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目的建立巢式PCR技术检测RhD阴性孕妇血浆中游离胎儿DNA(cffDNA)的RHD基因型,以预测胎儿RhD血型。方法采用QIAamp DNA Blood Mini Kit提取32例RhD阴性孕妇血浆游离DNA,针对RHD外显子7和10分别设计外侧、内侧2组特异性引物,巢式PCR方法检测孕妇血浆游离胎儿DNA的RHD型,测序验证PCR产物的序列特异性。结果孕妇血浆游离DNA经巢式PCR扩增后,有27例成功扩增出RHD外显子7、10特异性条带,5例未检测到RHD基因特异性扩增,32例中有30例胎儿RHD型与出生后血型相符,检测准确率为93.1%。结论采用巢式PCR技术检测RhD阴性孕妇血浆游离胎儿DNA来判定胎儿RHD型,具有良好的准确性、敏感性和特异性,为RhD新生儿溶血病的早期诊断提供了一种新的、可靠的检测手段。
Objective To establish a nested PCR technique to detect RHD genotypes of free fetal DNA (cffDNA) in plasma of RhD-negative pregnant women to predict fetal RhD blood group. Methods Plasma DNA was extracted from 32 cases of RhD-negative pregnant women using QIAamp DNA Blood Mini Kit. Two sets of specific primers were designed for the exon 7 and 10 of RHD respectively. The nested PCR was used to detect the RHD phenotype of plasma free fetal DNA. Sequencing verified the sequence specificity of the PCR product. Results After plasma DNA was amplified by nested PCR in pregnant women, 27 cases of RHD exon 7 and 10 were amplified successfully, 5 cases did not detect RHD gene specific amplification, 30 cases of 32 cases Fetal RHD type and blood type after birth, the detection accuracy was 93.1%. Conclusion The detection of fetal free plasma DNA in RhD-negative pregnant women by nested-PCR technique has good accuracy, sensitivity and specificity for the detection of fetal RHD type. It provides a new and reliable method for the early diagnosis of RhD neonatal hemolytic disease testing method.