Genotyping of RHD c.1227G>A allele by melting curve analysis

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.