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Setup of SYBR green real-time PCR method to detect the HLA-DQ alleles in patients with celiac disease
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Kazem Mashayekhi   , Mohammad Rostami-Nejad , Pedram Azimzadeh , Davar Amani , Shabnam Kazemian , Shaghayegh Derakhshani , Hadi Hasanzadeh , Mohammad Reza Zali |
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Abstract: (7429 Views) |
| 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. |
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| Keywords: Celiacdisease, Real-Time PCR Technique, HLA-Typing |
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Full-Text [PDF 1859 kb]
(2259 Downloads)
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Type of Study: Research |
Subject:
General
Received: 2015/01/18 | Accepted: 2015/03/17 | Published: 2015/06/10
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