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

Kazem Mashayekhi; Mohammad Rostami-Nejad; Pedram Azimzadeh; Davar Amani; Shabnam Kazemian; Shaghayegh Derakhshani; Hadi Hasanzadeh; Mohammad Reza Zali

Koomesh

Journal of Semnan University of Medical Sciences

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

Author(s):

Kazem Mashayekhi,

Mohammad Rostami-Nejad,

Pedram Azimzadeh,

Davar Amani^,*,

Shabnam Kazemian,

Shaghayegh Derakhshani,

Hadi Hasanzadeh,

Mohammad Reza Zali

Koomesh:Vol. 16, issue 4; 527-535

Published online:Sep 28, 2015

Article type:Research Article

Received:Jan 01, 2015

Accepted:Mar 01, 2015

How to Cite:Kazem MashayekhiMohammad Rostami-NejadPedram AzimzadehDavar AmaniShabnam KazemianShaghayegh DerakhshaniHadi HasanzadehMohammad Reza Zaliet al.Setup of SYBR green real-time PCR method to detect the HLA-DQ alleles in patients with celiac disease.koomesh.16(4):e151237.

Abstract

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.

Keywords

Celiacdisease

Real-Time PCR Technique

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