Thermal Shock Viral RNA Release in COVID-19 Testing

Abstract

Abstract Introduction/Objective The public health emergency of the COVID-19 pandemic emphasized the crucial role of medical laboratory professionals and scientists in molecular diagnostics laboratories to ensure success in infection control strategies. The demand for laboratory testing using nucleic acid amplification tests to detect SARS-CoV-2 RNA imposed strains in laboratory supplies. Here, we explored an alternative cost-effective solution that will simplify the pre-PCR steps by using a simple heating method to release viral RNA. Methods/Case Report Samples tested using the reference automated extraction method were used:100 samples identified as positive for SARS-CoV-2 RNA and 500 samples tested negative for SARS-CoV-2 RNA were used for the study and sorted with equal distribution according to Ct values of (a) <20, (b) 20–30, and (c) >30.100 ul from swab preserved in Universal Transport Medium was treated with 30 μg of proteinase K, and another set was tested without proteinase K pre-treatment. All samples with or without proteinase K were diluted to minimize PCR inhibitors. The thermal shock protocol was set at (98°C, 5 minutes; 4°C, 2 minutes) and screened for purity. Performance and method verification studies were performed. Internal extraction, positive template, and no template controls were markers used for testing quality. The experimental study was performed by qualified testing personnel and all under the same experimental conditions. Results (if a Case Study enter NA) The Ct values from the thermal shock RNA release were compared to the automated extraction method and statistically analyzed.The criteria for acceptability for validation of this new RNA extraction proceeding were set to 100% concordance compared to the commercial kit using an automated extraction. PCR efficiency was at 98% and a slope of -3.3. Within run precision of 2% and limits of detection from 200 to 20,000 copies/uL The method 100% (50/50) concordance on samples previously identified as negative by automated methods and identified 86% (86/100) with a mean difference of 3 Ct. Conclusion Our findings suggest that the thermal shock treatment of nasopharyngeal swabs in viral transport media can successfully extract viral nucleic acid for nucleic acid amplification and is a reasonable alternative for chemical extraction methods when molecular diagnostic laboratories persistently encounter supply chain issues.