Portal de Periódicos da CAPES

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

2020; Public Library of Science; Volume: 15; Issue: 7 Linguagem: Inglês

10.1371/journal.pone.0236564

1932-6203

Mohammad R. Hasan, Faheem Mirza, Hamad Al-Hail, Sathyavathi Sundararaju, Thabisile Xaba, Muhammad Rafaih Iqbal, Hashim Alhussain, Hadi M. Yassine, Andrés Pérez‐López, Patrick Tang,

Molecular Biology Techniques and Applications

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.