VIRAL GENE AMPLIFICATION ASSAYS FOR DETECTION OF CONTAMINATING MURINE VIRUSES

广泛使用源自或经过的生物材料

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.