device combination proposed here will reach the underserved populations who can be much more fully protected when enabled with point of care diagnosis of genital chlamydial infections.