Application of real-time PCR and melting curve analysis in rapid detection of Ael and Bel blood types.

The ABO blood group is the most important blood group system in transfusion medicine. In addition to the normal levels of ABO antigen expression, Ael and Bel represent the two major blood types that have a weak expression of the A or B antigens on red blood cells. Due to the fact that typing of Ael and Bel by conventional serological methods is time consuming and sometimes gives false-positive and false-negative results, it is warranted to develop an additional technique for the identification of Ael and Bel individuals. Through genetic analysis we have previously identified Ael as possessing an A allele with IVS6+5G-->A mutation (Transfusion 2003;43:1138-1144) and Bel as possessing a B gene with 502C-->T mutation in Taiwan (Vox Sanguinis 2003;85:216-220). Hence, real-time PCR-based genotyping methods were developed in this study to facilitate the detection of Ael and Bel. For genotyping of Ael and Bel, the region of mutations was PCR amplified and subjected to the LightCycler (LC) real-time PCR assay using LC Red640-labeled hybridization probe. Melting curve analysis was performed to determine the melting temperature Tm that was used for genotype detection of Ael and Bel blood types. For Ael genotyping, the melting curve of the normal control appears as one peak at 59.19+/-0.07 degrees C (mean+/-SE) and that of Ael appears as 2 peaks at 59.21+/-0.07 degrees C and 64.39+/-0.07 degrees C, corresponding to the O and Ael alleles, respectively. For Bel genotyping, the melting curve of the normal control appears as one peak at 67.99+/-0.11 degrees C and that of Bel appears as 2 peaks at 59.99+/-0.12 degrees C and 68.1+/-0.13 degrees C, corresponding to the Bel and O alleles, respectively. This genotyping method was shown to be accurate, based on automated sequencing of the PCR-amplified products. It takes only 90 min to perform this genotyping test. Detecting the Ael and Bel blood types by combined LC-PCR and melting-curve analysis is a rapid, reliable, and easy method.