In this study a total of 98 confirmed nasopharyngeal swabs from COVID-19 symptomatic individuals were analysed. These samples were routinely collected in October 2020 and were eluted in the universal transport medium (UTM™, Copan Diagnostics Inc, US) and processed at the regional reference laboratory for COVID-19 diagnostic of the San Martino Policlinico Hospital, Hygiene Unit, Genoa, Liguria, Northwest Italy.
All samples were processed in parallel by using three methods: (i) standard extraction-based (EB) method performed with STARMag 96x4 Viral DNA/RNA 200C Kit (Seegene Inc., South Korea); (ii) RNA extraction-free (EF) method with a heating step (EFh+) and (iii) unheated RNA extraction-free technique (EFh–). For the EB method, total RNA was extracted and set up for RT-PCR by means of the Nimbus IVD Seegene platform using the Allplex™ 2019-nCoV Assay kit (Seegene Inc., South Korea), according to the manufacturer’s instructions. For the EFh + method, thermolysis was first performed at 95°C for 5 min and at 55°C for 3 min on Biorad CFX96™ thermal cycler (Bio-Rad Laboratories, US). EFh– method was identical to EFh + unless the thermolysis step.
The material thus obtained was tested for the identification of SARS-CoV-2 by means of a one-step real-time multiplex RT-PCR quantitative assay on Biorad CFX96™ thermal cycler, targeting the nucleoprotein region (N), RNA-dependent RNA-polymerase (RdRp)/spike (S) proteins and envelop (E) region. Samples showing a cycle threshold (Ct) value < 40 for at least two genes were considered positive.
The standard EB technique was considered a reference method against both EFh + and EFh–. A sensitivity with 95% confidence intervals (CIs) was calculated. As the observed Ct values were approximately normally distributed, the average target-specific difference in Ct values between the three techniques was computed and compared by applying the repeated measures analysis of variance (ANOVA) and follow-up Tukey honestly significant difference post-hoc test. Data were analysed in R stats packages, v. 4.0.3 [15].