A rapid and sensitive assay to identify HLA-DQ2/8 risk alleles for celiac disease using real-time PCR method.

Abstract

To perform a simple, rapid and sensitive Real-time PCR based SYBR Green method to determine the human leukocyte antigen (HLA)-DQ 2/8 alleles in celiac disease (CD) patients. Many molecular techniques are available to determine the HLA-DQ2 and DQ8 alleles, but they are too expensive and have many steps that make them difficult to use. To determine the HLA-DQ 2/8 alleles we have developed a new real-time PCR assay, using SYBR Green technique with melting curve analysis on genomic DNA isolated from 75 CD patients and 94 healthy controls. The specific primers to examine HLA-DQA1*05, HLA-DQB1*02 and HLA-DQB1*0302 alleles were used and results were compared with commercially available kits. Using this method, the presence of HLA-DQ2 and HLA-DQ8 alleles were determined with sensitivity and specificity 80% and 100% respectively and compared to low resolution commercially available kits, the results of this method were more efficient. The frequency of DQ2 and DQ8 in patients was 76% and 29%, respectively and overall 96% of patients were carries DQ2 and/or DQ8 alleles. The result of this study showed that Real-time PCR using SYBR Green method with melting curve analysis has good efficiency to identify the HLA-DQ2/8 risk alleles.