Real-time Fluorescent Quantitative PCR Detection Of Endogenous And Exogenous Expression Of IGF-Ⅰ

Objective:The problem of doping has always been one of the major issues that have plagued all sports events,The world anti-doping agency prohibits the use of stimulants in all sporting events.However, the effective detection of stimulants is far behind the doping development and use,Through the study of gene doping IGF-Ⅰ, to set up a method for detecting the expression of exogenous gene and endogenous gene,to provide a theoretical basis for gene doping detection method IGF- I.Methods:This experiment adopts the method of r AAV2 mediated IGF- Ⅰinjecting exogenous gene into the muscle of mice,to detection of endogenous and exogenous expression of IGF-Ⅰ.30 male ICR rats were used to be the experimental object and then randomly divided into the blank control group(n=6), the injection of 3 days group(n=6), the injection of 7d group(n=6), the injection of 14 d group(n=6), 30 d injection group(n=6)。Construct IGF-Ⅰplasmid containing the purpose gene, and packaging of the virus. Oblique in the right knee of mice, the needle was injected with a virus, according to different experimental days respectively extract DNA from muscle and blood of the two different organizations.Design specific fluorescent probes in IGF-Ⅰgene sequences exon 2-3 to identify exogenous IGF-Ⅰgene.Synthesis of ITC genes and corresponding ITC probes, as the internal threshold control templates.To extract the sample DNA as a template for real-time fluorescent quantitative PCR.During the experiment, the sample DNA, ITC plasmid, purpose probes, ITC probes, primers and other reaction in the same system.After the experiment by comparing purpose probe and the ITC probe the size of the Ct value,to exclude the interference of external factors in the experiment.By comparing the corresponding category of Ct value, to determine the expression of IGF-Ⅰ.Experiment results were analysed by the Excel and SPSS 17.0 statistics software package.Result:(1) In the process of real-time fluorescent quantitative PCR,through experimental instrument can capture the purpose probe fluorescence signal accumulation,description experiment injection of exogenous IGF-Ⅰwas expressed in mice,the experimental design method is possible to detect the expression of the exogenous gene.(2) Different time get muscle,DNA was extracted for PCR.The experimental results show that along with the increase number of days Ct value increased gradually,and Ct value between 3 d and 7 d group increased obviously,Ct values between 14 d and 30 d group tends to slow the rise;Different time get blood,DNA was extracted for PCR,The experimental results show that with the increase experimental days Ct value changing trend is not obvious. But the Ct value of Ct value is less than the blank control group.(3) Respectively the muscles and blood was used to extract DNA as a template for PCR, In 3 d, 7 d group, the Ct value of muscle DNA less than the Ct values of blood DNA, and significant difference between two group(P < 0.01);In 15 d,30d group,the Ct value of muscle DNA greater than the Ct values of blood DNA, and significant difference between two group(P < 0.01).(4) With different materials of muscle, blood extract DNA as a template for PCR,the experimental results show that the amount of different materials between muscle and blood,Ct value is no significant difference(P > 0.05).Conclusions:(1) The experiment established method can effectively detect the distinction between endogenous and exogenous expression of IGF-Ⅰ.(2) Different time has a great influence on the detection result of muscle, there is no effect on the blood test results.(3) Different based site has a larger effect on testing results. The end of the experiment showed that the gene injection within 7 days the muscle test is more beneficial than blood testing,after 7 days the blood test is more beneficial than muscle testing.(4) Amount of different materials has no effect on the testing results of the experiment.