Improved heat shock method for extracting total RNA from nasopharyngeal swab samples even with low viral load

• Published in: Diagnostic microbiology and infectious disease 2024-05, Vol.109 (1), p.116210-116210, Article 116210
• Main Authors: Khodaei, Hannan, Azimi, Leila, Akhavan Sepahy, Abbas, Ashrafi, Fatemeh, Rajabnejad, Maryam
• Format: Article
• Language: English
• Subjects: Clinical Laboratory Techniques - methods; COVID-19; COVID-19 Testing - methods; Extraction kit; Heat shock; Heat-Shock Response; Humans; Nasopharynx; RNA, Viral - analysis; RNA, Viral - genetics; SARS-COV-2; SARS-CoV-2 - genetics; Sensitivity and Specificity; Specimen Handling - methods; Total RNA; Viral Load
• ISSN: 0732-8893; 1879-0070
• DOI: 10.1016/j.diagmicrobio.2024.116210

This study aimed to improve the heat shock method as a cost-effective and time-efficient for total RNA extraction. We compared the effectiveness of two total RNA extraction methods by using Real-Time PCR for nasopharynx swabs. Include: I; use of a commercial total RNA extraction kit as a standard. II; utilized a modified heat shock method (MHS). Time, centrifuge speed and duration, proteinase K, and RNA carrier were optimized. The optimized parameters included treating the sample with 5 μg/μL at 56°C for 5 minutes, heating at 95°C for 5 minutes followed by thermal shock in ice for 3 minutes, adding 4 μg/μL RNA carrier at room temperature for 3 minutes, and centrifuging at 7000 rpm for 10 minutes. This optimization demonstrated a sensitivity and specificity of 100% (CI: 95%) even in samples with low viral load. Our in-house method presents a rapid, and cost-effective alternative for total RNA extraction.