Setup of SYBR green real-time PCR method to detect the HLA-DQ alleles in patients with celiac disease

 Introduction: HLA-DQ2 and HLA-DQ8 alleles are important genetic factors in development of celiac disease. There are many molecular techniques available to determine these alleles, but they are very complicated to perform and also cost highly. Therefore, the aim of this study was to set up a simple and quick real-time PCR based SYBR ® Green method to determine HLA-DQ alleles in patients with celiac disease. Materials and Methods: To determine HLA-DQ alleles and evaluate Real-time PCR using SYBR ® Green method for our aim, first, DNA extraction was performed for patients with celiac disease, whose diagnosis was confirmed by using serology and pathology tests. Later, the specific primers for detecting HLA-DQA1*05, HLA-DQB1*02 and HLA-DQB1*0302 alleles were used. Finally the results were compared with commercially available kits. Results: Presence of HLA-DQ2 and HLA-DQ8 alleles were determined by using this method with 100% sensitivity and specificity. Also, in comparison with low resolution commercially available kits, the results of this method were more efficient. Conclusion: Real-time PCR using SYBR® Green method with melting curve analysis is a well efficient method to identify the HLA-DQ2 and HLA-DQ8 alleles. Also, in compararison with conventional HLA-typing techniques in Iran, this method was faster and easier to performe and displayed higher sensitivity and specificity in distinguishing the alleles.