基于性染色体定量检测的伴性遗传病无创性产前筛查方法的研究

目的：利用孕妇血浆游离DNA对产前胎儿性别进行识别，为伴性遗传性疾病的无创性产前筛查提供参考。方法利用X、Y染色体的特异性引物和探针构建识别胎儿性别的实时荧光定量聚合酶链反应（PCR）检测技术，并对灵敏度、准确性等方法学指标进行评价，然后将构建好的方法用于24例未孕健康女性及临床上50例16～20孕周的孕妇血浆DNA的检测。结果在母体DNA胎儿Y染色体定性检测方法的灵敏度检测中，当胎儿DNA丰度为3％～6％时，母体DNA模板量下限是50 pg。当母体DNA模板量在0．5～50 ng时，Y染色体最低检测丰度为1％，相当于胎儿DNA丰度为2％；当母体DNA模板量在0．05～0．5 ng时，Y染色体最低检测丰度为2％～4％，相当于胎儿DNA丰度为4％～8％；当无母体DNA时，Y染色体模板量下限为0．5 pg。标本定量检测中妊娠女性的血浆DNA浓度明显高于未孕健康女性（P＜0．05）。50例孕妇静脉血标本中，除1例标本因提取失败未作检测外，其余49例标本初次试验在未考虑母体血浆DNA模板起始用量时，检测结果为17例男性，32例女性，与胎儿出生后性别不完全一致；但是当加大母体血浆DNA模板起始用量（＞50 pg）后，结果为23例男性，26例女性，准确率为100％。结论该试验所构建的利用母体血浆游离DNA进行的早期无创性产前筛查方法具有较高的准确性，可以为伴性遗传出生缺陷性疾病的早期筛查和预防提供重要的参考价值。

Quantitative detection of sex chromosomal for non-invasive prenatal screening of sex-linked inheritable disease

Objective To identify the fetal sex for the non-invasive prenatal screening of sex-linked inheritable diseases using free DNA in maternal plasma .Methods A method to identify fetal sex ,using the X and Y chromosome specific primers and TaqMan probes ,was developed and evaluated for the sensitivity .Then ,24 cases of healthy women and 50 cases of pregnant women with gestational age for 16-20 weeks were detected and analyzed .Results The results of sensitivity analysis for fetal Y chromosome detection in maternal DNA indicatd that when the fetal DNA abundance was 3% -6% ,the required amount of plasma DNA from pregnant women was at least 50 pg .When the template amount of plasma DNA from pregnant women was 0 .5-50 ng ,the limit of detection for fetal Y-chro-mosome was 1% ,indicating that the fetal DNA abundance should be 2% for detecting .When the template amount of plasma DNA from pregnant women was 0 .05-0 .5 ng ,the limit of detection for fetal Y-chromosome was 2% -4% , indicating that the fetal DNA abundance should be 4% -8% .When there were no maternal DNA as template ,the limit of detection for Y-chromosome could reach as low as 5 pg .The plasma DNA levels of pregnant women were sig-nificantly higher than that of healthy females in the quantitative detection （P〈0 .05） .One in the 50 cases of pregnant women samples could not be detected for the failure of DNA extraction .The result of the first detection showed 17 males and 32 females in the remaining 49 cases ,which was not highly consistent with clinical results .Then ,the detec-tion was performed again by increasing the quantity of initial DNA （〉 50 pg） .The results showed an accuracy of 100% compared with the clinical results ,including 23 males and 26 females .Conclusion The accuracy of this method for early non-invasive prenatal screening using free DNA in maternal plasma could be high enough for early diagnosis and prevention of sex-linked genetic diseases .