بهینه‌سازی تکثیر ژن apa در مایکوباکتریوم آویوم تحت گونه پاراتوبرکلوزیس با استفاده از دی متیل سولفوکسید

نوع مقاله : مقاله پژوهشی

نویسندگان
مصطفی روستا 1
زهرا همتی 2
عبدالله درخشنده 3
امین نعمت‌الهی 4
1 دانشجوی دکترای تخصصی باکتری شناسی، گروه پاتوبیولوژی، دانشکده دامپزشکی، دانشگاه شهرکرد، شهرکرد- ایران
2 گروه پاتوبیولوژی، دانشکده دامپزشکی، دانشگاه شهرکرد، شهرکرد- ایران
3 گروه پاتوبیولوژی، دانشکده دامپزشکی، دانشگاه شیراز، شیراز- ایران
4 گروه بهداشت و کنترل کیفیت مواد غذایی، دانشکده دامپزشکی، دانشگاه شهرکرد، شهرکرد- ایران
10.22034/IJVCS.2024.14931.1108
چکیده
مایکوباکتریوم آویوم تحت گونه پاراتوبرکلوزیس  (MAP)یک باکتری بسیار کندرشدی است که به‌عنوان عامل بیماری یون مطرح می‌باشد. این بیماری خسارت‌های اقتصادی فراوانی به صنعت دامپروری به‌خصوص گاوداری‌های شیری در سراسر دنیا وارد می‌کند. باتوجه‌به علائم بالینی و آسیب‌شناسی مشابه در بیماری یون و کرون، احتمال می‌رود این باکتری در بروز و پیشرفت بیماری کرون نقش داشته باشد، ازاین‌رو امکان انتقال  MAP از طریق شیر موجب ایجاد نگرانی‌های زیادی شده است؛ لذا تشخیص صحیح این باکتری با حساسیت و ویژگی بالا نظیر روش‌های بر پایه مولکولی از اهمیت ویژه‌ای برخوردار است. بررسی توالی ژن apa در پایگاه داده‌‌ای NCBI  نشان می‌دهد که دارای درصد بالایی از بازهای سیتوزین و گوانین یا GC می‌باشد. این امر منجر به اختلال در تکثیر این ژن در طی سیکلPCR  می‌گردد. ﺗﻜﺜﻴﺮ ﻗﻄﻌﺎﺕ ﻏﻨﻲ ﺍﺯ GC به ﻋﻠﺖ شکل‌گیری ﺳﺎﺧﺘﺎﺭﻫﺎﻱ ﺛﺎﻧﻮﻳﻪ ﻣﺎﻧﻨﺪ ﺳﺎﺧﺘﺎﺭﻫﺎﻱ ﺳﻨﺠﺎﻕﺳﺮﻱ ﻳﺎ ﺣﻠﻘﻮﻱ ﺩﺭ ﺭﺷﺘﻪﻱ ﺍﻟﮕﻮ، ﻫﻤﻮﺍﺭﻩ ﺑﺎ ﻣﺸﻜﻼﺕ ﺯﻳﺎﺩﻱ ﻫﻤﺮﺍﻩ ﺑﻮﺩﻩ ﺍﺳﺖ. مطالعة ﺣﺎﺿﺮ باهدف ﺑﺮﺭﺳﻲ ﺍﻣﻜﺎﻥ ﺗﻜﺜﻴﺮ ﺑﺨﺸﻲ ﺍﺯ ژن apa ﺑﺎ ﺑﻬﻴﻨﻪ‌ﺳﺎﺯﻱ غلظت دی متیل سولفوکسید (DMSO) ﺑﻪ ﺍﺟﺮﺍ ﺩﺭﺁﻣﺪ. ﺑﺮﺭﺳﻲ ﺑﻴﻮﺍﻧﻔﻮﺭﻣﺎﺗﻴﻚ ﺍﺯ ﻧﻈﺮ ﻭﺿﻌﻴﺖ GC ﻭ ﺳﺎﺧﺘﺎﺭﻫﺎﻱ ﺛﺎﻧﻮﻳﻪﻱ ﺍﺣﺘﻤﺎﻟﻲ، توسط CpG Plot و Mfold انجام ﮔﺮدید. در نهایت طبق طراحی PCR غلظت 3 میکرولیتر DMSO بدون حضور باندهای غیر اختصاصی و با دمای اتصال 55 درجه سانتی‌گراد ژن apa تکثیر گردید. نتایج این مطالعه نشان داد، تکثیر ژن apa در شرایط عدم حضور DMSO امکان‌پذیر نبود و اضافه‌کردن این ماده سبب بهبود تکثیر این ژن گردید.
کلیدواژه‌ها
مایکوباکتریوم آویوم تحت گونه پاراتوبرکلوزیس
بیماری یون
apa
PCR
دی متیل سولفوکسید

عنوان مقاله English

Optimization of apa gene replication in Mycobacterium avium subsp paratuberculosis the usage of dimethyl sulfoxide (DMSO)

نویسندگان English

Mustafa Roosta 1
Zahra Hemati 2
Abdollah Derakhshandeh 3
Amin Nematollahi 4
1 PhD Candidate of Bacteriology, Department of Pathobiology, Faculty of Veterinary Medicine, Shahrekord University, Shahrekord- Iran
2 Department of Pathobiology, Faculty of Veterinary Medicine, Shahrekord University, Shahrekord- Iran
3 Department of Pathobiology, Faculty of Veterinary Medicine, Shiraz University, Shiraz- Iran
4 Department of Food Hygiene and Quality Control, Faculty of Veterinary Medicine, Shahrekord University, Shahrekord- Iran
چکیده English

Mycobacterium avium subsp. paratuberculosis is a remarkably slow-growing bacterium identified as the causative agent of Johne’s disease. This disease causes economic losses especially in dairy cattle breeding, all over the world. Considering the similar clinical and pathological symptoms of Crohn's disease and Johne's disease, it is probable that this bacterium plays a role in the occurrence and progression of Crohn's disease. Hence the potential transmission of MAP through milk has raised significant concerns. Examination of the apa gene sequence in the NCBI database indicates a substantial prevalence of cytosine and guanine nucleotides, signifying elevated GC content. This leads to the disruption of gene amplification throughout the PCR cycle. The abundance of GC-rich regions has caused various problems due to the formation of secondary structures, such as needle-like or ring-shaped topologies in DNA pattern. The aim of this study was the feasibility of enhancing a portion of the apa gene through the modification of dimethyl sulfoxide (DMSO) concentration. A bioinformatics analysis utilizing CpG Plot and Mfold was performed to evaluate the GC content and probable secondary structures of the targeted region of the apa gene. According to the PCR design, the apa gene was amplified using 3μl of DMSO at an annealing temperature of 55 °C, without any unspecific bands. The results of this study demonstrated that apa gene amplification was unachievable without DMSO, and the application of this substance facilitated the gene's amplification.

کلیدواژه‌ها English

Mycobacterium avium subsp. paratuberculosis
Johne&‌rsquo؛ s disease
apa
PCR
DMSO
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علوم درمانگاهی دامپزشکی ایران
دوره 18، شماره 2 - شماره پیاپی 31
پاییز و زمستان 1403
دی 1403
صفحه 49-58