Rapid and Reliable One-Step ABO Genotyping Using Direct Real-Time Allele-Specific PCR and Melting Curve Analysis...

ABO genotyping is a molecular diagnostic technique important for transfusion and transplantation in medicine, and human identification in forensic science. Because ABO genotyping are labor intensive and time consuming, the genotyping cannot be firstly used to resolve the serological ABO discrepancy in blood bank. For rapid one-step ABO genotyping, we developed direct, real-time, allele-specific polymerase chain reaction (PCR), and melting curve analysis (DRAM assay) without DNA preparation. In DRAM assay, we used a special PCR buffer for direct PCR, a rapid RBC lysis buffer, white blood cells as template without DNA preparation, allele-specific primers for discriminating three ABO alleles (261G/del, 796C/A, and 803G/C), and melting curve analysis as a detection method. There was 100% concordance among the results of ABO genotyping by the DRAM assay, serologic typing, PCR-RFLP and PCR-direct sequencing of 96 venous blood samples. We were able to reduce the number of manual steps to three and the hands-on time to 12 min, compared to seven steps and approximately 40 min for conventional ABO genotyping using allele-specific PCR with purified DNA and agarose gel electrophoresis. We have established and validated the DRAM assay for rapid and reliable one-step ABO genotyping in a closed system. The DRAM assay with an appropriate number of allele-specific primers could help in resolving ABO discrepancies and should be valuable in clinical laboratory and blood bank.