Rapid Isothermal Nucleic Acid Amplification Assay and Device for HSV Testing

描述(由申请人提供)：该项目的目标是开发能够以简单、廉价和用户友好的形式实现基于核酸的人类病原体快速诊断的分析和设备。这样的系统将使医疗保健专业人员能够及时识别和治疗传染病，从而改善患者的预后并防止进一步传播。该项目的重点是1型和2型单纯疱疹病毒(HSV-1和HSV-2)，这种病毒可能导致新生儿和免疫功能受损的人受到危及生命的感染。在性病诊所和产科病房中存在对HSV快速诊断的需求。该项目涉及一个多学科的研究团队，拥有生物检测开发、工程和临床病理学方面的专业知识。该团队将开发一种系统，该系统应能够从疱疹皮损的拭子样本(据报道平均每毫升裂解缓冲液中含有8*104个病毒颗粒)进行常见或特定类型的HSV检测，而不会受到背景人类DNA或与口腔生殖系统损害相关的其他细菌或病毒病原体的干扰。该系统应采用与低成本手持电子模块相匹配的一次性和自给式样本输入-回答-输出墨盒格式，以提供易用性并避免交叉污染。包括样品制备和检测在内的总分析时间应不超过30分钟。该项目建立在先前工作的基础上，将新颖的等温DNA扩增反应与通过DNA功能化的金纳米球对寡核苷酸的比色检测相结合，并开发用于生物硫酸盐检测的微流控系统。该项目的第一个目标是进一步开发和系统优化单重和多重检测方法，用于从临床样本的DNA分离中快速、灵敏和可靠地检测HSV，包括等温扩增和视觉读出。第二个目标是建立和优化简单但有效的样本制备方案，适合与根据目标1开发的等温分析相结合，以促进从疱疹皮损拭子样本中进行可靠的HSV检测。第三个目标侧重于开发设备原型，以执行根据目标1和目标2开发的样品制备和等温放大方法。该项目提供了一个独特的机会，将多学科的专门知识结合在一起，开发用于核酸测试的下一代分析和分析器。设想的系统将能够以与当前(主要基于聚合酶链式反应的)方法相同的稳健性和灵敏度在护理点环境中快速检测病原体，同时与目前用于护理点核酸检测的台式系统相比，成本降低1-2个数量级。从长远来看，这里开发的分析和设备可以应用于其他临床病原体，包括艾滋病毒和流感等RNA病毒，用于生物治疗检测，以及资源有限的环境。

DESCRIPTION (provided by applicant): The goal of this project is the development of assays and devices that will enable rapid nucleic acid-based diagnosis of human pathogens in a simple, inexpensive, and user-friendly format. Such a system will allow health care professionals in a point-of-care setting to identify and treat infectious diseases in a timely manner, thereby improving patient outcomes and preventing further spread. The project focuses on Herpes Simplex Virus type 1 and 2 (HSV-1 and HSV-2), which can lead to life-threatening infections in newborns and immuno- compromised individuals. A need exists for rapid HSV diagnosis in STD clinics and in maternity wards. The project involves a multidisciplinary research team with expertise in bioassay development, engineering and clinical pathology. This team will develop a system which shall be capable of type-common or type-specific HSV detection from swab samples of herpetic lesions (reported to contain on average 8*104 virus particles per mL lysis buffer), with no interference from background human DNA or from other bacterial or viral pathogens associated with orogenital lesions. The system shall employ a disposable and self-contained sample-in answer-out cartridge format that mates to a low cost handheld electronics module, to provide ease-of-use and to avoid cross-contamination. The total assay time including sample preparation and detection shall be less than 30 minutes. The project builds on previous work combining novel isothermal DNA amplification reactions with colorimetric detection of oligonucleotides through DNA-functionalized gold nanospheres, and on the development of microfluidic systems for biothreat detection. The first aim of this project is to further develop and systematically optimize single- and multiplex assays for rapid, sensitive, and robust detection of HSV from DNA isolates of clinical samples, involving isothermal amplification followed by visual read-out. The second aim is to establish and optimize simple but efficient sample preparation protocols suitable for coupling to the isothermal assays developed under aim 1, to facilitate robust HSV detection from swab samples of herpetic lesions. The third aim focuses on developing device prototypes to execute the sample preparation and isothermal amplification methods developed under aims 1 and 2. This project presents a unique opportunity to bring together multidisciplinary expertise to develop a next generation of assays and analyzers for nucleic acid testing. The envisioned system will enable rapid pathogen detection in point of care settings with the same robustness and sensitivity as current (mostly PCR-based) methods, while reducing the cost by 1-2 orders of magnitude compared to current benchtop systems for nucleic acid testing at the point of care. In the long term, the assays and devices developed herein can be applied to other clinical pathogens, including RNA viruses such as HIV and influenza, to biothreat detection, and to resource-limited settings.