Molecular Beacon Assay Development for Severe Acute Respiratory Syndrome Coronavirus 2 Detection.

Josué Carvalho,Conceição Faria,Fani Sousa,Micaela Riscado,Sílvia Socorro,Cândida Teixeira Tomaz,Jéssica Lopes-Nunes,Joana Figueiredo,Carla Cruz,André Miranda,Ana Paula Duarte,Rui Teixeira,Tiago Santos,Mafalda Felgueiras

doi:10.3390/s21217015

Josué Carvalho, Conceição Faria + Show 12 more

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https://doi.org/10.3390/s21217015

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Abstract

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. The novelty of this work lies in the design and optimization of two MBs for detection of SARS-CoV-2, namely, concentration, fluorescence plateaus of hybridization, reaction temperature and real-time results. We also identify putative G-quadruplex (G4) regions in the genome of SARS-CoV-2. A total of 458 nasopharyngeal and throat swab samples (426 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.

Highlights

Coronavirus disease 2019 (COVID-19) has instigated a global effort toward the development of fast and accurate detection techniques that would allow a more effective control of the global pandemic [1].Reverse transcription-PCR (RT-PCR) is the mainstay of SARS-CoV-2 detection in which two or three target genes, mainly the open reading frame 1ab (ORF1ab), nucleocapsid protein (N), and envelope protein (E), are simultaneously amplified, according to WHO guidance [2]
Various alternative approaches are currently being developed to reduce the time for COVID19 diagnosis [4,5], namely, loop-mediated isothermal amplification (LAMP), which works at a constant temperature and has already been used to successfully detect and identify viruses such as Zika [6]
putative G4 sequences (PQS) were scored based on their propensity to fold into

Summary

Introduction

Coronavirus disease 2019 (COVID-19) has instigated a global effort toward the development of fast and accurate detection techniques that would allow a more effective control of the global pandemic [1].Reverse transcription-PCR (RT-PCR) is the mainstay of SARS-CoV-2 detection in which two or three target genes, mainly the open reading frame 1ab (ORF1ab), nucleocapsid protein (N), and envelope protein (E), are simultaneously amplified, according to WHO guidance [2]. Various alternative approaches are currently being developed to reduce the time for COVID19 diagnosis [4,5], namely, loop-mediated isothermal amplification (LAMP), which works at a constant temperature and has already been used to successfully detect and identify viruses such as Zika [6]. It is a less accurate procedure, and only few samples can be run at a time in a low throughput setting [7]. CRISPR-Cas12/13-based SHERLOCK, DETECTR, CARVER, and PAC-MAN have been applied to detect SARS-CoV-2 [7]

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