Fast SARS-CoV-2 detection by RT-qPCR in preheated nasopharyngeal swab samples

Julia Alcoba-Florez; Rafaela Gonzalez-Montelongo; Antonio Inigo-Campos; Diego Garcia-Martinez de Artola; Helena Gil-Campesino; Laura Ciuffreda; Agustin Valenzuela-Fernandez; Carlos Flores

Samples: We selected nasopharyngeal swabs from four COVID-19/SARS-CoV-2 patients and four COVID-19 negative controls collected in 2 mL volume of VTM (Deltalab). Sample manipulation was performed under a biosafety class II cabinet (TELSTAR bio-II-A). Diagnosis of COVID-19/SARS-CoV-2 infection was conducted with 200 μL of VTM with standard RNA extractions, which used the MagCore viral Nucleic Acid Extraction kit (RBC Bioscience) in a MagCore liquid handler (RBC Bioscience) (lasting approximately 1 h) or the Nuclisens easyMAG kit (bioMérieux) with the eMAG liquid handler (bioMérieux) (lasting approximately 1 h and 20 min), and a one-step RT-qPCR using the LightMix® Modular SARS and Wuhan CoV E-gene kit (Roche). This commercial mix was supplemented with primers and probes for detecting the human actin (ACTB) gene expression to serve as an internal control of the reaction as described elsewhere (Fenollar et al., 2016) (Table 1) . The experiments included a non-template control and a positive control for the E-gene amplification (LightMix® kit Sarbecovirus ivRNA control +, Roche). The RT-qPCR was performed in 10 μL final volume reactions (5 μL of sample) using a CFX96 Touch Real-Time PCR Detection System (Bio-Rad). Following the specifications, thermal cycling was performed at 55 o C for 5 min for the RT step, followed by an amplification step with initial denaturation at 95 o C for 5 min and 45 cycles of 95 o C for 5 sec, 60 o C for 15 sec, and 72 o C for 15 sec. A final cooling step at 40 o C for 30 sec was also included. Thermal cycling took 1 h and 14 min in total.