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

Abstract

The fast spread of SARS-CoV-2 has led to a global pandemic, calling for\nfast and accurate assays to allow infection diagnosis and prevention of\ntransmission. We aimed to develop a molecular beacon (MB)-based\ndetection assay for SARS-CoV-2, designed to, detect the ORF1ab and S\ngenes, proposing a two-stage COVID-19 testing strategy, using MBs to\ndetect the presence of target amplicons by fluorescence analysis. Two\nMBs were designed, optimized in terms of concentration, fluorescence\nplateaus of hybridization, reaction temperature and best real-time\nresults. A total of 450 nasopharyngel and throat swab samples (418\npositive and 32 negative) were tested with the MB assay and the\nfluorescence levels compared with the cycle threshold (Ct) values\nobtained from a commercial RT-PCR test in terms of test duration,\nsensitivity and specificity. Our results show that the samples with\nhigher fluorescence levels correspond to those with low Ct values,\nsuggesting a correlation between viral load and increased MB\nfluorescence. The proposed assay represents a fast (total duration of 2\nh 20 min including amplification and fluorescence reading stages) and\nsimple way of detecting SARS-CoV-2 in clinical samples from the upper\nrespiratory tract. Our two-stage testing strategy is suitable for\nfurther development into a point-of-care assay and potentially scalable\nto population level.