Molecular Detection of Sars-Cov-2 RNA in Nasopharyngeal/Oropharyngeal Swab of Patient Without RNA Extraction

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.