ABSTRACT
BackgroundA new coronavirus (SARS-CoV-2) caused the current Covid-19 epidemic. Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) is used as the gold standard for clinical detection of SARS-CoV-2. Under ideal conditions RT-qPCR Covid-19 assays have analytical sensitivity and specificity greater than 95%. However, when the sample panel is enlarged including asymptomatic individuals, the sensitivity decreases and false-negative are reported. Moreover, RT-qPCR requires up to 3-6 hours with most of the time involved in RNA extraction from swab samples. MethodsWe introduce CovidArray, a microarray-based assay, to detect SARS-CoV-2 markers N1 and N2 in the nasopharyngeal swabs. The method is based on solid phase hybridization of fluorescently labelled amplicons upon RNA extraction and reverse transcription. This approach combines the physical-optical properties of the silicon substrate with the surface chemistry used to coat the substrate to obtain a diagnostic tool of great sensitivity. Furthermore, we used an innovative approach, RNAGEM, to extract and purify viral RNA in less than 15 minutes. To validate the CovidArray results, we exploited the high sensitivity of the droplet digital PCR (ddPCR) technique. ResultWe correctly assigned 12 nasopharyngeal swabs, previously analyzed by RT-qPCR. Thanks to the CovidArray sensitivity that matches that of the ddPCR, we were able to identify a false-negative sample. ConclusionsCovidArray is the first DNA microarray-based assay to detect viral genes in the swabs. Its high sensitivity and the innovative viral RNA extraction by RNAGEM allows to reduce both the amount of false negative results and the total analysis time to about 2 hours.
ABSTRACT
Since the outbreak of COVID-19 crisis, the handling of biological samples from confirmed or suspected SARS-CoV-2 positive individuals demanded the use of inactivation protocols to ensure laboratory operators safety. While not standardized, these practices can be roughly divided in two categories, namely heat inactivation and solvent-detergent treatments. As such, these routine procedures should also apply to samples intended for Extracellular Vesicles (EVs) analysis. Assessing the impact of virus inactivating pre-treatments is therefore of pivotal importance, given the well-known variability introduced by different pre-analytical steps on downstream EVs isolation and analysis. Arguably, shared guidelines on inactivation protocols tailored to best address EVs-specific requirements will be needed among the EVs community, yet deep investigations in this direction havent been reported so far. In the attempt of sparking interest on this highly relevant topic, we here provide preliminary insights on SARS-CoV-2 inactivation practices to be adopted prior serum EVs analysis by comparing solvent/detergent treatment vs. heat inactivation. Our analysis entailed the evaluation of EVs recovery and purity along with biochemical, biophysical and biomolecular profiling by means of Nanoparticle Tracking Analysis, Western Blotting, Atomic Force Microscopy, miRNA content (digital droplet PCR) and tetraspanin assessment by microarrays. Our data suggest an increase in ultracentrifugation (UC) recovery following heat-treatment, however accompanied by a marked enrichment in EVs-associated contaminants. On the contrary, solvent/detergent treatment is promising for small EVs (< 150 nm range), yet a depletion of larger vesicular entities was detected. This work represents a first step towards the identification of optimal serum inactivation protocols targeted to EVs analysis.
ABSTRACT
A workflow for SARS-CoV-2 epitope discovery on peptide microarrays is herein reported. The process started with a proteome-wide screening of immunoreactivity based on the use of a high-density microarray followed by a refinement and validation phase on a restricted panel of probes using microarrays with tailored peptide immobilization through a click-based strategy. Progressively larger, independent cohorts of Covid-19 positive sera were tested in the refinement processes, leading to the identification of immunodominant regions on SARS-CoV-2 Spike (S), Nucleocapsid (N) protein and Orf1ab polyprotein. A summary study testing 50 serum samples highlighted an epitope of the N protein (region 155-171) providing 92% sensitivity and 100% specificity of IgG detection in Covid-19 samples thus being a promising candidate for rapid implementation in serological tests.
