5 NUCLEASE ASSAYS FOR RODENT HEALTH MONITORING

用于啮齿动物健康监测的 5 种核酸酶测定

• 批准号: 2839489
• 负责人: DAVID G BESSELSEN
• 金额: $ 15.6万
• 依托单位: UNIVERSITY OF ARIZONA
• 依托单位国家: 美国
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-06-15 至 2003-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2839489
• 关键词: SCID mouse; biomaterial evaluation; communicable disease diagnosis; diagnosis quality /standard; diagnostic tests; epizootiology; fluorescent dye /probe; laboratory mouse; laboratory rat; microorganism culture; nuclease; nucleic acid quantitation /detection; nucleic acid sequence; polymerase chain reaction; postmortem; technology /technique development; veterinary medicine

The value of laboratory animal health monitoring to the performance of reliable biomedical research has been well established. Although serologic assays have provided a cornerstone for the detection of viruses and several bacterial species in laboratory rodents, there is an increasing need to develop other methods to detect these agents. High-throughput assays that detect nucleic acid sequences specific for individual and/or groups of infectious agents would provide an ideal supplement to serologic testing and could be used to evaluate whole animals and biomaterials such as transplantable tumors and cell lines. Traditional polymerase chain reaction (PCR) and reverse transcriptase polymerase chain reaction (RT-PCR) assays have been developed for multiple infectious agents and have demonstrated the usefulness of this type of methodology in rodent diagnostics. However, currently available PCR and RT-PCR assays generally target one infectious agent, vary greatly in their thermocycling parameters, and require post-PCR processing for the detection of PCR products, all of which preclude high-throughput evaluation of samples for multiple agents. Fluorogenic 5' prime nuclease assays (FFPNA) have been developed that amplify and detect specific DNA and RNA sequences without the need for post-PCR processing. This technology lends itself to the development of high-throughput assays that can be used to efficiently monitor for multiple infectious agents from a variety of biological specimens. The overall goal of this project is to develop FFPNA for the direct identification of infectious agents known to naturally infect laboratory rodents and/or contaminate biomaterials that are inoculated into rodents. The specific aims of this project are to develop genus- and/or species-specific FFPNA for most viruses and several non-viral agents that infect laboratory rodents, and to evaluate these assays as an alternative to rodent antibody production (RAP) testing and as an adjunct methodology for laboratory rodent health monitoring. FFPNA will greatly enhance the diagnostic capabilities for routine health monitoring of laboratory rodents and will provide a viable, cost-effective alternative for RAP testing.

实验室动物健康监测对可靠生物医学研究绩效的价值已得到充分确立。 尽管血清学检测为实验室啮齿动物中病毒和几种细菌的检测提供了基础，但越来越需要开发其他方法来检测这些病原体。 高通量检测，检测核酸序列的具体个人和/或群体的传染性病原体将提供一个理想的补充血清学测试，并可用于评估整个动物和生物材料，如可移植的肿瘤和细胞系。 传统的聚合酶链反应（PCR）和逆转录酶聚合酶链反应（RT-PCR）检测已开发用于多种传染性病原体，并已证明这种方法在啮齿动物诊断中的有用性。 然而，目前可用的PCR和RT-PCR测定通常针对一种传染性病原体，它们的热循环参数变化很大，并且需要PCR后处理以检测PCR产物，所有这些都排除了对多种病原体的样品的高通量评价。已经开发了荧光5'引物核酸酶测定（FFPNA），其扩增和检测特异性DNA和RNA序列而不需要PCR后处理。 该技术有助于开发高通量检测方法，可用于有效监测多种生物标本中的多种感染因子。 本项目的总体目标是开发FFPNA，用于直接鉴定已知自然感染实验室啮齿动物和/或污染接种到啮齿动物中的生物材料的传染性病原体。 本项目的具体目标是开发用于感染实验室啮齿动物的大多数病毒和几种非病毒因子的属和/或种特异性FFPNA，并评估这些检测方法作为啮齿动物抗体产生（RAP）检测的替代方法和实验室啮齿动物健康监测的辅助方法。FFPNA将大大提高实验室啮齿动物常规健康监测的诊断能力，并将为RAP测试提供可行的，具有成本效益的替代方案。