Rapid Isothermal Nucleic Acid Amplification Assay and Device for HSV Testing

用于 HSV 检测的快速等温核酸扩增测定和装置

• 批准号: 7523579
• 负责人: Angelika Niemz
• 金额: $ 60.67万
• 依托单位: KECK GRADUATE INST OF APPLIED LIFE SCIS
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-07-15 至 2012-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7523579
• 关键词: Address; Biological Assay; Buffers; Classification; Clinic; Clinical; Clinical Engineering; Clinical Sensitivity; Communicable Diseases; Complex; Condition; Coupled; Coupling; Cytolysis; DNA; DNA amplification; Detection; Development; Devices; Diagnosis; Diagnostic; Electronics; Engineering; Environment; Genomics; Goals; Gold; Grant; HIV; Health Professional; Herpesvirus 1; Human; Human Herpesvirus 2; Human Identifications; Human Resources; Individual; Infection; Influenza; Interdisciplinary Study; Laboratories; Lateral; Lead; Lesion; Life; Liquid substance; Methods; Microfluidics; Molecular; Nanosphere; Newborn Infant; Nucleic Acid Amplification Tests; Nucleic Acids; Numbers; Oligonucleotides; Operative Surgical Procedures; Outcome; Partner in relationship; Pathogen detection; Pathology; Patients; Pregnant Women; Preparation; Protocols documentation; RNA Viruses; Range; Reaction; Reading; Reagent; Relative (related person); Reporting; Resources; Sampling; Science; Sensitivity and Specificity; Series; Simplexvirus; Staging; Standards of Weights and Measures; Swab; System; Testing; Time; Viral; Virion; Virus Diseases; Visual; Work; assay development; base; biothreat; cost; design; improved; instrumentation; internal control; magnetic beads; multidisciplinary; next generation; novel; pathogen; point of care; prevent; prototype; rapid detection; seal; technology validation; user-friendly; viral DNA; viral detection; virus identification; ward

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.

描述（由申请人提供）：该项目的目的是开发分析和设备，这些测定和设备将以简单，廉价且用户友好的格式以基于核酸的快速诊断。这样的系统将允许在护理点环境中的医疗保健专业人员及时识别和治疗传染病，从而改善患者的结局并防止进一步传播。该项目着重于单纯疱疹病毒1型和2型（HSV-1和HSV-2），这可能导致新生儿和免疫受损的个体威胁生命的感染。在性病诊所和产妇病房中，需要快速的HSV诊断。该项目涉及一个具有生物测定发展，工程和临床病理学专业知识的多学科研究团队。该团队将开发一个系统，该系统应能够从疱疹病变的拭子样品（据报道平均包含8*104个病毒颗粒颗粒），不受背景人类DNA的干扰或来自与骨质病变相关的其他细菌或病毒病原体。该系统应采用一次性且具有独立的样品开启式墨盒格式，该格式与低成本的手持电子模块配合，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测定时间应少于30分钟。该项目基于以前的工作，将新型的等温DNA扩增反应与通过DNA官能化的金纳米球以及生物疗法检测的微流体系统的开发结合了对寡核苷酸的比色检测。该项目的第一个目的是进一步开发并系统地优化单单和多重测定，以从临床样品的DNA分离株中快速，敏感和可靠的HSV检测，涉及等温扩增，然后进行视觉读取。第二个目的是建立和优化简单但有效的样品制备方案，适合于AIM 1下开发的等温测定方案，以促进从疱疹病变的拭子样品中稳健的HSV检测。第三个目标着重于开发设备原型以执行样品制备和AIM 1和2下开发的等温扩增方法。该项目提供了一个独特的机会，可以将多学科专业知识汇总在一起，以开发下一代核酸测试的测定法和分析师。所设想的系统将在护理设置中以与当前（主要是基于PCR的）方法相同的鲁棒性和敏感性在护理设置中进行快速病原体检测，同时与当前的基准台式系统相比，在核酸测试的核酸测试中，成本降低了1-2个数量级护理点。从长远来看，本文开发的分析和设备可以应用于其他临床病原体，包括HIV和流感等RNA病毒，以进行生物治疗检测以及资源有限的设置。