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

被引:5
|
作者
Carvalho, Josue [1 ]
Lopes-Nunes, Jessica [1 ]
Figueiredo, Joana [1 ]
Santos, Tiago [1 ]
Miranda, Andre [1 ]
Riscado, Micaela [1 ]
Sousa, Fani [1 ,2 ]
Duarte, Ana Paula [1 ,2 ]
Socorro, Silvia [1 ]
Tomaz, Candida Teixeira [1 ,2 ]
Felgueiras, Mafalda [3 ]
Teixeira, Rui [3 ]
Faria, Conceicao [3 ]
Cruz, Carla [1 ]
机构
[1] Univ Beira Interior, UBI Ctr Invest Ciencias Saude, CICS, Av Infante D Henrique, P-6200506 Covilha, Portugal
[2] Univ Beira Interior, C4 Cloud Comp Competence Ctr, UBIMed, Estr Municipal 506, P-6200284 Covilha, Portugal
[3] Hosp Univ Cova Beira CHUCB, Serv Patol Clin Ctr, P-6200251 Covilha, Portugal
关键词
RNA viruses; SARS-CoV-2; COVID-19; fluorescence detection; diagnosis; G-QUADRUPLEXES;
D O I
10.3390/s21217015
中图分类号
O65 [分析化学];
学科分类号
070302 ; 081704 ;
摘要
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.
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页数:13
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