Severe cutaneous adverse drug reactions (SCARs) are rare but deadly drug reactions with severe damages to patients. One of the most well-known SCARs risk factors is the human leukocyte antigen (HLA) genes polymorphism. Among the HLA polymorphic alleles, the HLA-A*33:03 allele has been found in association with SCARs induced by various drugs, especially in Asian people. There has not been any report on the specific detection protocol of the HLA-A*33:03 allele. Objective: This study aimed to

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... n a nested AS-PCR protocol for detecting and distinguishing diplotype genotype of the HLA-A*33:03 allele. Methods: A nested allele-specific (AS)-PCR protocol with four primer sets was designed. The method was compared with the Sanger sequencing method on 100 samples of unknown genotypes of unrelated Vietnamese people. Results: The nested AS-PCR method could identify the HLA-A*33:03 allele and the HLA-A*33:03 diplotype genotypes. Comparison with the Sanger sequencing method showed an absolute agreement (ĸ = 1.00, p < 0.001). The nested AS-PCR protocol had a sensitivity of 100% (95%CI: 92.13-100%) and a specificity of 100% (95%CI: 93.51-100%). The protocol was used for the determination of HLA-A*33:03 allele distribution in 810 unrelated Vietnamese Kinh people, showing a frequency of HLA-A*33:03 carriers of 19.6% and an allele frequency of 10.55%. Conclusions: A novel nested AS-PCR method with a hundred-percent sensitivity and a specificity for the HLA-A*33:03 allele detection was reported. The protocol can be applied for the stratification of patients at SCAR risks with various drugs.