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构建

发布时间：2018-04-03 22:21

  本文选题：游离胎儿DNA　切入点：无创性产前诊断　出处：《第三军医大学》2014年硕士论文

【摘要】：随着社会的迅速发展及医疗水平的不断提高，感染性疾病已经得到了有效的控制，遗传性疾病逐渐成为造成婴儿疾患的主要原因。目前，研究者已经发现300多种伴性遗传病，如血友病、色盲和肾性糖尿病等。伴性遗传病不仅影响患者的生活质量，而且给社会造成了极大的负担。早期识别胎儿性别可以提前预测伴性遗传病的发生情况，达到对伴性遗传病进行产前诊断的目的，这无疑对患者家庭及社会具有非常重要的意义。 目前，国内各大医院已经将产前诊断(prenatal diagnosis，PD)作为胎儿出生前的必检项目，诊断方法主要有绒膜绒毛取样、羊膜穿刺术、经皮脐血抽样、胎儿活组织检查及超声波诊断等。但是上述诊断方法存在诸多不足之处，如容易导致孕妇流产和感染，敏感度与准确性低等。 近年来，孕妇血浆遗传物质的发现为伴性遗传病的无创性产前诊断提供了新的思路。随着对母体血浆中胎儿遗传物质研究的不断深入，进一步发现孕妇血浆中存在着游离胎儿DNA (cell-free fetal DNA，cffDNA)。CffDNA在无创性产前诊断方面具有很多优势，但是其在母体外周血中含量很少，对检测方法的灵敏度要求较高。尽管如此，目前研究者借助现有的分子学诊断技术对cffDNA进行了检测分析，已经成功用于胎儿的RhD血型分析、妊娠相关疾病及染色体疾病的筛查。 此外，随着新技术新方法的不断发展，灵敏度更高、重复性更好的一些生物学技术也在不断被研发出来，基于MGB-TaqMan探针的实时荧光定量PCR就是近年来在普通的TaqMan探针基础上发展起来的一项新技术，其具有荧光本底低、分辨率更高、杂交特异性更强、重现性好、探针长度缩短等优点，更适合上述游离胎儿DNA这样的低丰度DNA的检测。 目的： 本文拟构建一种MGB-TaqMan探针实时荧光定量PCR技术方法，对母体血浆胎儿游离DNA进行检测分析，，并将其用于胎儿性别早期识别与伴性遗传病的无创性产前筛查。 方法： 1.合成X、Y染色体特异性的引物和探针；对引物、探针浓度及PCR的退火温度等反应条件进行优化，构建识别胎儿性别的实时荧光定量PCR检测技术；并且利用1例女性和1例男性的临床标本对筛选的最佳PCR反应条件进行验证。 2.通过男性与女性基因组DNA标准品不同比例的混合，模拟孕妇血浆游离胎儿DNA低丰度的情况；根据不同比例的混合DNA模板对构建的荧光定量PCR方法的灵敏度、准确性等指标进行评价。 3.随机选取门诊50例孕周为16-20周的孕妇标本及体检的20例健康未孕的女性标本，利用其对构建方法的临床检测能力进行验证与评估。 结果： 1.实时荧光定量PCR (Real-time PCR)确定的最优反应条件为：引物浓度为0.4M，探针浓度为0.1M，退火温度为60℃。 2.灵敏度检测结果显示能够检测到Y染色体的母体DNA模板量下限是50pg。母体DNA模板量在0.5-50ng之间时，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0.05-0.5ng之间时，染色体最低检测丰度为2-4%，相当于胎儿DNA丰度为4-8%。无母体DNA时，Y染色体模板量下限为0.5pg。 3.血浆游离DNA的定量检测中，健康未孕女性的血浆DNA浓度值最高为1.75ng/mL，最低为0.50ng/mL，平均值为1.13ng/mL；妊娠女性的血浆DNA浓度值最高为6.50ng/mL，最低为0.25ng/mL，平均值为3.38ng/mL。妊娠女性比正常女性的浓度均值高2.25ng/mL，具有显著的统计学差异(P0.05)。 4.在49例进行胎儿性别定性检测的妊娠女性中，初次实验，在未考虑母体血浆DNA模板起始用量时，结果为17例男性，32例女性，与胎儿出生后性别不完全一致；但加大DNA模板起始用量后(50pg)，结果为23例男性，26例女性，准确率为100%。 结论： 1.本文成功构建一种用于母体血浆胎儿游离DNA分析的MGB-TaqMan实时荧光定量PCR技术方法，可以通过对胎儿性别进行早期识别，为伴性遗传病的无创性产前筛查提供有价值的参考。 2.通过对不同比例的男性与女性基因组DNA标准品进行混合，成功模拟了孕妇血浆中的游离胎儿DNA的低丰度情况。 3.本研究构建的用于母体血浆胎儿游离DNA分析的实时荧光定量PCR技术方法具有较高的灵敏度。能够检测到Y染色体的母体DNA模板量下限是50pg。当母体DNA模板量大于50pg时，Y染色体DNA检测的灵敏度可以达到1%，即胎儿DNA丰度为2%。无母体DNA时，Y染色体模板量下限为0.5pg。 4.本研究构建的用于母体血浆胎儿游离DNA分析的实时荧光定量PCR技术方法鉴别胎儿性别的准确率为100%。标本定量检测中妊娠女性的血浆DNA含量明显高于正常女性。
[Abstract]:With the rapid development of society and the improvement of health level, infectious diseases have been effectively controlled, genetic disease has become the main cause of infant disease. At present, researchers have found that 300 kinds of sex linked genetic diseases, such as hemophilia, blindness and kidney of diabetes. Sex linked genetic disease not only affects patients the quality of life, but also to society caused great burden. Early identification of fetal sex partners can predict the occurrence of genetic diseases in advance to prenatal diagnosis of sex linked genetic disease, it is very important to the patient's family and society.
At present, the domestic large hospitals have prenatal diagnosis (prenatal diagnosis PD) as the fetus before birth must be detected, diagnosis methods mainly include amniocentesis, chorionic villus sampling, percutaneous umbilical blood sampling, fetal biopsy and ultrasonic diagnosis. But there are many deficiencies of the diagnosis methods, such as easily lead to pregnant women abortion and infection, sensitivity and low accuracy.
In recent years, genetic material found in plasma of pregnant women with genetic diseases of non-invasive prenatal diagnosis provides a new way of thinking. With the study of fetal genetic material in maternal plasma deepening further revealed the existence of fetal DNA in maternal plasma (cell-free fetal DNA, cffDNA.CffDNA) has many advantages in the noninvasive diagnosis but in the prenatal, maternal peripheral blood content rarely, sensitivity of detection method has higher requirement. In spite of this, the researchers used molecular current diagnostic techniques were tested on cffDNA, has been successfully used in the analysis of fetal RhD blood type, screening and disease related chromosomal disease in pregnancy.
In addition, with the continuous development of new technology and new method, higher sensitivity, better repetition of some biological technology is being developed, a new technique of real-time fluorescence quantitative PCR MGB-TaqMan probe is in recent years based on the common TaqMan probe is developed based on the fluorescence background with low resolution hybrid high, more specific, reproducible, the advantages of the probe length shorter, more suitable for the detection of fetal DNA in low abundance like DNA.
Objective:
In this paper, a real-time fluorescent quantitative PCR technique based on MGB-TaqMan probe was developed to detect and analyze fetal free DNA in maternal plasma, and it was used for noninvasive prenatal screening of fetal sex early and sex linked genetic diseases.
Method锛

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