Molecular beacon assay development for Severe Acute Respiratory Syndrome Coronavirus 2 detection

The fast spread of SARS-CoV-2 has led to a global pandemic, calling for fast and accurate assays to allow infection diagnosis and prevention of transmission. We aimed to develop a molecular beacon (MB)-based detection assay for SARS-CoV-2, designed to, detect the ORF1ab and S genes, proposing a two-stage COVID-19 testing strategy, using MBs to detect the presence of target amplicons by fluorescence analysis. Two MBs were designed, optimized in terms of concentration, fluorescence plateaus of hybridization, reaction temperature and best real-time results. A total of 450 nasopharyngel and throat swab samples (418 positive and 32 negative) were tested with the MB assay and the fluorescence levels compared with the cycle threshold (Ct) values obtained from a commercial RT-PCR test in terms of test duration, sensitivity and specificity. Our results show that the samples with higher fluorescence levels correspond to those with low Ct values, suggesting a correlation between viral load and increased MB fluorescence. The proposed assay represents a fast (total duration of 2 h 20 min including amplification and fluorescence reading stages) and simple way of detecting SARS-CoV-2 in clinical samples from the upper respiratory tract. Our two-stage testing strategy is suitable for further development into a point-of-care assay and potentially scalable to population level.