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目的:利用孕妇血浆中游离胎儿DNA建立产前非创伤性诊断Kidd血型基因型的实时荧光PCR技术(RQ-PCR)。方法:采用QIAamp试剂盒抽提孕妇血浆DNA,利用短串联重复序列(STR)确认提取物存在胎儿DNA,利用RQ-PCR检测胎儿Kidd基因型。采用血清学方法检测婴儿脐带血Kidd表型,回顾性评价基因分型方法的准确性。结果:在182例孕妇中筛选出96例纯合子Kidd表型,进一步通过鉴定其婴儿脐带血Kidd表型,从中筛选得到46例标本孕妇纯合子表型而婴儿脐带血JK(a+b+)表型。在这46例孕妇血浆DNA标本中能检出父源性等位基因的标本为38例,孕妇表型分别为6例JK(a+b-)和32例JK(a-b+)。38例孕妇血浆DNA标本用RQ-PCR法均检测到了孕妇所不具备的JK*B或JK*A父源性等位基因,其结果与婴儿脐带血Kidd表型相吻合。结论:本实验建立的RQ-PCR非创伤性产前诊断胎儿Kidd血型基因型是可行的,当孕妇为纯合子时可通过产前非创伤性基因分型辅助诊断和预防新生儿溶血病。
OBJECTIVE: To establish a real-time fluorescence PCR (RQ-PCR) method for the nonnatal diagnosis of Kidd blood group genotypes in pregnant women using free fetal DNA. METHODS: Plasma DNA was extracted from pregnant women using QIAamp kit. Fetal DNA was identified by short tandem repeat (STR) and Kidd genotype was detected by RQ-PCR. The serological method was used to detect the cord blood Kidd phenotype, and the accuracy of the genotyping method was retrospectively evaluated. RESULTS: 96 cases of homozygous Kidd phenotype were screened out from 182 pregnant women. The Kidd phenotype of umbilical cord blood of infants was further screened. 46 cases of homozygous phenotype of pregnant women were screened out and the cord blood cord blood JK (a + b +) type. In the 46 cases of pregnant women plasma DNA samples can be detected in 38 cases of paternal alleles, the pregnant women were 6 cases of JK (a + b-) and 32 cases of JK (a-b +). Thirty-eight plasma DNA samples of pregnant women were tested by RQ-PCR method for the JK * B or JK * A parental alleles that were not found in pregnant women. The results were in good agreement with the infant cord blood Kidd phenotype. Conclusion: The RQ-PCR established in our study is feasible for the diagnosis of fetal Kidd blood group genotypes. When pregnant women are homozygous, it can be used to diagnose and prevent neonatal hemolytic disease by prenatal non-invasive genotyping.