VIRAL GENE AMPLIFICATION ASSAYS FOR DETECTION OF CONTAMINATING MURINE VIRUSES

用于检测污染性鼠病毒的病毒基因扩增测定

• 批准号: 6122943
• 负责人: CYNTHIA L. BESCH-WILLIFORD
• 金额: --
• 依托单位: UNIVERSITY OF MISSOURI-COLUMBIA
• 依托单位国家: 美国
• 财政年份: 1997
• 资助国家: 美国
• 起止时间: 1997-06-01 至 1999-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6122943
• 关键词: DNA virus; Polyomavirus muris 1; RNA virus; antiviral antibody; communicable disease diagnosis; cytomegalovirus; diagnosis design /evaluation; laboratory mouse; laboratory rat; lymphocytic choriomeningitis; method development; nucleic acid hybridization; nucleic acid probes; nucleic acid sequence; polymerase chain reaction; virus diseases; virus genetics; zoonosis

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.

广泛使用来源于或通过 用于研究调查或人类治疗方案的啮齿动物 越来越多的人开始关注 材料被啮齿动物病毒污染。 诊断测试 老鼠经常用来筛选病毒污染的这些 生物材料是修饰的小鼠或大鼠抗体生产（MAP 或RAP）测试，涉及用于抗体的活动物成分的生物测定 生产到病毒污染物，和病毒分离组件。 的 耗时的MAP/RAP测试受到可变灵敏度的困扰， 特殊性 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， 使用核酸扩增和杂交技术。 的 该提案的总体目标是发展基因扩增， 用于直接鉴定啮齿动物病毒的杂交测定 生物材料。 具体目标如下：（1）扩增基因 通过聚合酶链反应（PCR）从每个选定的 已知污染生物制品的鼠RNA和DNA病毒列表 样品 通过杂交评价各试验的专属性 使用高特异性、非放射性寡核苷酸的实验 探针和嵌套引物组。 2.）的情况。开发复杂的PCR检测， 一个样本中可以检测出多种病毒， 含有对每种病毒特异的引物巴黎的扩增测定。 在 使用酶标记的附加液体杂交方法 将开发寡核苷酸探针以使扩增子自动化 序列确认程序。 3.）第三章比较特异性和 PCR检测的灵敏度与抗体产生的灵敏度 试验. 简单和复杂PCR测定的灵敏度将是 根据已知病毒感染的细胞样本计算 浓度，并与相同样品的结果进行比较， MAP/RAP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 评估试剂中的鼠病毒污染，从而防止 这些材料在人类临床治疗和生物医学中的应用 research.