/pdf/rt-lamp-crispr-cas12-13-based-sars-cov-2-detection-methods-fy0db6j2gv.pdf

RT-LAMP CRISPR-Cas12/13-Based SARS-CoV-2 Detection Methods.

ABSTRACT Introduction Symptomatic testing and asymptomatic screening for SARS-CoV-2 continue to be essential tools for mitigating virus transmission. Though COVID-19 diagnostics initially defaulted to oropharyngeal or nasopharyngeal sampling, the worldwide urgency to expand testing efforts spurred innovative approaches and increased diversity of detection methods. Strengthening innovation and facilitating widespread testing remains critical for global health, especially as additional variants emerge and other mitigation strategies are recalibrated. Areas covered A growing body of evidence reflects the need to expand testing efforts and further investigate the efficiency, sensitivity, and acceptability of saliva samples for SARS-CoV-2 detection. Countries have made pandemic response decisions based on resources, costs, procedures, and regional acceptability – the adoption and integration of saliva-based testing among them. Saliva has demonstrated high sensitivity and specificity while being less invasive relative to nasopharyngeal swabs, securing saliva’s position as a more acceptable sample type. Expert opinion Despite the accessibility and utility of saliva sampling, global implementation remains low compared to swab-based approaches. In some cases, countries have validated saliva-based methods but face challenges with testing implementation or expansion. Here, we review the localities that have demonstrated success with saliva-based SARS-CoV-2 testing approaches and can serve as models for transforming concepts into globally-implemented best practices.

Saliva as a sample type for SARS-CoV-2 detection: implementation successes and opportunities around the globe

Severe acute respiratory syndrome coronavirus -2 (SARS-CoV-2), is a novel Betacoronavirus variant that emerged in December 2019 causing the coronavirus disease 19 (COVID19) pandemic. It is reported that asymptomatic and presymptomatic individuals can transmit the virus and this silent transmission has been a major obstacle for the control of the pandemic. To overcome this obstacle, widespread testing with a rapid turnaround time is required. Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) is currently the golden standard for the diagnosis of COVID19 worldwide. Even though RT-qPCR is an efficient method in terms of sensitivity and specificity, the need for elaborate instrumentation and skilled personnel restricts its widespread use. Restriction of RT-qPCR to a limited number of laboratories makes it further time-consuming. Many approaches are present to address the requirement for a rapid and accurate COVID19 diagnosis. In this review, different CRISPR-based approaches for the point-of-care diagnosis of COVID19 are compared. Among these approaches, CRISPR-FDS on-chip assay is found to be the best option as it is reported to be highly sensitive and specific, has a short turnaround time (15 min), does not need RNA isolation or special tools, and simple to perform. In terms of clinical validation, SHERLOCK, STOPCovid, and DETECTR were the most extensively studied ones and they are also reported to be highly sensitive and specific compared to RT-qPCR.

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CRISPR-based Approaches for the Point-of-Care Diagnosis of COVID19

Early detection of SARS-CoV-2 using molecular techniques is paramount to the fight against COVID-19. Due to its high sensitivity and specificity, RT-qPCR is the “gold standard” method for this purpose. However, its technical requirements, processing time and elevated costs hamper its use towards massive and timely molecular testing for COVID-19 in rural and socioeconomically deprived areas of Latin America. The advent and rapid evolution of CRISPR-Cas technology has boosted the development of new pathogen detection methodologies. Recently, DETECTR -a combination of isothermal RT-LAMP amplification and Cas12a-mediated enzymatic detection-has been successfully validated in the Netherlands and the USA as a rapid and low-cost alternative to RT-qPCR for the detection of SARS-CoV-2 from nasopharyngeal swabs. Here, we evaluated the performance of RCSMS, a locally adapted variant of DETECTR, to ascertain the presence of SARS-CoV-2 in saliva samples from 276 patients in two hospitals in Lima, Peru (current status over a total of 350 samples). We show that a low-cost thermochemical treatment with TCEP/EDTA is sufficient to inactivate viral particles and cellular nucleases in saliva, eliminating the need to extract viral RNA with commercial kits, as well as the cumbersome nasopharyngeal swab procedure and the requirement of biosafety level 2 laboratories for molecular analyses. Our clinical validation shows that RCSMS detects up to 5 viral copies per reaction in 40 min, with sensitivity and specificity of 93.8% and 99.0% in the field, respectively, relative to RT-qPCR. Since CRISPR-Cas biosensors can be easily reprogrammed by using different guide RNA molecules, RCSMS has the potential to be quickly adapted for the detection of new SARS-CoV-2 variants. Notably, estimation of its negative and positive predictive values suggests that RCSMS can be confidently deployed in both high and low prevalence settings. Furthermore, our field study validates the use of lateral flow strips to easily visualize the presence of SARS-CoV-2, which paves the way to deploy RCSMS as a “point of care” test in environments with limited access to state-of-the-art diagnostic laboratories. In sum, RCSMS is a fast, efficient and inexpensive alternative to RT-qPCR for expanding COVID-19 testing capacity in low- and middle-income countries.

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Angélica Quintana Reyes

Papers

Clinical validation of RCSMS: a rapid and sensitive CRISPR-Cas12a test for the molecular detection of SARS-CoV-2 from saliva