单细胞实时荧光定量PCR结合比较阈值法在诊断唐氏综合征中的应用

Application of real-lime fluorescence quantitative PCR accompanied with comparison of Delta CT for diagnosis of Down＇s syndrome from a single cell

目的探讨单细胞水平实时荧光定量PCR技术对于诊断唐氏综合征的应用价值。方法22例唐氏综合征患者及胎儿、40名正常对照外周血或脐血经梯度离心、显微操作分离单个淋巴细胞，应用引物延伸预扩增及实时荧光定量PER技术扩增基因组12号染色体上GAPDH基因、21号染色体上S100B基因和DSCR1基因。比较病例组与对照组ACT值有无差异，计算病例组S100B／GAPDH、DSCR1／GAPDH的值。结果两组间ACT值差异有统计学意义（P〈0．01），病例组低于对照组。病例组S100B／GAPDH、DSCR1／GAPDH值分别为1．891（1．563～2．287）、1．840（I．562～2．168）。结论实时荧光定量PCR是一种可信的诊断方法，为唐氏综合征的无创性产前诊断及植入前遗传学诊断等提供了新的思路。

Objective To evaluate the use of real-time fluorescence quantitative PCR （FQ-PCR） accompanied with comparison of Delta CT as a method for diagnosis of Down＇s syndrome from a single cell. Methods Single lymphocyte was isolated from the peripheral or umbilical cord blood samples of 22 clinically diagnosed Down＇s syndrome patients or fetus and 40 normal controls by mieromanipulation techniques. Primer extension preamplifieation （PEP） and real-time FQ-PCR were used to amplify the SIODB and DCSR1 located on chromosome 21, and GAPDH located on ehromosome 12. Two pairs of ACT values were compared between the two groups. The ratios of S1OOB/GAPDH and DSCR1/GAPDH products were calculated in trisomy 21 group. Results The Acr values of Down＇s 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 S100B/GAPDH and DSCR1/GAPDH products for trisomy 21 were 1. 891 （ 1.563-2.287 ） and 1. 840（ 1.562-2. 168）, respectively. Conclusion Real-time FQ-PCR is a reliable method that may provide a new way for non-invasive prenatal diagnosis and preimplantation genetic diagnosis for Down＇s syndrome.