Reviewer response for version 1

Abstract

Introduction: Diagnosis of COVID-19 is best performed with real-time PCR (qPCR), the most sensitive method for detection and quantification of viral RNA. Using the CDC protocol, for e ach sample tested for the virus, three qPCR tests are performed, targeting the viral genes N1, and N2, in addition to the internal control gene RNase P. Samples in which internal control fail to amplify should be labelled ‘invalid’. Methods: Thus, this study aims to determine the frequency of inhibition of the RNase P gene used as an internal control in qPCR tests for SARS-CoV-2 in a reference hospital in Southern Brazil during COVID-19 pandemic (01/FEB/2021 to 31/MAR/2021) Results: A total 10,311 samples were available for analysis. Mean Ct value for the RNAse P gene was 26.65 and the standard deviation was 3.18. A total of 252 samples were inhibited (2.4%) during the study period: amongst these, 77 (30.5%) showed late amplifications (beyond 2 standard deviations from mean Ct value), and 175 (69.4%) revealed no fluorescence at all for the RNase P gene. Conclusions: In conclusion, this study shows a low percentage of inhibition using RNase P as an internal control in COVID-19 PCR reactions using the CDC protocol. Thus, proving the effectiveness of this protocol for identification of SARS-CoV-2 in clinical samples. Re-extracting was efficacious for samples that showed little or no fluorescence for the RNase P gene. Keywords: SARS-CoV-2; Covid-19; qPCR; Inhibition; Internal control