CAJ | 학술논문

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。
목적：이용잉부혈장유리DNA대산전태인성별진행식별，위반성유전성질병적무창성산전사사제공삼고。방법이용X、Y염색체적특이성인물화탐침구건식별태인성별적실시형광정량취합매련반응（PCR）검측기술，병대령민도、준학성등방법학지표진행평개，연후장구건호적방법용우24례미잉건강녀성급림상상50례16～20잉주적잉부혈장DNA적검측。결과재모체DNA태인Y염색체정성검측방법적령민도검측중，당태인DNA봉도위3％～6％시，모체DNA모판량하한시50 pg。당모체DNA모판량재0．5～50 ng시，Y염색체최저검측봉도위1％，상당우태인DNA봉도위2％；당모체DNA모판량재0．05～0．5 ng시，Y염색체최저검측봉도위2％～4％，상당우태인DNA봉도위4％～8％；당무모체DNA시，Y염색체모판량하한위0．5 pg。표본정량검측중임신녀성적혈장DNA농도명현고우미잉건강녀성（P＜0．05）。50례잉부정맥혈표본중，제1례표본인제취실패미작검측외，기여49례표본초차시험재미고필모체혈장DNA모판기시용량시，검측결과위17례남성，32례녀성，여태인출생후성별불완전일치；단시당가대모체혈장DNA모판기시용량（＞50 pg）후，결과위23례남성，26례녀성，준학솔위100％。결론해시험소구건적이용모체혈장유리DNA진행적조기무창성산전사사방법구유교고적준학성，가이위반성유전출생결함성질병적조기사사화예방제공중요적삼고개치。
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 .