5 NUCLEASE ASSAYS FOR RODENT HEALTH MONITORING

实验动物健康监测对开展可靠的生物医学研究的价值已得到充分证实。尽管血清学分析为检测实验室啮齿动物中的病毒和几种细菌物种提供了基础，但越来越需要开发其他方法来检测这些病原体。检测单个和/或群体感染因子特异性核酸序列的高通量检测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，如可移植肿瘤和细胞系。传统的聚合酶链反应（PCR）和逆转录聚合酶链反应（RT-PCR）检测方法已被用于多种感染因子，并已证明这种方法在啮齿动物诊断中的有效性。然而，目前可用的PCR和RT-PCR检测通常针对一种感染因子，其热循环参数差异很大，并且需要PCR后处理才能检测PCR产物，所有这些都妨碍了对多种病原体的样品进行高通量评估。荧光5'引物核酸酶测定法（FFPNA）已经开发出来，可以扩增和检测特定的DNA和RNA序列，而无需进行pcr后处理。该技术有助于开发高通量分析，可用于有效监测来自各种生物标本的多种感染因子。该项目的总体目标是开发FFPNA，用于直接鉴定已知的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感染因子。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，并评估这些测定法作为啮齿动物抗体产生（RAP）测试的替代方法和作为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检测提供一种可行的、具有成本效益的替代方法。

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.