شناسایی ویروس هپاتیت B در بیماران همودیالیزی با استفاده از روش تکثیر هم‌دمایی به واسطه حلقه (LAMP)
محورهای موضوعی : میکروب شناسی مولکولی
محمد حسن شاه حسینی
1
,
زهره اسماعیلی
2
,
الهام مسلمی
3
,
پریچهر یغمایی
4
1 - دانشگاه آزاد اسلامی ، واحد شهر قدس ، گروه میکروبیولوژی
2 - دانشگاه آزاد اسلامی ، واحد علوم تحقیقات تهران، گروه زیست شناسی
3 - دانشگاه آزاد اسلامی ، واحد تهران شرق، گروه زیست شناسی
4 - دانشگاه آزاد اسلامی ، واحد علوم تحقیقات تهران، گروه زیست شناسی
کلید واژه: PCR, هپاتیت B, همودیالیز, LAMP,
چکیده مقاله :
سابقه و هدف: شناسایی بیماری هپاتیت B یکی از مشکلات اساسی مراکز همودیالیز محسوب می‌شود. هدف از این پژوهش راه‌اندازی و بهینه‌سازی روش مولکولی تکثیر هم‌دمایی به‌واسطه حلقه (LAMP) برای ارزیابی شیوع ویروس هپاتیت B در نمونه‌های سرمی افراد همودیالیزی است. مواد و روش‌ها: این پژوهش به‌صورت مقطعی-توصیفی بر روی سرم 136 بیمار مراجعه کننده به 3 مرکز همودیالیز بیمارستان مصطفی خمینی (53 نمونه)، بیمارستان چمران (54 نمونه) و بیمارستان عرفان (31 نمونه) انجام شد. پس از استخراج DNA با استفاده از 6 پرایمر اختصاصی ژن HBsAg شرایط واکنش و غلظت مواد برای تشخیص HBV بهینه سازی گردید. همچنین محصولات واکنش با استفاده از روش ژل الکتروفورز با استفاده از اتیدیوم بروماید و سایبرگرین ارزیابی گردید. یافته ها: در این مطالعه حساسیت روش بهینه‌سازی شده LAMP، 4 ذره ویروسی بود. همچنین این روش ویژگی بسیار بالایی داشت. از افراد مراجعه کننده به بیمارستان مصطفی خمینی 13 مورد به‌وسیله تکنیک LAMP و 7 مورد به‌وسیله PCR و از افراد مراجعه کننده به بیمارستان چمران 4 مورد به‌وسیله تکنیک LAMP و 2 مورد به‌وسیله PCR مثبت بودند. اما میزان شناسایی ویروس در افراد مراجعه کننده به بیمارستان عرفان با استفاده از هر دو تکنیک یکسان (2 مورد) بود. نتیجه گیری: برای شناسایی ویروس هپاتیت B، روش LAMP در مقایسه با روش PCR معمولی بسیار سریع‌تر، مقرون به صرفه‌تر و دقیق‌تر است.
Background and Objectives: Infections with Hepatitis B virus in hemodialysis centers still remain as a major problem. The propose of this study is optimization and development of LAMP technique for assessment of HBV infection in serum of hemodialysis patients. Material and Methods: This cross sectional study was performed on totally, 136 serum samples were obtained from the patients how referred to hemodyalis center in Tehran as: 52 serum samples from Mastafa khomaini hospital, 54 serum samples from Chamran hospital and 31 serum samples from Erfan hospital. After DNA extraction, 6 HBsAg specific primers were used for optimizing of reaction conditions and material concentration for LAMP. LAMP products were evaluated by adding 1% SYBER Green and electrophoresis stained by Ethidium-Bromide. Results: LAMP sensitivity was determined as 4 particles in this study. In addition, LAMP technique Showed a high specify for detecting the viruses. Totally, 13and 7 cases of the serum samples of Mastafa khomaini were positive by LAMP and PCR respectfully. Also, 4 and 2 samples for second group and 2 and 2 samples for third group were positive respectfully by LAMP and PCR. Conclusion: The LAMP technique is a more sensitive and cost friendly system to detect HBV in comparison to conventional PCR.
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