Research And Application Of Nucleic Acid Extraction Technology From The Pathogen Of Infected Animal Tissues

In recent years, the situation of animal disease prevention and control in our country was very severe, cross-border incoming risk of exotic animal diseases was increasing. Molecular biology techniques were more and more used in the animal disease detection and diagnosis for its characters of fasten, sensitivity, specificity, etc. The quality and quantity of nucleic acids (DNA/RNA) extracted from animal tissue may affect subsequent species molecular identification, molecular biology diagnostic of epidemic disease and pathogenic classification and identification. In animal quarantine, the sensitivity and specificity of molecular detection methods were largely determined by the sample preparation (ie, process of nucleic acid extraction). So nucleic acid extraction techniques from the pathogen of animal tissue were studied, to meet the requirements of rapid nucleic acid extraction in the port animal quarantine and molecular detection in the laboratoryThe study consists of four parts as below:1. Nucleic acid was extracted by classic nucleic acid extraction method, such as phenol extraction method, the salt wash method, TriZol method and commercial kits.2. On the basis of the classical method, a new rapid method was improved and assembed for extracting high quality nucleic acid.3. Instance of DNA extraction: Using positive tissues infected by perkinsus as a template to optimize the formulation and process from the organization liquefaction, digestion and cell lysis, and the final purification. Then these selected techniques were assembled to determine the one-step combination method of DNA extraction. Lastly, quality testing was carried out with real-time fluorescence quantitative PCR on DNA extracted.4. Instance of RNA extraction:Using peste des petits ruminants virus (PPRV) inactivated cellculture and foot-and-mouth disease virus (FMDV) inactivated cellculture as a template, stability and sensitivity of RNA extracted by this method was tested by real-time fluorescence quantitative PCR technology, combined with the silica column purification technology.In summary, a method of nucleic acid extraction was established, which not only can meet rapid detection in the port, but also meets accurate detection in the laboratory. And it was the first time to carry out samples pretreatment techniques that were suitable for port rapid, highly sensitive detection of animal tissue samples. The method would broaden developing spaceof clinical application and animal disease detection.