COVID-19 active case findings based on self-collected saliva samples with CRISPR-Cas12a detection

Abstract

COVID-19 is an infectious disease caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus affecting the world population. Early detection has become one of the most successful strategies to alleviate the epidemic and pandemic of this contagious coronavirus. Surveillance testing programs have been initiated in many countries worldwide to prevent the outbreak of COVID-19. In this study, we demonstrated that our previously established clustered regularly interspaced short palindromic repeats (CRISPR)-Cas12a-based assay could detect variants of concern during 2021 in Thailand, including Alpha, Beta, and Delta strains as well as Omicron strain in early 2022. In combination with the newly designed saliva collection funnel, we established a safe, simple, economical, and efficient self-collection protocol for the COVID-19 screening process. We successfully utilized the assay in an active case finding with a total number of 578 asymptomatic participants to detect the SARS-CoV-2 in saliva samples. We finally demonstrated that the validation and evaluation in a large-scale setting could provide valuable information and elaborate the practicality of the test in real-world settings. Our optimized protocol yielded effective results with high sensitivity, specificity, and diagnostic accuracy (96.86%). In addition, this study demonstrates COVID-19 active case findings in low-resource settings, which would be feasible and attractive for surveillance and outbreak prevention in the future.

Keywords: COVID-19; CRISPR-Cas12a; RPA; active case findings; qRT-PCR; saliva samples.

Figure 1.

The representative result obtained from CRISPR-Cas12a assay targeting S gene for three SARS-CoV-2 variants of concern detection. The assay was tested against the RNA extracted from patients infected with different strains of SARS-CoV-2 (verified by Sanger sequencing). The Ct values of tested samples range between 16 and 30. The positive control is the RNA of the SARS-CoV-2 wild-type strain.

Figure 2.

Saliva collection vial and funnel. (A) Design of the assembled device and (B) self-collected saliva collection package.

Figure 3.

Flow chart demonstrating the experimental plan.

Figure 4.

The representative of positive and negative results from CRISPR-Cas12a-based assay targeting SARS-CoV-2 S gene tested with RNA extracted from saliva specimens. The Ct values of positive representative samples range between 23 and 29.

Figure 5.

The results of CRISPR-Cas12a-based assay tested with a total of 22 samples (labeled 1 to 22) of SARS-CoV-2 (Omicron variant) positive nasopharyngeal swab samples. The representative samples contain different mean Ct values ranging from 20 to 28 based on qRT-PCR analysis. (A color version of this figure is available in the online journal.)