Molecular Detection of Sars-Cov-2 RNA in Nasopharyngeal/Oropharyngeal Swab of Patient Without RNA Extraction
Date
2022
Authors
Journal Title
Journal ISSN
Volume Title
Publisher
Department of Biotechnology
Abstract
RT-PCR is the gold standard method used till date for covid19 detection. Owing to the
limited supply of SARS-CoV-2 RNA extraction kits in different health care facilities of Nepal,
it results in enormous pressure to optimize reagent use, thereby affecting the overall
diagnostic quality. This proposed research aimed to detect SARS-CoV-2 RNA from NPS
through direct RT-qPCR technique omitting entire RNA extraction process. For this, 184
clinical NPS samples were obtained from Covid19 suspected patients who visited the
Kirtipur Municipality-TU Biotech Corona Laboratory, and all subsequent steps were
carried out there. These corresponding sample was subjected to RNA extraction followed
by RT-qPCR as well as heat inactivated- RT-qPCR for validation. Eventually, their Ct values
were compared wherein, the impact of heating temperatures and sample volume on
assay sensitivity was also studied. The overall efficacy of these techniques was
comparatively analyzed based on their Ct values. Heating NPS samples (n=184) for 20 min
at 70 °C yielded a sensitivity, specificity, and accuracy of 93.3%, 96.7%, and 91.3%
respectively. According to our paired T-test analysis, the mean Ct values of the N1 and
RNase P genes were statistically significant at 95% CI (p<0.001), whereas the N2 genes
were not (p>0.001). The results thus obtained was also compared with that of
conventional RT-qPCR technique. Thus, a strong agreement using Cohen’s Kappa
(κ=0.803) was found between two methods indicating reliability of heat inactivation
assay. Therefore, direct RT-PCR might be a useful method for quickly identifying COVID19
suspects. This research offers a quick fix for the RNA extraction supply crunch.
Furthermore, this emerging concept could even drastically lower costs and accelerate
assay TAT by omitting the RNA extraction step.
Description
Keywords
Covid19, Direct RT-qPCR, Heat-shock, RNA extraction, SARS-CoV-2, RT-qPCR validation, Ct value