Abstract
A new coronavirus (SARS-CoV-2) caused the current coronavirus disease (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 negatives are reported. Moreover, RT-qPCR requires up to 3–6 h with most of the time involved in RNA extraction from swab samples. We 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-labeled 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 min. We correctly assigned 12 nasopharyngeal swabs, previously analyzed by RT-qPCR. Thanks to the CovidArray sensitivity we were able to identify a false-negative sample. CovidArray 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 the reduction of both the amount of false-negative results and the total analysis time to about 2 h.
Highlights
In December 2019, an unexplained acute respiratory disease, Covid-19, was first reported in Wuhan, China [1]
We propose an approach to detect SARS-CoV-2 endowed with high sensitivity, accuracy, and multiplexing capability based on microarray technology, named CovidArray
Data intensities were extracted with the Mapix software and the data analysis was performed for each experiment
Summary
In December 2019, an unexplained acute respiratory disease, Covid-19, was first reported in Wuhan, China [1]. RT-qPCR Covid-19 assays have analytical sensitivity and specificity greater than 95% [8] This number refers to tests validated under ideal conditions with hospital samples containing viral loads higher than those from asymptomatic individuals. RPP30, but, compared to the commercial RT-qPCR kits, our system has the additional potential to distinguish the SARS-CoV-2 variants or to differentiate it from other viral and bacterial respiratory tract infections by adding new primers and capture probes to the same array Another key improvement introduced by CovidArray is the faster analysis time. The introduction of rapid antigen detection (RAD) tests based on immunoassays for qualitative determination of SARS-CoV-2 antigens allows one to obtain a result in about 15–30 min Due to their low sensitivity especially for asymptomatic subjects [25], the actual negativity of the sample must in any case be confirmed by the nucleic acid test. Here we demonstrated that combination of single-tube extraction by RNAGEM with highly sensitive multiplex microarray substrate with optimal properties allows to reduce the number of PCR cycles from 40 to 25 and to lead to an overall increase in accuracy and a reduction in analysis time
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