Detection of severe acute respiratory syndrome coronavirus 2 and influenza viruses based on CRISPR-Cas12a

Abstract

Due to the common symptoms of COVID-19, patients are similar to influenza-like illness. Therefore, the detection method would be crucial to discriminate between SARS-CoV-2 and influenza virus-infected patients. In this study, CRISPR-Cas12a-based detection was applied for detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), influenza A virus, and influenza B virus which would be a practical and attractive application for screening of patients with COVID-19 and influenza in areas with limited resources. The limit of detection for SARS-CoV-2, influenza A, and influenza B detection was 10, 103, and 103 copies/reaction, respectively. Moreover, the assays yielded no cross-reactivity against other respiratory viruses. The results revealed that the detection of influenza virus and SARS-CoV-2 by using RT-RPA and CRISPR-Cas12a technology reaches 96.23% sensitivity and 100% specificity for SARS-CoV-2 detection. The sensitivity for influenza virus A and B detections was 85.07% and 94.87%, respectively. In addition, the specificity for influenza virus A and B detections was approximately 96%. In conclusion, the RT-RPA with CRISPR-Cas12a assay was an effective method for the screening of influenza viruses and SARS-CoV-2 which could be applied to detect other infectious diseases in the future.

Comparative performance of CRISPR-Cas12a assays for SARS-CoV-2 detection tested with RNA extracted from clinical specimens

“…According to our study ( Table 2 & Supplementary Table 1), the CRISPR-Cas12a assays based on N1 ( Ding et al, 2020 ) and S ( Mayuramart et al, 2020 ) genes yielded diagnostic accuracy (≥ 95%) comparable to the qRT-PCR results). The CRISPR-Cas12a assays within E, N2 ( Broughton et al, 2020 ) and S ( Mayuramart et al, 2020 ) genes provided suitable sensitivity of detection (≥ 95%) while N1 ( Ding et al, 2020 ) and S ( Mayuramart et al, 2020 ) genes provided excellent specificity of detection (100%). Ideally, multiple target genes should be detected by using CRISPR-Cas12a to ensure the most effective SARS-CoV-2 detection.…”

“…The results revealed that detection of the E gene ( Broughton et al, 2020 ) of SARS-CoV-2 based on CRISPR-Cas12a provides the highest sensitivity (97.7%). On the other hand, S ( Mayuramart et al, 2020 ) and N1 ( Ding et al, 2020 ) genes yielded the highest specificity (100%). Interestingly, S ( Mayuramart et al, 2020 ) and N1 ( Ding et al, 2020 ) genes represented acceptable diagnostic accuracy as high as 98.1% and 97.2%, respectively ( Table 2 ).…”

“…On the other hand, S ( Mayuramart et al, 2020 ) and N1 ( Ding et al, 2020 ) genes yielded the highest specificity (100%). Interestingly, S ( Mayuramart et al, 2020 ) and N1 ( Ding et al, 2020 ) genes represented acceptable diagnostic accuracy as high as 98.1% and 97.2%, respectively ( Table 2 ).…”

“…Meanwhile, Ding et al established the method based on a pair of primers and 2 regions of crRNAs within the N gene ( Ding et al, 2020 ). Mayuramart et al reported the assay based on 2 pairs of primers and 2 regions of crRNAs for the S gene ( Mayuramart et al, 2020 ). The current study evaluated the performance of these primers and crRNAs for SARS-CoV-2 detection based on CRISPR-Cas12a assays with RNA extracted from the same clinical specimens.…”

“…The current study evaluated the performance of these primers and crRNAs for SARS-CoV-2 detection based on CRISPR-Cas12a assays with RNA extracted from the same clinical specimens. Although the conditions of RPA, Cas12a reaction, fluorescent detection and signal scoring system used in our study were slightly different from the optimal conditions described in previous reports ( Broughton et al, 2020 ; Ding et al, 2020 ; Mayuramart et al, 2020 ), all the assays were performed under the same conditions and tested against samples from the same cohort to ensure appropriate evaluation of the assays.…”