تمییز آلل‏ های MHC سگ (DLA-DRB1) با روش تحلیل دقیق دمای شکافت DNA

نوع مقاله: هماتولوژی و بیوشیمی درمانگاهی

نویسندگان

1 گروه بیماریهای داخلی، دانشکده دامپزشکی دانشگاه تهران، تهران، ایران

2 گروه میکروبیولوژی و ایمونولوژی، دانشکده دامپزشکی دانشگاه تهران، تهران، ایران

چکیده

زمینه مطالعه: نقش مهم مولکول‏های مجتمع عمده پذیرش بافتی (MHC) اتصال به پپتید‌های پادگنی و عرضه آن‌ها به لنفوسیت‏های T است. از اینرو تنوع MHC با حساسیت و یا مقاومت در برابر بسیاری از بیماری‏های عفونی و بیماری های خودایمن مرتبط است. یک روش نوین بررسی تنوع ژنتیکی، تحلیل دقیق دمای شکافت (HRM-High Resolution Melting) DNA است که می توان آنرا برای بررسی تنوع MHC به کار برد. هدف: تمییز آلل‏ های MHC سگ (DLA-DRB1) با روش تحلیل دقیق دمای شکافت DNA روش کار: تعداد 40 نمونه خون از سگ های ارجاعی به بیمارستان دام‏های کوچک دانشکده دامپزشکی دانشگاه تهران جمع آوری گردید. پس از استخراج DNA و افزوده سازی اگزون دوم ژن DLA-DRB1، تحلیل HRM و منحنی ذوب صورت گرفت. برای تطبیق الگوهای HRM با توالی نوکلئوتیدی آلل ها، از روش تعیین توالی مستقیم استفاده شد. نتایج: بر اساس تنوع مشاهده شده در نتایج HRM و منحنی ذوب، 40 نمونه به 8 ژنوتیپ (A تا H) قابل تقسیم‏بندی بودند. بیشترین فراوانی مربوط به تیپ A (00/25 %) و سپس تیپ C و E (هر کدام 00/15 %) بود. در مجموع 5/82 % نمونه ها هتروزیگوت و 5/17 % نمونه ها هوموزیگوت بودند. نتیجه گیری: مطالعه حاضر اولین مطالعه‏ای است که برای تایپیگ آلل‏های ژن DLA-DRB1 از تکنیک HRM استفاده شده است. تطبیق نتایج HRM با توالی آلل ها نشان داد که این روش می‏تواند برای تفکیک آلل‏های DLA-DRB1 به کار رود.

کلیدواژه‌ها


عنوان مقاله [English]

Discrimination of dog MHC (DLA-DRB1) alleles by high-resolution melt analysis

نویسندگان [English]

  • Seyed Milad Vahedi 1
  • Shahram Jamshidi 1
  • Leila Lankari Mohaje 1
  • Gholamraza Nikbakhat Brujeni 2
1 1Department of Internal Medicine, Faculty of Veterinary Medicine, University of Tehran, Tehran, Iran
2 2Department of Microbiology and Immunology, Faculty of Veterinary Medicine, University of Tehran, Tehran, Iran
چکیده [English]

BACKGROUND: The function of molecules of major histocompatibility complex (MHC) is to bind antigenic peptides and display them on the cell surface for recognition by T cells. MHC polymorphism is related to suseptibility or resistance to immune-mediated and infectious diseases. High Resolution Melt (HRM) analysis is a novel and powerful technique in molecular biology for the detection of genetic polymorphisms and can be used for discrimination of MHC alleles and genotyping. OBJECTIVES: Discrimination of dog MHC alleles (DLA-DRB1) by high-resolution melt analysis. METHODS: Forty blood samples were collected from dogs referred to Small Animal Teaching Hospital of faculty of veterinary medicine, University of Tehran. After DNA extraction and amplification the exon 2 of DLA-DRB1, HRM and Melt Curve analysis was carried out. Direct sequencing was used for checking the HRM patterns. RESULTS: Following the HRM and Melt curve analysis, forty samples could be classified into 8 genotypes (A to H). Most frequent was A (25.00%) and then C and E (each one 15.00%). In total, 82.5% and 17.5% of samples were heterozygote and homozygote, respectively. CONCLUSIONS: In this study, the HRM technique was used for DLA-DRB1 gene alleles typing for the first time. Confirmation HRM results through sequencing results revealed that HRM can be used for discrimination of DLA-DRB1 alleles.

کلیدواژه‌ها [English]

  • MHC
  • Dog
  • DLA-DRB1
  • HRM
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