Application Of SYBR Green I Real-time PCR In ABO Genotyping

Object To establish a genotyping method for ABO blood group and screening for common alleles. Method We collected samples from 66 health donors whose blood phenotype were identical between forward and reverse testing and common loci of ABO gene (261/467/802/803/1061) were confirmed by classical PCR and sequencing. Results We screened 8 common genotypes: A101/ O01,02 (7 cases), A102/ O01,02 (8 cases), A101/A102 (3 cases), B101/B101 (3 cases), B101/ O01,02 (14 cases), O01,02/ O01,02 (18 cases), A101/B101 (3 cases), A102/B101 (10 cases), which were consistent with corresponding phenotypes. Conclusion The genotyping method we established for ABO grouping was reliable and can be regarded as a standard reference for the method of SYBR Green I PCR assessment.Objective To investigate the value of real-time PCR for blood genotyping. Methods By samples collected from the first part of our experiments, we designed primers with an artificial mismatch base at the last but one of 3′terminus for 261, 467 and 803 in real-time PCR and used melting curve to analyze the genotype. Results The primers of 261nor-260 (Aâ†'T), 261mut-260 (Câ†'T), 467mut-466 (Tâ†'G), 803nor-804 (Tâ†'G) and 803mut-804 (Tâ†'G) were optimal for real-time PCR with no unspecific reaction-derived interferences. Real-time PCR was a rapid and robust method with a highest sensitivity of 12.5ng. Conclusion: Compared with classical PCR which would be influenced by template and conditions, SYBR Green I real-time PCR can be an accurate and potential technique in blood genotyping by melting curve analysis.