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

基本信息

批准号：
6540625
负责人：
DAVID G BESSELSEN
金额：
$ 12.52万
依托单位：
UNIVERSITY OF ARIZONA
依托单位国家：
美国
项目类别：
财政年份：
1999
资助国家：
美国
起止时间：
1999-06-15 至 2004-05-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/6540625
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

项目摘要

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的性能的价值已经确定。尽管血清学测定为检测病毒和实验室啮齿动物的几种细菌物种提供了基石，但越来越需要开发其他方法来检测这些药物。高通量测定法检测了针对个体和/或传染性剂组的核酸序列，将为血清学测试提供理想的补充，可用于评估全动物和生物材料，例如移植肿瘤和细胞系。传统聚合酶链反应（PCR）和逆转录酶聚合酶链反应（RT-PCR）测定已为多种传染剂开发，并证明了这种方法在啮齿动物诊断中的有用性。但是，当前可用的PCR和RT-PCR分析通常针对一种感染剂，其热循环参数差异很大，并且需要PCR后处理以检测PCR产物，所有这些都排除了多种药物的样品的高通量评估。已经开发了荧光5'蛋白核酸酶测定（FFPNA），可以放大和检测特定的DNA和RNA序列，而无需PCR后处理。这项技术可用于开发高通量测定法，可用于有效监测来自各种生物标本的多种感染剂。该项目的总体目标是开发FFPNA，以直接鉴定已知的自然感染实验室啮齿动物和/或接种啮齿动物的生物材料的传染药。该项目的具体目的是为大多数病毒和几种感染实验室啮齿动物的非病毒药物开发属和/或物种特异性的FFPNA，并评估这些测定方法作为啮齿动物抗体生产（RAP）测试的替代方法，并作为实验室啮齿动物健康监测的辅助方法。 FFPNA将大大增强实验室啮齿动物的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。