Detection of SARS-CoV-2 RNA by direct RT-qPCR on nasopharyngeal specimens without extraction of viral RNA

• Published in: PloS one 2020-07, Vol.15 (7), p.e0236564-e0236564
• Main Authors: Hasan, Mohammad Rubayet, Mirza, Faheem, Al-Hail, Hamad, Sundararaju, Sathyavathi, Xaba, Thabisile, Iqbal, Muhammad, Alhussain, Hashim, Yassine, Hadi Mohamad, Perez-Lopez, Andres, Tang, Patrick
• Format: Article
• Language: English
• Subjects: Automation; Betacoronavirus; Biology and life sciences; Clinical Laboratory Techniques - methods; Coronavirus Infections - diagnosis; Coronaviruses; Correlation coefficient; Correlation coefficients; COVID-19; COVID-19 Testing; Diagnosis; Disease transmission; Engineering and Technology; Genetic aspects; Humans; Incubation; Laboratories; Mathematical analysis; Medicine; Medicine and health sciences; Methods; Nasopharynx - virology; Pandemics; Pathogens; Pathology; Pneumonia, Viral - diagnosis; Polymerase chain reaction; Reagents; Real-Time Polymerase Chain Reaction; Reproducibility of Results; Research and analysis methods; Ribonucleic acid; RNA; RNA sequencing; RNA, Viral - isolation & purification; SARS-CoV-2; Sensitivity analysis; Sensitivity and Specificity; Severe acute respiratory syndrome; Severe acute respiratory syndrome coronavirus 2; Specimen Handling - methods; Thermal cycling; Viral diseases; Viruses
• ISSN: 1932-6203; 1932-6203
• DOI: 10.1371/journal.pone.0236564

To circumvent the limited availability of RNA extraction reagents, we aimed to develop a protocol for direct RT-qPCR to detect SARS-CoV-2 in nasopharyngeal swabs without RNA extraction. Nasopharyngeal specimens positive for SARS-CoV-2 and other coronaviruses collected in universal viral transport (UVT) medium were pre-processed by several commercial and laboratory-developed methods and tested by RT-qPCR assays without RNA extraction using different RT-qPCR master mixes. The results were compared to that of standard approach that involves RNA extraction. Incubation of specimens at 65°C for 10 minutes along with the use of TaqPath™ 1-Step RT-qPCR Master Mix provides higher analytical sensitivity for detection of SARS-CoV-2 RNA than many other conditions tested. The optimized direct RT-qPCR approach demonstrated a limit of detection of 6.6x103 copy/ml and high reproducibility (co-efficient of variation = 1.2%). In 132 nasopharyngeal specimens submitted for SARS-CoV-2 testing, the sensitivity, specificity and accuracy of our optimized approach were 95%, 99% and 98.5%, respectively, with reference to the standard approach. Also, the RT-qPCR CT values obtained by the two methods were positively correlated (Pearson correlation coefficient r = 0.6971, p = 0.0013). The rate of PCR inhibition by the direct approach was 8% compared to 9% by the standard approach. Our simple approach to detect SARS-CoV-2 RNA by direct RT-qPCR may help laboratories continue testing for the virus despite reagent shortages or expand their testing capacity in resource limited settings.