RhD 纯合性的测定：双重扩增阻滞突变系统方法与多重实时定量 PCR 方法的比较
Determination of RhD zygosity: comparison of a double amplification refractory mutation system approach and a multiplex real-time quantitative PCR approach.
作者信息
Chiu R W, Murphy M F, Fidler C, Zee B C, Wainscoat J S, Lo Y M
机构信息
Departments of Chemical Pathology and Clinical Oncology, The Chinese University of Hong Kong, Prince of Wales Hospital, Shatin, New Territories, Hong Kong SAR.
出版信息
Clin Chem. 2001 Apr;47(4):667-72.
BACKGROUND
Rh isoimmunization and hemolytic disease of the newborn still occur despite the availability of Rh immunoglobulin. For the prenatal investigation of sensitized RhD-negative pregnant women, determination of the zygosity of the RhD-positive father has important implications. The currently available molecular methods for RhD zygosity assessment, in general, are technically demanding and labor-intensive. Therefore, at present, rhesus genotype assessment is most commonly inferred from results of serological tests. The recent elucidation of the genetic structure of the prevalent RHD deletion in Caucasians, as well as the development of real-time PCR, allowed us to explore two new approaches for the molecular determination of RhD zygosity.
METHODS
Two methods for RhD zygosity determination were developed. The first was based on the double Amplification Refractory Mutation System (double ARMS). The second was based on multiplex real-time quantitative PCR. For the double ARMS assay, allele-specific primers were designed to directly amplify the most prevalent RHD deletion found in RhD-negative individuals in the Caucasian population. The multiplex real-time quantitative PCR assay, on the other hand, involved coamplification and quantification of RHD-specific sequences in relation to a reference gene, albumin, in a single PCR reaction. A ratio, DeltaCt, based on the threshold cycle, was then determined and reflects the RHD gene dosage.
RESULTS
The allele-specific primers of the double ARMS assay reliably amplified the RHD-deleted allele and therefore accurately distinguished homozygous from heterozygous RhD-positive samples. The results were in complete concordance with serological testing. For the multiplex real-time quantitative PCR assay, the DeltaCt values clearly segregated into two distinct populations according to the RHD gene dosage, with mean values of 1.70 (SD, 0.17) and 2.62 (SD, 0.29) for the homozygous and heterozygous samples, respectively (P: <0.001, t-test). The results were in complete concordance with the results of serological testing as well as with the double ARMS assay.
CONCLUSION
Double ARMS and real-time quantitative PCR are alternative robust assays for the determination of RhD zygosity.
背景
尽管有Rh免疫球蛋白，但新生儿Rh血型同种免疫和溶血病仍有发生。对于致敏的RhD阴性孕妇的产前检查，确定RhD阳性父亲的合子性具有重要意义。目前可用的用于RhD合子性评估的分子方法通常技术要求高且劳动强度大。因此，目前，恒河猴基因型评估最常从血清学检测结果中推断得出。最近对白种人中普遍存在的RHD缺失的基因结构的阐明，以及实时PCR的发展，使我们能够探索两种新的分子方法来确定RhD合子性。
方法
开发了两种确定RhD合子性的方法。第一种基于双重扩增不应突变系统（双重ARMS）。第二种基于多重实时定量PCR。对于双重ARMS检测，设计等位基因特异性引物直接扩增白种人群中RhD阴性个体中最普遍的RHD缺失。另一方面，多重实时定量PCR检测涉及在单个PCR反应中与参考基因白蛋白一起共扩增和定量RHD特异性序列。然后根据阈值循环确定一个比率DeltaCt，它反映了RHD基因剂量。
结果
双重ARMS检测的等位基因特异性引物可靠地扩增了RHD缺失等位基因，因此准确地区分了纯合和杂合RhD阳性样本。结果与血清学检测完全一致。对于多重实时定量PCR检测，DeltaCt值根据RHD基因剂量明显分为两个不同的群体，纯合和杂合样本的平均值分别为1.70（标准差，0.17）和2.62（标准差，0.29）（P：<0.001，t检验）。结果与血清学检测结果以及双重ARMS检测结果完全一致。
结论
双重ARMS和实时定量PCR是用于确定RhD合子性的可靠替代检测方法。
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