Blood Group Research Laboratory, Dalian Blood Center, Dalian 116001, China.
Blood Group Research Laboratory, Dalian Blood Center, Dalian 116001, China.
Transfus Apher Sci. 2021 Jun;60(3):103078. doi: 10.1016/j.transci.2021.103078. Epub 2021 Feb 5.
DEL is the weakest known D-positive phenotype and is detectable only by adsorption and elution tests. RHD c.1227G>A is an important marker for DEL phenotype in East Asians. The aim of this study was to develop a method for RHD c.1227G>A genotyping by single-tube PCR with melting curve analysis.
Two GC-rich tails of different lengths were attached to the 5'-end of allele-specific primers for RHD 1227G and 1227A alleles, such that RHD c.1227G>A could be distinguished by the melting temperature. A total of 145 D-negative Chinese Han blood donors were genotyped for RHD c.1227G>A by melting curve analysis, conventional polymerase chain reaction with sequence-specific primers (PCR-SSP), and sequencing.
In 143 subjects (143/145, 98.6%), PCR-SSP and melting curve analysis produced consistent results with RHD exon 9 sequencings. Two samples were genotyped as RHD 1227G/A by PCR-SSP, but as RHD 1227A/A or A/- by melting curve analysis. These two samples were confirmed to be RHD 1227A/A or A/-. Based on RHD exon 9 sequencing, the accuracy, sensitivity, specificity, positive predictive value, and negative predictive value of the melting curve analysis for detecting both RHD 1227A and 1227G were all 100%. In contrast, the accuracy, specificity and positive predictive value of PCR-SSP for RHD 1227G detection were 98.62%, 98.21% and 94.29%, respectively, which were lower than those observed with the melting curve analysis.
Melting curve analysis for RHD c.1227G>A genotyping is a simple, rapid, and reliable method, superior to conventional PCR-SSP.
DEL 是已知的最弱的 D 阳性表型，只能通过吸附洗脱试验检测。RHD c.1227G>A 是东亚 DEL 表型的重要标志物。本研究旨在建立一种通过单管 PCR 熔解曲线分析进行 RHD c.1227G>A 基因分型的方法。
在 RHD 1227G 和 1227A 等位基因的等位基因特异性引物的 5'端分别连接两个不同长度的 GC 丰富尾巴，使得 RHD c.1227G>A 可以通过熔解温度来区分。对 145 名中国汉族 D 阴性献血者进行 RHD c.1227G>A 的熔解曲线分析、常规聚合酶链反应与序列特异性引物（PCR-SSP）和测序。
在 143 名受试者（143/145，98.6%）中，PCR-SSP 和熔解曲线分析与 RHD 外显子 9 测序结果一致。有 2 个样本通过 PCR-SSP 鉴定为 RHD 1227G/A，但通过熔解曲线分析鉴定为 RHD 1227A/A 或 A/-。这两个样本被确认为 RHD 1227A/A 或 A/-。基于 RHD 外显子 9 测序，熔解曲线分析对检测 RHD 1227A 和 1227G 的准确性、灵敏度、特异性、阳性预测值和阴性预测值均为 100%。相比之下，PCR-SSP 对 RHD 1227G 检测的准确性、特异性和阳性预测值分别为 98.62%、98.21%和 94.29%，低于熔解曲线分析。
RHD c.1227G>A 基因分型的熔解曲线分析是一种简单、快速、可靠的方法，优于常规 PCR-SSP。
