Analytical sensibility and specificity of two RT-qPCR protocols for SARS-CoV-2 detection performed in an automated workflow

Barra, Gustavo Barcelos; Santa Rita, Ticiane Henriques; Mesquita, Pedro Goes; Jacomo, Rafael Henriques; Nery, Lidia Freire Abdalla

The World Health Organization declared that COVID-19 outbreak constituted a Public Health Emergency of International Concern and the development of reliable laboratory diagnosis of SARS-CoV-2 became mandatory to identify, isolate and provide optimized care for patients early. RT-qPCR testing of respiratory secretions is routinely used to detect causative viruses in acute respiratory infection. RT-qPCR in-house protocols to detect the SARS-CoV-2 have been described. Validations of these protocols are considered a key knowledge gap for COVID-19, especially if executed in a high throughput format. Here, we investigate the analytical sensitivity and specificity of two interim RT-qPCR protocols for the qualitative detection of SARS-CoV-2 executed in a fully automated platform. Under our conditions, the N1 and RdRP (modified) showed the highest analytical sensitivity for their RNA targets. E assay, in its original concentration, was considered a tertiary confirmatory assay. Taken together, N1, RdRP (optimized) and E presented appropriated analytical sensibility and specificity in our automated RT-qPCR workflow for COVID-19 virus, E being at least 4-fold less sensitive than the others. This study highlights the importance of local validation of in-house assays before its availability to the population. The use of the synthetic RT-qPCR target to investigate novel assays diagnostic parameters in automated workflows is a quick, simple effective way to be prepared for upcoming threats. The proposed assay detected the first SARS-CoV-2 infection in Brazilian Central-West.