Monday article #35: A highly sensitive, specific, simpler and faster method for Covid-19 detection



The current gold-standard for the detection of Covid-19 infection is real-time RT-PCR (Reverse Transcriptase Polymerase Chain Reaction) of samples collected from nasopharyngeal or oropharyngeal swabs [1]. This method is both sensitive and specific, making it a reliable way for the detection of Covid-19 infection in suspected individuals [2]. However, this diagnostic method possesses some disadvantages such as needing specialised laboratory instruments and trained/professional manpower, hence it is usually only available at certain hospitals or diagnostic labs. Another major drawback of the PCR-based approach is that it takes around 24-48 hours to obtain the results [3], making it inconvenient for real-time patient management. In this article, we discuss an emerging diagnosis method that does not need sophisticated equipment nor skilled manpower, and is able to give results much more rapidly than RT-PCR.


Clustered regularly interspaced short palindromic repeats (CRISPR) is widely known as a valuable tool that allows researchers to edit DNA sequences and alter genetic functions or defects [4]. However, various new diagnostic methods have been developed based on the CRISPR system, including SHERLOCK and DETECTR.


*SHERLOCK = Specific High-sensitivity Enzymatic Reporter un-LOCKing

DETECTR = DNA Endonuclease Targeted CRISPR Trans Reporter


Zhang and colleagues [5] recently used the SHERLOCK system for Covid-19 detection. Firstly, RNA will be purified from patient nasopharyngeal or oropharyngeal samples and will undergo amplification using recombinase polymerase amplification (RPA). A guide RNA is designed to match certain nucleic acid sequences, such as E (envelope) and N (nucleoprotein) genes of the coronavirus. This guide RNA will help guide the Cas13a nuclease to the site of interest. If this target sequence is present, i.e., the coronavirus is present in the sample, the Cas13a will become activated and cleave the sequence to release a signal molecule. This signal molecule will act as an indicator for a positive test result using lateral flow visualisation, such as those seen in the widely available at-home rapid test kits. The test results can be obtained within around 70 minutes.



The DETECTR system is another method that was developed into potential Covid-19 test kits [6]. This system is similar to SHERLOCK, but uses the Cas12a enzyme instead of Cas13a. Firstly, the patient's nasopharyngeal or oropharyngeal sample will undergo RNA extraction and the extracted RNA will undergo isothermal amplification using RT-lamp. Next, Cas12a nuclease recognizes and cleaves the predefined SARS-Cov-2 RNA sequences, such as the E and N genes used in SHERLOCK, which are detected by cleavage of a reporter molecule. The Cas12a will be activated and subsequently cleave the sequence to release a reported signal. The DETECTR assay can be run in approximately 30–40 min and conveniently visualised on a lateral flow strip.


The systems mentioned above are both highly sensitive and highly specific methods for the detection of Covid-19 infection. An exciting fact of CRISPR-based systems is their ability to provide results within 30-70 minutes, instead of having to wait 1-2 days for PCR-based methods. This will help to curb the spread of coronavirus infection and aid in real-time patient management, reducing morbidity and mortality. Another major advantage of the CRISPR-based diagnostic methods is that they can be used in economically restricted areas due to their lack of requirement for specialised laboratory instruments and reagents. In addition, results can be visualised with the naked eye and simply interpreted through lateral flow strips, removing the need of skilled manpower for the interpretation of results. Furthermore, during the early phase of infection, the sensitivity of RT-PCR test is quite low, leading to false-negative results when the viral load is still below detectable levels. In contrast, CRISPR-based systems will be capable of detecting the viral genome in various stages of infection, including the early incubation period [7].


Therefore, CRISPR-based diagnostic methods discussed today fulfils the criterias of being sensitive, specific, simple to use and rapid without the need for sophisticated laboratory instruments or skilled personnels. These CRISPR-based systems are in the process of becoming commercially available and have the potential to even replace conventional RT-PCR tests!




REFERENCES:


[1] Cheng, M.P., Papenburg, J., Desjardins, M., Kanjilal, S., Quach, C., Libman, M., Dittrich, S. and

Yansouni, C.P. (2020) ‘Diagnostic testing for severe acute respiratory syndrome–related coronavirus 2: a narrative review’, Annals of internal medicine, 172(11), pp.726-734. [Online] DOI: 10.7326/M20-1301 (Accessed: 26 October 2022).


[2] Maia, R., Carvalho, V., Faria, B., Miranda, I., Catarino, S., Teixeira, S., Lima, R., Minas, G. and

Ribeiro, J. (2022) ‘Diagnosis methods for COVID-19: A systematic review’, Micromachines, 13(8), p.1349. [Online] DOI: 10.3390/mi13081349 (Accessed: 26 October 2022).


[3] Hillary, V.E., Ignacimuthu, S. and Ceasar, S.A. (2021) ‘Potential of CRISPR/Cas system in the

diagnosis of COVID-19 infection’, Expert Review of Molecular Diagnostics, 21(11), pp.1179-1189. [Online] DOI: 10.1080/14737159.2021.1970535 (Accessed: 26 October 2022).


[4] Safari, F., Afarid, M., Rastegari, B., Borhani-Haghighi, A., Barekati-Mowahed, M. and

Behzad-Behbahani, A. (2021) ‘CRISPR systems: novel approaches for detection and combating COVID-19’, Virus Research, 294, p.198282. [Online] DOI: 10.1016/j.virusres.2020.198282 (Accessed: 26 October 2021).


[5] Zhang, F., Abudayyeh, O.O. and Gootenberg, J.S. (2020) ‘A protocol for detection of COVID-19

using CRISPR diagnostics’, A protocol for detection of COVID-19 using CRISPR diagnostics, 8. [Online] Available at: https://go.idtdna.com/rs/400-UEU-432/images/Zhang%20et%20al.%2C%202020%20COVID-19%20detection%20(updated).pdf (Accessed: 26 October 2022).


[6] Broughton, J.P., Deng, X., Yu, G., Fasching, C.L., Servellita, V., Singh, J., Miao, X., Streithorst,

J.A., Granados, A., Sotomayor-Gonzalez, A. and Zorn, K. (2020) ‘CRISPR–Cas12-based

detection of SARS-CoV-2’, Nature biotechnology, 38(7), pp.870-874. [Online] DOI: 10.1038/s41587-020-0513-4 (Accessed: 26 October 2022).


[7] Metsky, H.C., Freije, C.A., Kosoko-Thoroddsen, T.S.F., Sabeti, P.C. and Myhrvold, C. (2020)

‘CRISPR-based surveillance for COVID-19 using genomically-comprehensive machine learning design’, BioRxiv. [Online] DOI: 10.1101/2020.02.26.967026 (Accessed: 26 October 2022).


 

This article was prepared by Phoebe Tee

 






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