Otimização do diagnóstico molecular de SARS-CoV-2 : RT-qPCR direta e amplificação isotérmica

Abstract

COVID-19 pandemic onset led to a need for mass testing to control SARS-CoV-2 spread. This study assessed the performance of direct RT-qPCR and RT-LAMP detecting virus in clinical samples to optimize SARS-CoV-2 molecular detection. Initially, many methodologies to direct sample use in RT-qPCR, without RNA extraction, were assessed and compared to silica column extraction. High sensitivity (97.44%) and specificity (100%) were observed using nuclease-free water or DMSO 1.5% dilution of clinical samples with high viral load (CT ≤ 30). RT-LAMP fluorometric and colorimetric assays were standardized and evaluated for diagnostic performance using extracted RNA or direct use of sample in reaction. In initial assessment stage set of primers target to N2 presented better performance and their genome region present few alterations in main variants of SARS-CoV-2, so they were chosen to next clinical validation stage. Both protocols showed high sensitivity (98.25% to fluorometric and 94.94% to colorimetric), and specificity (90.91% to fluorometric and 100% to colorimetric) using extracted RNA from clinical samples with CT ≤ 30. Although direct sample use in RT-qPCR evidenced a promise performance in high viral load samples (CT ≤ 30), this method did not present a good performance in RT-LAMP with low sensitivity, specificity, and reproducibility through replicates. Among the visualization techniques for RT-LAMP, colorimetric method showed an intermediate color result that could cause an operator misinterpretation. On the other hand, fluorometric assay allowed an easy and indubitable interpretation of results. Even it was necessary RNA extraction to a better diagnosis performance, characteristics how fast execution, low cost and simplified interpretation of results provide to RT-LAMP a potential application in mass test of COVID-19 suspicious patients, mainly in localities with no complex laboratorial resources.