Molecular Beacon Assay Development for Severe Acute Respiratory Syndrome Coronavirus 2 Detection
Abstract
:1. Introduction
2. Materials and Methods
2.1. Bioinformatics Analysis of SARS-CoV-2 Genome
2.2. FRET Melting Assay
2.2.1. Circular Dichroism (CD) Spectroscopy
2.2.2. Nuclear Magnetic Resonance (NMR) Spectroscopy
2.2.3. Hybridization Temperature Determination
2.3. Clinical Samples
2.3.1. cDNA Synthesis and Amplification
2.3.2. cDNA Detection Using Molecular Beacon
2.4. Real-Time PCR for Clinical Samples
3. Results
4. Discussion
- i.
- Ease of result interpretation, requiring less training and enabling health care staff to use the test correctly. The current RT-qPCR tests employ multiplex reactions that render a multitude of amplification plots for two to four different genes, which are not readily interpreted by health care staff with limited training in molecular biology techniques or molecular diagnosis. Furthermore, despite OMS recommendations, the cycle threshold (Ct) and copy number values provided by RT-qPCR are not being used to drive the routine clinical practice. Therefore, the extra unused information provided by RT-qPCR is creating a needless and expensive bottleneck. End-point RT-PCR provides the same Positive/Negative result for the presence/absence of SARS-CoV-2 RNA at a potentially lower cost and higher throughput, with easier interpretation.
- ii.
- Less expensive lab apparatus and reagents needed. The proposed test does not require expensive real-time thermocyclers, being possible to execute using a simple end-point thermocycler or programmable heating block, and a plate reader or fluorescence reading apparatus. This enables the laboratories of developing countries with poor access to heavy lab machinery to implement the method in their facilities using existent equipment as end-point PCR is already used to diagnose other infectious diseases such as Hepatitis B, Ebola, and HIV. Furthermore, as the reactions are performed using a single set of primers and a single molecular beacon, the users can easily implement the protocol using existent enzyme master mixes and reagents, without the need to purchase expensive RT-qPCR SARS-CoV-2 detection kits.
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Conflicts of Interest
References
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Genome Region | Position | Length | QGRS 1 | G-Score | Total of Genomes Analyzed | Genomes with PQS | Conservation Level (%) |
---|---|---|---|---|---|---|---|
ORF1ab | 1,574 | 26 | GGTGTTGTTGGAGAAGGTTCCGAAGG | 19 | 211,072 | 207,006 | 98.07 |
13,385 | 20 | GGTATGTGGAAAGGTTATGG | 18 | 210,888 | 99.91 | ||
S | 24,215 | 20 | GGTTGGACCTTTGGTGCAGG | 17 | 211,072 | 210,803 | 99.87 |
24,268 | 24 | GGCTTATAGGTTTAATGGTATTGG | 19 | 210,999 | 99.97 | ||
25,197 | 22 | GGCCATGGTACATTTGGCTAGG | 17 | 207,837 | 98.47 | ||
N | 28,903 | 15 | GGCTGGCAATGGCGG | 18 | 211,072 | 191,696 | 90.82 |
Variant | SARS-CoV-2 Gene | SARS-CoV-2 G4 Sequence | Total of Sequences Analyzed | Number of Sequences with PQS | Conservation (%) |
---|---|---|---|---|---|
Alpha | ORF1ab | GGTGTTGTTGGAGAAGGTTCCGAAGG | 153,813 | 151,813 | 99.02 |
S | GGCTTATAGGTTTAATGGTATTGG | 153,259 | 99.96 | ||
Delta | ORF1ab | GGTGTTGTTGGAGAAGGTTCCGAAGG | 184,757 | 180,642 | 97.77 |
S | GGCTTATAGGTTTAATGGTATTGG | 184,691 | 99.96 | ||
Beta | ORF1ab | GGTGTTGTTGGAGAAGGTTCCGAAGG | 13,185 | 12,942 | 98.16 |
S | GGCTTATAGGTTTAATGGTATTGG | 13,175 | 99.92 | ||
Gamma | ORF1ab | GGTGTTGTTGGAGAAGGTTCCGAAGG | 36,027 | 35,930 | 99.73 |
S | GGCTTATAGGTTTAATGGTATTGG | 36,012 | 99.96 | ||
Lambda | ORF1ab | GGTGTTGTTGGAGAAGGTTCCGAAGG | 211 | 211 | 100 |
S | GGCTTATAGGTTTAATGGTATTGG | 211 | 100 | ||
Mu | ORF1ab | GGTGTTGTTGGAGAAGGTTCCGAAGG | 3,266 | 3,247 | 99.42 |
S | GGCTTATAGGTTTAATGGTATTGG | 3,263 | 99.91 |
MB1 | MB2 | |||||
---|---|---|---|---|---|---|
Temperature (°C) | t = 0 Min | t = 5 Min | t = 21 Min | t = 0 Min | t = 5 Min | t = 21 Min |
65 | 51.05 | 45.09 | 42.31 | 61.29 | 66.81 | 65.94 |
63.8 | 58.65 | 57.64 | 56.51 | 60.09 | 62.01 | 60.86 |
62 | 59.68 | 57.81 | 56.75 | 78.69 | 72.64 | 64.07 |
59.1 | 64.42 | 63.26 | 62.18 | 82.31 | 73.63 | 65.91 |
55.7 | 59.58 | 57.75 | 55.69 | 49.42 | 48.48 | 45.86 |
52.9 | 72.69 | 68.76 | 63.37 | 77.06 | 68.49 | 62.01 |
51 | 68.03 | 66.23 | 61.45 | 76.58 | 69.99 | 66.78 |
50 | 60.36 | 62.92 | 60.34 | 70.81 | 65.35 | 60.38 |
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Carvalho, J.; Lopes-Nunes, J.; Figueiredo, J.; Santos, T.; Miranda, A.; Riscado, M.; Sousa, F.; Duarte, A.P.; Socorro, S.; Tomaz, C.T.; et al. Molecular Beacon Assay Development for Severe Acute Respiratory Syndrome Coronavirus 2 Detection. Sensors 2021, 21, 7015. https://doi.org/10.3390/s21217015
Carvalho J, Lopes-Nunes J, Figueiredo J, Santos T, Miranda A, Riscado M, Sousa F, Duarte AP, Socorro S, Tomaz CT, et al. Molecular Beacon Assay Development for Severe Acute Respiratory Syndrome Coronavirus 2 Detection. Sensors. 2021; 21(21):7015. https://doi.org/10.3390/s21217015
Chicago/Turabian StyleCarvalho, Josué, Jéssica Lopes-Nunes, Joana Figueiredo, Tiago Santos, André Miranda, Micaela Riscado, Fani Sousa, Ana Paula Duarte, Sílvia Socorro, Cândida Teixeira Tomaz, and et al. 2021. "Molecular Beacon Assay Development for Severe Acute Respiratory Syndrome Coronavirus 2 Detection" Sensors 21, no. 21: 7015. https://doi.org/10.3390/s21217015
APA StyleCarvalho, J., Lopes-Nunes, J., Figueiredo, J., Santos, T., Miranda, A., Riscado, M., Sousa, F., Duarte, A. P., Socorro, S., Tomaz, C. T., Felgueiras, M., Teixeira, R., Faria, C., & Cruz, C. (2021). Molecular Beacon Assay Development for Severe Acute Respiratory Syndrome Coronavirus 2 Detection. Sensors, 21(21), 7015. https://doi.org/10.3390/s21217015