VIRAL GENE AMPLIFICATION ASSAYS FOR DETECTION OF CONTAMINATING MURINE VIRUSES

广泛使用源自或通过的生物材料 用于研究调查或人类治疗方案的啮齿动物 引发了人们对检测到不经意的 被啮齿动物病毒污染的材料。诊断性测试 经常用来筛查这些病毒污染的老鼠 生物材料是改良的小鼠或大鼠抗体产生(MAP 或RAP)测试，一种涉及活体动物成分的抗体生物检测 生产病毒污染物，以及病毒分离组件。这个 耗时的MAP/RAP测试受不同敏感度和 具体细节。一种更准确和灵敏的检测方法 生物标本中啮齿动物病毒污染的影响因素 利用核酸扩增和杂交技术。这个 这项建议的总体目标是发展基因扩增和 杂交法用于鼠类病毒的直接鉴定 生物材料。具体目标如下：1)扩增基因 通过聚合酶链式反应(PCR)对所选的每个 已知可污染生物的小鼠RNA和DNA病毒清单 样本。用杂交法评价每种方法的特异性 利用高特异性、非放射性寡核苷酸的实验 探针和套式引物组。2.)开发复杂的聚合酶链式反应分析方法 在一个样本中可以检测到多种病毒 扩增试验包含针对每种病毒的特异引物PARIS。在……里面 此外，使用酶标记的液体杂交方法 将开发寡核苷酸探针以使扩增子自动化 序列确认程序。3.)比较特异度和 聚合酶链式反应检测与抗体产生的敏感性 测试。简单和复杂的聚合酶链式反应分析的灵敏度将是 根据感染了已知病毒的细胞样本计算得出 浓度，并与相同样品的结果进行比较 地图/说唱测试。通过应用基因扩增和 杂交技术，我们将能够快速准确地 评估试剂中的鼠类病毒污染，从而防止 这些材料在人类临床治疗和生物治疗中的应用 研究。

Extensive use of biologic materials originating from or passaged through rodents for use in research investigations or human therapeutic regimes has prompted increasing concern about detecting inadvertent contamination of materials with rodent viruses. The diagnostic test mouse frequently used to screen for virus contamination of these biologic materials is the modified mouse or rat antibody production (MAP or RAP) test, a bioassay involving a live animal component for antibody production to viral contaminates, and a virus isolation component. The time-consuming MAP/RAP test is plagues by variable sensitivities and specificities. A more accurate and sensitive approach for the detection of rodent viral contaminates in biologic specimens can be effected by use of nucleic acid amplification and hybridization technologies. The overall goal of this proposal is to develop gene amplification and hybridization assays for the direct identification of rodent viruses in biologic materials. The specific aims are as follows: 1.) Amplify gene sequences by polymerase chain reaction (PCR) from each of a selected list of murine RNA and DNA viruses known to contaminate biological samples. The specificity of each assay evaluated by hybridization experiments utilizing high-specificity, nonradioactive oligonucleotide probes and nested primer sets. 2.) Develop complex PCR assays such that multiple viruses can be detected in a single specimen by an amplification assay containing primer paris specific for each virus. In addition, liquid hybridization methods using enzyme-labelled oligonucleotide probes will be developed to automate the amplicon sequence confirmation procedures. 3.) Compare the specificity and sensitivity of the PCR assays with that of the antibody production tests. The sensitivity of the simple and complex PCR assays will be calculated from cellular samples infected with known virus concentrations, and compared with results of identical samples subjected to the MAP/RAP test. Through the application of gene amplification and hybridization technology, we will be capable of quickly and accurately assessing murine virus contamination in reagents, and thus prevent the use of these materials in human clinical treatments and in biological research.