یک روش مقرون به صرفه و سریع برای استخراج RNA کل با استفاده از پروتئیناز K و تیمارحامل RNA و به دنبال آن شوک حرارتی
کد: G-1047
نویسندگان: Hannan Khodaei © ℗, Leila Azimi, Abbas Akhavan Sepahy, Fatemeh Ashrafi, Maryam Rajabnejad
زمان بندی: زمان بندی نشده!
دانلود: دانلود پوستر
خلاصه مقاله:
خلاصه مقاله
Introduction and Objectives: The global outbreak of SARS-CoV-2 has underscored the importance of clinical detection methods. This study proposes an alternative RNA extraction technique that is both time-efficient and economical during the COVID-19 pandemic, particularly in contrast to conventional commercial RNA extraction kits. Materials and Methods: We assessed the efficacy of two distinct RNA extraction protocols for detecting SARS-CoV-2 via Real-Time PCR (RT-PCR) from nasopharyngeal swabs. The protocols compared were: I) the standard method utilizing a commercial total RNA extraction kit and II) a suggested heat shock protocol. Key variables, including extraction time, centrifugation speed and duration, Proteinase K treatment, and RNA carrier addition, were optimized through a three-step process. Results: Total RNA was extracted using both the commercial kit and the heat shock protocol. The proposed method involved treating samples with 5 μg/μL of Proteinase K at 56°C for 5 minutes, followed by heating at 95°C for an additional 5 minutes, and a subsequent thermal shock on ice for 3 minutes. After adding 4 μg/μL RNA carrier at room temperature for 3 minutes, samples were centrifuged at 7000 rpm for 10 minutes. The optimized method exhibited a sensitivity and specificity of 100% (CI: 95%). Conclusion: Our findings demonstrate that this in-house extraction method is a viable, rapid, and cost-effective alternative for total RNA extraction, particularly beneficial in areas lacking access to commercial kits. Additionally, it serves as a reliable option during urgent situations.
کلمات کلیدی
SARS-CoV-2, Total RNA, Extraction method, Heat shock
