Direct determination of ABO blood group genotypes from whole blood using PCR-amplification of specific alleles method

ABO antigens are known to be oligosaccharide antigens, and are widely expressed on the membranes of red blood cells and tissue cells. Therefore, the determining ABO blood group antigens is important in both transfusion and organ transplantation, and is one of the markers used for personal identification in forensics. The ABO blood group is currently determined by the presence of A and B antigens on red blood cells using serological tests in clinical laboratories. The gene sequences of the ABO blood group in chromosome 9q34.1-q34.2 have also been determined. Accordingly, it has become possible to genetically analyze the ABO blood group using molecular biological techniques. We recently developed an ABO genotyping method based on PCR amplification of specific alleles (PASA) using DNA extracted from blood and saliva. However, the extraction and purification of DNA is necessary prior to PCR because blood and saliva contain various substances that inhibit PCR. Furthermore, the PCR amplification of specific alleles (PASA) method requires specificity and stability for allele-specific amplification. Therefore, it is very difficult to use whole blood directly. Here, we described the development and use of ABO genotyping from whole blood using a commercially available reagent kit, which can effectively neutralize inhibitory substances present in the blood. In this study, for all genes of the six major ABO genotypes and cisA2B3 genotype of the AB variant, only specific bands were clearly amplified, whereas non-specific bands were not amplified at all. In addition, this method was able to determine ABO genotyping using 5-fold diluted fresh whole blood, or 5-fold diluted whole blood that was freeze-stored in 100 μL aliquots at -20 C by subdivided for a maximum of 30 days. This analysis method to determine ABO blood group genotyping is simple and useful, and is expected to be used widely throughout research and clinical laboratories and forensic fields.