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：
6394721
负责人：
DAVID G BESSELSEN
金额：
$ 12.21万
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 2003-05-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/6394721
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.

实验动物健康监测对可靠的生物医学研究的价值已得到充分证实。尽管血清学测定为实验室啮齿动物中病毒和几种细菌的检测提供了基石，但越来越需要开发其他方法来检测这些物质。检测个体和/或感染源群体特异性核酸序列的高通量测定将为血清学检测提供理想的补充，并可用于评估整个动物和生物材料，例如可移植肿瘤和细胞系。传统的聚合酶链式反应 (PCR) 和逆转录酶聚合酶链式反应 (RT-PCR) 检测已针对多种传染源而开发，并已证明此类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 检测通常针对一种传染性病原体，其热循环参数差异很大，并且需要 PCR 后处理来检测 PCR 产物，所有这些都妨碍了对多种病原体样品的高通量评估。荧光 5' 引物核酸酶测定 (FFPNA) 已被开发出来，可以扩增和检测特定的 DNA 和 RNA 序列，无需进行 PCR 后处理。该技术有助于开发高通量检测，可用于有效监测各种生物样本中的多种感染因子。该项目的总体目标是开发 FFPNA，用于直接识别已知自然感染实验室啮齿动物和/或污染接种到啮齿动物体内的生物材料的传染原。该项目的具体目标是为感染实验室啮齿动物的大多数病毒和几种非病毒制剂开发属和/或种特异性 FFPNA，并评估这些测定作为啮齿动物抗体产生 (RAP) 测试的替代方法和实验室啮齿动物健康监测的辅助方法。 FFPNA 将大大增强实验室啮齿动物常规健康监测的诊断能力，并将为 RAP 检测提供可行、经济高效的替代方案。