| Real time fluorescent quantitative PCR is a fluorescent probe or a fluorescent dye is added in the PCR reaction system,using the fluorescence signal accumulation,real-time monitoring of the entire PCR reflect the process, through the standard curve produced, to carry out quantitative analysis of unknown template. Because it has the advantages of simple operation, fast and efficient, high sensitivity, is widely used in molecular diagnosis, molecular biology research,animal and food safety inspection etc..The test of separating the peripheral blood lymphocytes in whole blood of dog,the success of PCR gene was amplified by IL-4, IL-10, IL-13, IFN-α several immune cytokines, connected to the pMD-18 T vector, transformed into competent cells of DH5 α, screening, PCR and sequencing verified in Amp resistant medium, construction the standard plasmid. Finally,successfully established a real-time fluorescent quantitative PCR method for detection of canine whole blood IL-4, IL-10, IL-13, IFN-α cell factor, the method of template in 102~108 copies/ L range shows a good linear relationship, the correlation coefficient R2 were above 0.992. By using the method of canine distemper virus(CDV), canine parvovirus(CPV), canine adenovirus type 1(CAV-1) triple viable cell immunity level of immunity in beagle dogs after the vaccine was evaluated.The test results show that the dog dogs immunized with triple live vaccine, immune canine peripheral blood lymphocytes and blank outside the control of peripheral blood lymphocytes compared, IL-4, IL-10, IL-13, IFN-α several cellular immune factor mRNA levels were elevated significantly between 3~14 days(p<0.05); there was no significant difference between 5kinds of cellular immune level of factor m RNA of the control group(p>0.05).The results showed that the real-time fluorescence quantitative PCR method established in this study is feasible, IL-4, IL-10, IL-13, IFN-α cell immune factor can be used as cell immune index of evaluation. |
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