Design of a Rapid and Reversible Fluorescence Assay to Detect Covid-19 and other Pathogens

Main Article Content

Suzanne Scarlata V. Siddartha Yerramilli

Abstract

We describe a rapid and reusable biophysical method to assay COVID-19.  The method uses fluorescent sensors (i.e., molecular beacons) designed to detect a specific RNA sequence from COVID-19 but is general to any RNA of interest.  The assay can be used concurrently with an internal control without the need for amplification.  Molecular beacons are stem-loop structures in which a ~10 nucleotide loop region has the complementary sequence of a region of the target RNA, and a fluorophore and quencher are placed on the 5’ and 3’ ends of the stem. The energy of hybridization of the loop with its target is designed to be greater than the hybridization energy of the energy of the stem so that when the beacon encounters its target RNA, the structure opens resulting in dequenching of the fluorophore.  Here, we designed a beacon to different COVID-19 variants that is completely quenched in its native form and undergoes a 50-fold increase in fluorescence when exposed to nanomolar amounts of synthetic viral oligonucleotide.  No changes in intensity are seen when a control RNA (hGAPDH) is added. This increase in fluorescence with beacon opening can be completely reversed upon addition of single stranded DNA complementary to COVID-19 beacon loop region.  Beacons can be attached to an inert matrix allowing their use and reuse in concentrated form and can be made from morphilino oligonucleotides that are resistant to RNases. We present an analysis of the parameters that will allow the development of test strips to detect virus in aerosol, body fluids and community waste. 

Article Details

How to Cite
SCARLATA, Suzanne; YERRAMILLI, V. Siddartha. Design of a Rapid and Reversible Fluorescence Assay to Detect Covid-19 and other Pathogens. Medical Research Archives, [S.l.], v. 10, n. 4, apr. 2022. ISSN 2375-1924. Available at: <https://esmed.org/MRA/mra/article/view/2730>. Date accessed: 29 mar. 2024. doi: https://doi.org/10.18103/mra.v10i4.2730.
Section
Research Articles

References

1. Prinzi A. How the SARS-CoV-2 EUA Antigen Tests Work. American Society of Microbiology. 2020.
2. Tyagi S, Kramer FR. Molecular Beacons: Probes that Fluoresce upon Hybridization. Nature Biotechnology. 1996;14(3):303-308.
3. Guo Y, Lu Z, Cohen IS, Scarlata S. Development of a Universal RNA Beacon for Exogenous Gene Detection. Stem Cells Translational Medicine. 2015;4(5):476-482.
4. Kim JM, Chung YS, Jo HJ, et al. Identification of Coronavirus Isolated from a Patient in Korea with COVID-19. Osong Public Health Res Perspect. 2020;11(1):3-7.
5. Ranoa DRE, Holland RL, Alnaji FG, et al. Saliva-Based Molecular Testing for SARS-CoV-2 that Bypasses RNA Extraction. 2020:2020.2006.2018.159434.
6. Paraskevis D, Kostaki EG, Magiorkinis G, Panayiotakopoulos G, Sourvinos G, Tsiodras S. Full-genome evolutionary analysis of the novel corona virus (2019-nCoV) rejects the hypothesis of emergence as a result of a recent recombination event. Infect Genet Evol. 2020;79:104212.
7. Lorenz R, Bernhart SH, Honer Zu Siederdissen C, et al. ViennaRNA Package 2.0. Algorithms Mol Biol. 2011;6:26.
8. Kumar SV, Hurteau GJ, Spivack SD. Validity of messenger RNA expression analyses of human saliva. Clin Cancer Res. 2006;12(17):5033-5039.
9. Yoon JG, Yoon J, Song JY, et al. Clinical Significance of a High SARS-CoV-2 Viral Load in the Saliva. J Korean Med Sci. 2020;35(20):e195-e195.
10. Albani JR. Chapter 3 - Fluorophores: Descriptions and Properties. In: Albani JR, ed. Structure and Dynamics of Macromolecules: Absorption and Fluorescence Studies. Amsterdam: Elsevier Science; 2004:99-140.
11. Tinsley JN, Molodtsov MI, Prevedel R, et al. Direct detection of a single photon by humans. Nature Communications. 2016;7(1):12172.
12. Runnels LW, Scarlata SF. Theory and application of fluorescence homotransfer to melittin oligomerization. Biophysical Journal. 1995;69(4):1569-1583.
13. Esbin MN, Whitney ON, Chong S, Maurer A, Darzacq X, Tjian R. Overcoming the bottleneck to widespread testing: a rapid review of nucleic acid testing approaches for COVID-19 detection. Rna. 2020;26(7):771-783.
14. Gopal A, Zhou ZH, Knobler CM, Gelbart WM. Visualizing large RNA molecules in solution. RNA. 2012;18(2):284-299.
15. Wyllie AL, Fournier J, Casanovas-Massana A, et al. Saliva is more sensitive for SARS-CoV-2 detection in COVID-19 patients than nasopharyngeal swabs. medRxiv. 2020:2020.2004.2016.20067835.
16. Wozniak A, Cerda A, Ibarra-Henriquez C, et al. A simple RNA preparation method for SARS-CoV-2 detection by RT-qPCR. bioRxiv. 2020:2020.2005.2007.083048.
17. Summerton J, Weller D. Morpholino Antisense Oligomers: Design, Preparation, and Properties. Antisense and Nucleic Acid Drug Development. 1997;7(3):187-195.
18. Chen J, Wu J, Hong Y. The morpholino molecular beacon for specific RNA visualization in vivo. Chemical Communications. 2016;52(15):3191-3194.
19. Nitin N, Santangelo PJ, Kim G, Nie S, Bao G. Peptide‐linked molecular beacons for efficient delivery and rapid mRNA detection in living cells. Nucleic Acids Research. 2004;32(6):e58-e58.