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, A(el) and B-el 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 A(el) 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 A(el) and B-el individuals. Through genetic analysis we have previously identified A(el) as possessing an A allele with IVS6+5G -> A mutation (Transfusion 2003;43:1138-1144) and B-el 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 A(el) and B-el. For genotyping of A(el) and B-el, 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 T-m that was used for genotype detection of A(el) and B-el blood types. For A(el) genotyping, the melting curve of the normal control appears as one peak at 59.19 +/- 0.07 degrees C (mean +/- SE) and that of A(el) appears as 2 peaks at 59.21 +/- 0.07 degrees C and 64.39 +/- 0.07 degrees C, corresponding to the O and A(el) alleles, respectively. For B-el genotyping, the melting curve of the normal control appears as one peak at 67.99 +/- 0.11 degrees C and that of B-el appears as 2 peaks at 59.99 +/- 0.12 degrees C and 68.1 +/- 0.13 degrees C, corresponding to the B-el 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 A(el) and B-el blood types by combined LC-PCR and melting-curve analysis is a rapid, reliable, and easy method.