孕妇外周血胎儿ABO血型基因分型技术的建立

本研究目的是建立孕妇外周血胎儿ABO血型基因分型技术，用于ABO血型不合引起的新生儿溶血病的产前诊断。根据ABO血型基因DNA全序列和mRNA序列设计4对引物，选择20例健康供者血浆，提取血浆中DNA进行扩增，探索最佳的血浆DNA提取及PCR扩增条件，初步建立单人ABO血型基因分型技术。将O型血浆DNA与A型或B型血浆DNA按1：1、2：1、4：1、8：1、10：1、20：1、40：1、100：1进行混合，模拟孕妇外周血胎儿与孕妇自身ABO基因混合状态，建立混合ABO血型基因分型技术。选取14例孕30周以上的孕妇外周血标本，进行胎儿ABO血型基因型鉴定，并对孕妇进行追踪，尽量获取胎儿出生以后的外周血标本进行ABO血型鉴定，以评价孕妇外周血胎儿ABO血型基因分型技术的灵敏度与准确性。结果表明：单人血浆进行准确血型鉴定的最少模板DNA量约为0．625ng，500μl血浆提取的DNA量即可达到PCR扩增要求；当混合血浆中O型DNA所占比例≤10时，可以准确检测出非0基因的存在；14名O型孕妇外周血标本中9例标本扩增出非O型基因，5例未扩增出非0基因；通过血清学方法对5例胎儿出生后外周血进行ABO血型鉴定，其中A型3例，B型1例，O型1例，与其基因分型结果一致，符合率100％。结论：本研究建立的孕妇外周血胎儿ABO血型基因提取、分型技术，可以对妊娠中晚期胎儿ABO血型基因型进行准确鉴定，从而为新生儿溶血病的产前诊断与预防提供指导意见。

Establishment of Genotyping Method for Fetal ABO Group from Pregnant Maternal Peripheral Blood

This study was aimed to establish a genotyping method to detect ABO group gene of fetus from peripheral blood of pregnant women for prenatal diagnosis of hemolytic disease of newborn （HDN） resulting from ABO blood group incompatibility. 4 pairs of primers were designed according to ABO blood group gene DNA and mRNA sequences. 20 plasma DNA samples from healthy donors were extracted and amplified to explore the best conditions for plasma DNA extraction and PCR amplification. The O group plasma DNA was mixed with A group or B group plasmas by the ratios of 1： 1,2： 1,4： 1,8： 1,10： 1,20： 1,40： 1,100：1 to simulate the status of mixed ABO gene from pregnant maternal blood and to establish the mixed blood group ABO genotyping technology. The pregnant maternal blood samples with more than 30 weeks of gestation were selected for detecting the fetal ABO blood group genotype. The blood samples should be taken as possible as after birth for identification of ABO blood group and evaluation of sensitivity and accuracy of fetal ABO blood group genotyping technology through peripheral blood of pregnant women. The results indicated that the minimal amount of template DNA from single blood plasma for accuracy identification was at least about 0. 625 ng, the DNA amount extracted from 500μl of plasma could meet the requirement for PCR amplification. When the proportion of O group plasma DNA in mixed plasma DNA was ≤ 10, the non-O group gene could be accurately detected. Among 14 peripheral blood samples from O-group pregnant women, the non-O group gene was amplified in 9 samples; the non-O group gene was not amplified in 5 samples. The identification of peripheral blood ABO group for 5 newborns using serologic method showed that the A group 3 cases, B group 2 cases, 0 group 1 case,which consisted with genotyping results with consistant rate 100%. It is concluded that in middle and late pregnancy the fetal ABO group gene can be detected accurately by means of established fetal ABO group gene extraction and typing technology, that provides some guidances for the prenatal diagnosis and prevention of HDN.