双管聚合酶链反应技术在检测RHD基因中的应用

目的 探讨采用简易的分子生物学方法检测RHD基因纯合子和杂合子的有效性。方法 按美国基因序列数据库中的RHD盒子序列设计两对引物,分别检测RHD基因下游盒子和融合盒子,并引入一对内对照引物,建立双管聚合酶链反应(PCR)技术用于检测RHD基因合子型。同时以经典的限制性片段长度多态性(RFLP)方法作对照。共检测30份Rh血型D抗原(RhD)阳性、阴性及弱D表型个体的全血DNA样品。结果 30份血DNA样品中,29份样品双管PCR法和RFLP法检测结果完全一致,并分别与样品的血清学血型相吻合;对其中的1例弱D表型血DNA样品,PCR和RFLP法均鉴定为RHD阴性纯合型(RHD^-／RHD^-),显示该个体拥有一条RHD^-单倍体,但RHD下游盒子检测出现假阴性。结论 双管PCR法是简易而有效的基因检测方法,可用于RHD合子型鉴定,在临床上可以预测胎儿的RhD血型。

Applications of RHD zygosity test through polymerase chain reaction for prediction of fetus Rh D-positive phenotype

OBJECTIVE: To explore a molecular method for the determination of RHD zygosity. METHODS: Two pairs of primers were designed specific for downstream rhesus box and hybrid rhesus box according to the sequences in GenBank. Together with a pair of internal control primer, a dual-tube polymerase chain reaction (PCR) method was established for determination of the RHD zygosity. Thirty RhD-positive, D-negative and weak D phenotype samples were evaluated by taking a recent restriction fragment length polymorphism (RFLP) method as reference. RESULTS: The results of the dual-tube PCR and RFLP are identical in 29 samples, which are also in concordance with the Rh phenotype, respectively. In one weak D sample, however, both methods tested as RHD(-)/RHD(-), which showed that there was one RHD(-) haplotype in this individual, but a false negative result in testing downstream rhesus box. CONCLUSIONS: The dual-tube PCR is a less-labored method for RHD zygosity detection. It can be used clinically in prenatal test to determine RHD zygosity of pregnant women's partner for prediction of fetus RhD phenotype.