Practical application of fluorescent quantitative PCR on Trisomy 21 in Chinese Han population

Objective To apply the fluorescent quantitative PCR method on the detection of Trisomy 21 by D21S11 locus and make a foundation for rapid prenatal diagnosis of Trisomy 21. Methods About 409 controls (39 amniotic fluid samples and 370 peripheral blood samples) and 35 patients (4 amniotic fluid samples and 31 peripheral blood samples) with Trisomy 21 were tested using fluorescent quantitative PCR by amplification of DNA fragment on D21S11 STR locus. The results were compared with conventional cytogenetic analysis to confirm the utility of this method. And the allele frequency distributions of D21S11 STR locus were analyzed. Results The 95% reference interval of fluorescent intensity ratios of peak heights of PCR products amplified from two alleles on D21S11 locus ranged from 0.84 to 1.42 (1.13 ± 0.29) in heterozygous controls. About 19 out of 35 patients showed a “diallelic“ pattern and their height ratio of fluorescent peaks of PCR products amplified from two alleles in patients with “diallelic” patterns were all outside of the 95% reference range of controls. The PCR products of DNA from 12 patients presented the third allele. No sample with the “monoallelic“ pattern was found. Four chimeras diagnosed by cytogenetic method could not be diagnosed by this method. There were 17 and 11 alleles found in controls and patients, respectively. About 343 out of 409 controls were heterozygous and the heterozygosity was 83.86%. We did not find any significant differences in the frequency distributions of alleles on D21S11 locus between controls and patients. But there were significant differences in the frequency distributions of alleles on D21S11 locus between controls and patients. But there were significant differences in the frequency distributions of alleles on D21S11 locus among different populations. Conclusions The fluorescent quantitative polymerase chain reaction method was rapid, accurate, and only small amount of starting material was needed, it could be applied in rapid prenatal diagnosis of Trisomy 21. D21S11 was a good marker with high heterozygosity for the screening of Trisomy 21. And the frequency distributions of alleles on D21S11 locus were significantly related to ethnic background. This work was supported by grants from the National Natural Science Foundation of China (30200107) as well as the Dominant Youth Fund from Wuhan University School of Medicine