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ACADEMIA Letters Literature Review: A potential detection method for SARS-CoV-2 Ananna Arna Austin Mardon Since late 2019, SARS-CoV-2 has claimed over a million lives. While another millions of infections are still active across the globe, many are silently carrying the virus without showing any visible symptoms. Although a common asymptomatic period is experienced among the infected when the virus sheds and undergoes exponential reproduction in the upper airways and lungs, they start to develop evident symptoms soon after. It is during this period that the viral load is highest at the upper respiratory tract, which undergoes a steep decline as symptoms appear. Individuals experiencing this asymptomatic period are termed as presymptomatic. However, some of them, though infected, may not show any symptoms at all – they are asymptomatic carriers. Both pre-symptomatic and asymptomatic patients are major contributors in exacerbating the community transmission. Thus, it is important to develop a cost and time effective, robust point of care diagnostics to identify these pre-symptomatic and asymptomatic carriers who often go undetected by the conventional testing methods and increase transmission. As laboratory-based tests like qPCR demands professional personnel to handle samples, carry out protocol, and interpret data, they become rather time-consuming to be used as a point-of-care diagnostics. A robust point-of-care diagnostics would be a technology/ protocol that gives specific and sensitive detection results rapidly with very minimal false-positives; however, its required equipment and reagents should be readily available. To develop such, isothermal amplification was coupled with CRISPR-based detection to generate a new workflow: DISCOVER. A comparative study of enzyme activity was done between Cas13 and Cas12. Lateral flow assay was performed where readout was taken of the freed reporter molecules during nucleic Academia Letters, August 2021 ©2021 by the authors — Open Access — Distributed under CC BY 4.0 Corresponding Author: Ananna Arna, arnaananna17@gmail.com Citation: Arna, A., Mardon, A. (2021). Literature Review: A potential detection method for SARS-CoV-2. Academia Letters, Article 2778. https://doi.org/10.20935/AL2778. 1 acid cleavage. There, Cas13 was found to have significantly higher nucleic acid detection ability at a relatively faster speed. However, Cas13’s detection duration was 60 minutes at a femtomolar level, but this is not sufficient for a point-of-care diagnostic. To tackle this, costeffective Loop-mediated isothermal amplification (LAMP) was used to amplify nucleic acid with high sensitivity in the attomolar level in under 20 minutes. Despite the rapid, sensitive nucleic acid detection of LAMP, they have non-specific interaction resulting in non-template control (NTC) signal that needs to be addressed. This NTC signal is due to the dimerization of LAMP primer sets which might lead to high false-positive. To work around this, Cas13 was used as a secondary probe for specific target recognition. For Cas13 to avoid false-positive detections, the guide RNAs must not overlap with the primers. Short and highly overlapping LAMP primer raised amplicons – size ranged from 1-60 nt – to avoid non-specific detection of Cas13. Since LAMP is only compatible with DNA substrates, a reverse transcriptase is incorporated to enable viral RNA to DNA conversion for proper targeting for isothermal amplification. Cas13 however works with single-stranded RNA only; so, transcription of DNA LAMP products into RNA (rLAMP) becomes important for substrate compatibility. A T7 polymerase promoter is incorporated into the primer to enable rLAMP. To confirm amplification only in the targeted primer regions, isoschizomer AfeI was used for a restriction enzyme digestion on LAMP products where only the Cas13 guide RNA-targeted amplicon regions were digested and NTC signals were not as they did not match the guide RNA. To confirm T7 promoter insertion, in vitro transcription was performed which resulted in 85 nt RNA products. These rLAMP products were then screened for Cas13 cleavage activity where the mid region of backward inner primer (mBIP) showed fastest detection. In the mBIP over NTC background, 10-fold change in signal was achieved in under two minutes whereas a saturated signal with 40-fold change was achieved within five minutes of detection that stayed stable well over five minutes. The result was then replicated in other mBIP rLAMP amplicons to evaluate the reliability and replicability of DISCOVER pipeline. Subsequently, saliva samples were processed with heat (denaturation) and reduced with chemical treatment (TCEP, EDTA, QuickExtract buffers, and DNA/ RNA shield) to inactivate virus and decrease endogenous RNase activity. When checked for compatibility, a low concentration of TCEP-EDTA reagents was shown to protect RNA from digestion by dampening RNase activity, as well as lowering viscosity and mucin gel formation in the saliva sample for easier processing. Lastly, the DISCOVER protocol on saliva sample was tested using FDA’s Emergency Use Authorization guidelines. RT-qPCR was performed to find the detection limit of the workflow in terms of specificity and quantification of the sample concentration. The workflow found to have a 40 cp/uL of lowest viral concentration in sample for detection without any significant false positive signal. In the protocol, human RNase P gene was used as an internal control Academia Letters, August 2021 ©2021 by the authors — Open Access — Distributed under CC BY 4.0 Corresponding Author: Ananna Arna, arnaananna17@gmail.com Citation: Arna, A., Mardon, A. (2021). Literature Review: A potential detection method for SARS-CoV-2. Academia Letters, Article 2778. https://doi.org/10.20935/AL2778. 2 and multiplexed with SARS-Cov-2 detection to confirm inactivation of virus and RNase in the sample. By combining the speed and sensitivity of LAMP and specificity of Cas13-mediated probe, DISCOVER workflow achieves a rapid detection protocol with attomolar sensitivity avoiding false-positives. The readout is ready in under five minutes due to rapid amplification of rLAMP substrates. Further, the ability of DISCOVER to accommodate both DNA and RNA virus makes it a strong candidate for point-of-care diagnostics to detect a wide range of pathogens. As the internal process control is in place for readout confirmation, multicolor detection using different fluorescents and higher-order multiplexing with other respiratory viruses integrated in a single test can also be achieved by making certain adjustments to the workflow. Developing a saliva-based assay has many advantages; collecting saliva sample does not require any medical personnel which minimizes contact between patients and healthcare providers and decrease PPE demand, and create opportunities for frequent testing for community surveillance and increase patient compliance. This is a reliable method as the viral load present in saliva and nasopharyngeal swabs are very similar. Additionally, saliva samples have been reported to have 97% concordance to RT-qPCR based nasopharyngeal swab detection. Compared to other detection methods like, DETECTR and STOPCovid12, DISCOVER does not rely on RNA extraction kits. They use readily available reagents and chelators to manipulate the sample (lysis). Even any CRISPR-based assay requires RNA extraction and purification for efficient readouts which DISCOVER achieves with its simple chemical extraction and heat inactivation of the sample. Usage of extraction -free saliva sample, accommodating sample detection of multiple origins (nose, saliva, etc.), target multiplexing to detect a human gene control or other related pathogens, and most importantly highly specific and sensitive readouts with rapid kinetics while avoiding false positives makes DISCOVER a good candidate for point-of-care diagnostics. The use of point-of-care would help in community surveillance and lower spreading of the virus. Further, simple programmability and fast readout without any professionals’ assistance would incentivize frequent testing. However, their use of RT-qPCR to validate their result seems time-consuming and counterproductive. Also, back and forth conversion between DNA and RNA may introduce genetic variations that may affect the findings. An amplification-free viral detection may be a good approach for the future diagnostics. Academia Letters, August 2021 ©2021 by the authors — Open Access — Distributed under CC BY 4.0 Corresponding Author: Ananna Arna, arnaananna17@gmail.com Citation: Arna, A., Mardon, A. (2021). Literature Review: A potential detection method for SARS-CoV-2. Academia Letters, Article 2778. https://doi.org/10.20935/AL2778. 3