用于无创产前诊断的SRY基因高灵敏高特异检测方法研究

通过检测母体外周血中胎儿游离DNA（cffDNA）的SRY基因,确定胎儿性别,可评估胎儿性连锁遗传病的发病风险,降低病儿出生率。本研究建立了高灵敏、高特异、闭管检测不易污染的实时荧光PCR偶联核酸侵入反应方法用于SRY基因的检测。通过优化反应体系中的检测探针浓度、FEN1酶用量、Taq酶用量及预扩增退火温度,确定了最佳的反应条件,即检测探针浓度为250 nmol/L、FEN1酶用量为7.5 U、Taq酶用量为0.5 U、预扩增退火温度为67℃。在最佳反应条件下,实现对含量低至4‰（4 copies/μL）的模拟样本的检测,并成功检测两例孕期分别为9周和10周的临床实际样本。结果表明,所建立的方...

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Bibliographic Details
Published in分析化学 Vol. 45; no. 10; pp. 1448 - 1454
Main Author 刘琳琳 郑梦琳 邹秉杰 宋沁馨 周国华
Format Journal Article
LanguageChinese
Published 南京军区南京总医院药理科,南京210002 2017
药物质量与安全预警教育部重点实验室,中国药科大学,南京210009%南京军区南京总医院药理科,南京,210002%药物质量与安全预警教育部重点实验室,中国药科大学,南京210009
Subjects
SRY基因
实时荧光PCR
无创产前诊断
核酸侵入反应
胎儿游离DNA
SRY基因
核酸侵入反应
胎儿游离DNA
Cell-free fetal DNA
SRY gene
Invader assay
实时荧光PCR
无创产前诊断
Non-invasive prenatal diagnosis
Real-time polymerase chain reaction
Online AccessGet full text
ISSN0253-3820
DOI10.11895/j.issn.0253-3820.170332

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Summary:通过检测母体外周血中胎儿游离DNA（cffDNA）的SRY基因,确定胎儿性别,可评估胎儿性连锁遗传病的发病风险,降低病儿出生率。本研究建立了高灵敏、高特异、闭管检测不易污染的实时荧光PCR偶联核酸侵入反应方法用于SRY基因的检测。通过优化反应体系中的检测探针浓度、FEN1酶用量、Taq酶用量及预扩增退火温度,确定了最佳的反应条件,即检测探针浓度为250 nmol/L、FEN1酶用量为7.5 U、Taq酶用量为0.5 U、预扩增退火温度为67℃。在最佳反应条件下,实现对含量低至4‰（4 copies/μL）的模拟样本的检测,并成功检测两例孕期分别为9周和10周的临床实际样本。结果表明,所建立的方法可用于母体外周血cffDNA的SRY基因检测,为临床开展基于SRY基因的无创产前诊断提供了新方法。
Bibliography:By detecting SRY gene of cell-free fetal DNA（cffDNA） in maternal peripheral blood,the sex of fetuses was determined,the risks of sex-linked genetic disorders were assessed and the birth rate of sick fetuses was decreased.A method of real-time polymerase chain reaction（PCR） coupled with invader assay was established to detect SRY gene.This method possessed the advantages such as high sensitivity,high specificity and non-contaminated with closed tube detection.Under the optimized reaction conditions such as 250 nmol/L detection probes,7.5 U FEN1 enzyme,0.5 U Taq polymerase and 67℃ of annealing temperature in pre-amplification,the simulated samples as low as 4‰（4 copies/μL） were detected and two clinical samples with the gestation age of 9 weeks and 10 weeks were successfully detected.The detection results showed that this method could be used to detect SRY gene of cffDNA in maternal peripheral blood,providing an effective technique for clinical non-invasive prenatal diagnosis based on SRY gene.
LIU Lin-Lin1,ZHEN
ISSN:0253-3820
DOI:10.11895/j.issn.0253-3820.170332