Apply Of Real-time Fluorescence Quantitative Polymerase Chain Reaction For The Diagnosis Of Down Syndrome From A Single Cell

Objective To investigate the feasibility and practicality of the application of real-time fluorescence quantitative PCR (FQ-PCR) as a method for diagnosis of Down syndrome form a single cell. To supply the experimental evidence of a stable, accurate, simple and rapid diagnosis technique and non-invasive prenatal diagnosis for Down syndrome.Methods 21 peripheral blood samples of Down's patients, 1 cord blood sample of Down's fetus, and 40 peripheral blood samples of normal controls were collected. All of the blood samples were treated by density gradient centrifugation to collect the nucleated cell layer. Single lymphocytes were isolated from the smear with Wright's staining by micromanipulation techniques. Primer extension preamplification (PEP) which use the 15 bases random primer and real-time fluorescence quantitative PCR were used to amplify the S100B and DCSR1 gene locus located on chromosome 21, and GAPDH located on chromosome 12. They were used to be the objective genes and the interior reference house-keeping gene, then a relative quantitation of these genes between patient group and normal control group could be done. Two pairs ofΔCT values were analytically compared between the two groups. The ratios of S100Bpatient/S100Bcontrol,DSCR1patient/DSCR1control products were calculated in trisomy 21 group.Results 3 nucleated cells of each patient or control were isolated from the smear. The product of PEP was used to amplify three different gene loci. As a result, at least 2 cells of each group objects were successfully amplified. And 79.0%(147/186) of the cells were totally amplified with three different loci, which means that 21%(39/186) of the cells were failed to amplify three loci. TheΔCT values of Down syndrome patients were significantly lower than those of normal controls. The difference between the two groups was statistically significant (P<0.01). The ratios of S100Bpatient/S100Bcontrol,DSCR1patient/DSCR1control products for trisomy 21 were 1.891(1.563~2.287)and 1.840(1.562~2.168), respectively.Conclusion PEP is a microamount genome amplification technique established in special reaction condition. It satisfies the detection of polygenic locus by the amplification of genome. With the use of random primer, PEP may keep a good balance in amplification procedure. Real-time fluorescence quantitative PCR is a quantitative detection of gene with high sensitivity, by which we could get the stable result of our study. Therefore, we distinguished the Down's patients from the normal control group. Real-time fluorescence quantitative PCR may provide a new way for non-invasive prenatal diagnosis and preimplantation genetic diagnosis for Down syndrome. Because of the real-time fluorescence quantitative PCR, a quick, simple, and accurate genetic diagnosis of Down's could be completed in 24hours. We can believe that it could be used widely in clinic in future.