一种新的RHD合子型直接测定方法

A new PCR method for direct determination of RHD zygosity

目的 建立简易的RHD合子型直接测定方法。方法 根据RHD基因序列和RHD基因缺失的原理设计两对引物 ,分别特异性针对RHD基因第一外显子和融合Rh盒子 ,再引入一对内对照引物 ,建立新的双管复式PCR方法 ;并用于检测 15 2份已知RHD基因序列和基因型的DNA样本、以及 35 9份已知Rh表型样本的RHD合子型。检测结果与使用近期报道的限制性片段长度多态性(RFLP)技术的鉴定结果相比较。结果 15 2份已知RHD基因序列和基因型的DNA样本包括 92份Rh阴性、2 8份D放散型、3例部分D表型、5例弱D型及 2 4例Rh阳性样品 ,全部样品的RHD合子型PCR检测结果均与已知的基因型相符合 ,可直接判断RHD(+) /RHD(+)、RHD(+) /RHD( )和RHD( ) /RHD( )三种合子型 ;35 9份已知Rh表型的样本 ,包括 12例孕妇配偶、18名家系成员、4 8例Rh阴性及2 81例Rh阳性捐血者 ,PCR检测结果 ,除一例外 ,均与RFLP结果一致。该个例PCR检测为RHD(+) /RHD( )杂合型 ,而RFLP结果为RHD( ) /RH1D( )纯合型 ,由于个例为Rh阳性表型 ,肯定存在至少一条RHD基因 ,所以显然RFLP检测下游Rh盒子出现假阴性。结论 双管PCR技术鉴定RHD合子型 ,简便、快速 ,具有较高的准确率 ,可常规用于临床或血库实验室。

Objective To establish a direct method for the determination of RHD zygosity. Methods Two pairs of primers were designed specific for hybrid Rhesus box and exon 1 of RHD,respectively. Combined with a pair of internal control primers,a new dual-tube PCR method was established. One hundred and fifty-two DNA samples,of which the sequence of RHD and RH genotypes were known,and samples from 359 donors or patients,of which the Rh phenotypes were determined,were evaluated by taking a recent restriction fragment length polymorphism (RFLP) method as reference. Results The RHD zygosities of 152 samples,including 92 Rh-negative,28 D el ,3 partial D,5 weak D and 24 Rh-positive,were determined in concordance with the known genotypes. It showed that the PCR method could detect RHD(+)/RHD(+),RHD(+)/RHD(-) and RHD(-)/RHD(-) zygosities in one test. The PCR results of 359 known phenotype samples including 12 husbands,18 family members,48 Rh-negative and 281 Rh-positive,were identical with RFLP except for one Rh-positive sample,which was tested as RHD(+)/RHD(-) heterozygote by PCR,whereas RHD(-)/RHD(-) homozygote by RFLP. Apparently,RFLP revealed a false negative result detecting downstream Rhesus box. Conclusions The dual-tube PCR is a less-labored and more rapid method for the determination of RHD zygosity comparing RFLP. It can be used routinely in the laboratories in clinics and blood banks.