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、廉价和用户友好的格式快速诊断人类病原体的检测方法和设备。这样的系统将使卫生保健专业人员在护理点环境中及时识别和治疗传染病，从而改善患者的治疗效果并防止进一步传播。该项目的重点是1型和2型单纯疱疹病毒（HSV-1和HSV-2），这两种病毒可在新生儿和免疫功能低下的个体中导致危及生命的感染。性病诊所和产科病房需要快速诊断单纯疱疹病毒。该项目涉及一个多学科研究团队，具有生物测定开发、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性病变的拭子样本（据报道，每mL裂解缓冲液平均含有8*104个病毒颗粒）中检测常见类型或特定类型的HSV，而不受人类DNA背景或与口腔生殖器病变相关的其他细菌或病毒病原体的干扰。该系统应采用一次性和自成一体的样本输入回答盒格式，与低成本手持式电子模块相匹配，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少于30分钟。该项目建立在先前的工作基础上，将新型等温DNA扩增反应与通过DNA功能化金纳米球进行的聚核苷酸比色检测相结合，并开发用于生物威胁检测的微流体系统。该项目的第一个目标是进一步发展和系统地优化从临床样品的DNA分离物中快速、灵敏和可靠地检测HSV的单次和多次检测方法，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品制备方案，适合与目标1中开发的等温分析相结合，以促进从疱疹病变拭子样本中进行稳健的HSV检测。第三个目标侧重于开发设备原型，以执行目标1和目标2下开发的样品制备和等温扩增方法。该项目提供了一个独特的机会，汇集多学科专业知识，开发下一代核酸检测方法和分析仪。设想的系统将能够在护理点环境中快速检测病原体，具有与当前（主要是基于pcr的）方法相同的稳健性和灵敏度，同时与目前在护理点进行核酸检测的台式系统相比，将成本降低1-2个数量级。从长远来看，本文开发的检测方法和设备可应用于其他临床病原体，包括RNA病毒，如艾滋病毒和流感，生物威胁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.