5 NUCLEASE ASSAYS FOR RODENT HEALTH MONITORING

实验动物健康监测对可靠的生物医学研究的绩效的价值已经得到了很好的确立。虽然血清学检测已经为检测实验室啮齿动物中的病毒和几种细菌提供了基石，但越来越有必要开发其他方法来检测这些病原体。高通量检测单个和/或一组感染性病原体的核酸序列将是血清学测试的理想补充，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。传统的聚合酶链式反应(PCR)和逆转录-聚合酶链式反应(RT-PCR)方法已被发展用于多种病原体的检测，并证明了这种方法在啮齿动物诊断中的有效性。然而，目前可用的聚合酶链式反应和逆转录-聚合酶链式反应检测方法一般只针对一种病原体，它们的热循环参数差异很大，而且需要对聚合酶链式反应产物的检测进行后处理，所有这些都阻碍了对多个病原体样本的高通量评估。荧光5‘末端核酸酶分析(FFPNA)已经被开发出来，它可以扩增和检测特定的DNA和RNA序列，而不需要PCR后处理。这项技术有助于高通量分析的发展，可以用来有效地从各种生物标本中监测多种感染源。该项目的总体目标是开发FFPNA，用于直接鉴定已知可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病病原体。该项目的具体目标是建立感染实验室啮齿动物的大多数病毒和几种非病毒制剂的属/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)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将极大地提高实验室啮齿动物常规健康监测的诊断能力，并将为RAP检测提供一种可行的、经济有效的替代方案。

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.