Detection and Differentiation of SARS-CoV-2, Influenza, and Respiratory Syncytial Viruses by CRISPR

Abstract

SARS-CoV-2, influenza, and respiratory syncytial viruses (RSVs) cause acute respiratory infections with similar symptoms. Since the treatments and outcomes of these infections are different, the early detection and accurate differentiation of the viruses are clinically important for the prevention and treatment of the diseases. We previously demonstrated that clustered regularly interspaced short palindromic repeats (CRISPR) could rapidly and precisely detect SARS-CoV-2. The objective of this study was to develop CRISPR as a test for simultaneously detecting and accurately distinguishing the viruses. The CRISPR assay with an RNA guide against each virus was performed in the reference standards of SARS-CoV-2, influenza A and B, and RSV. The CRISPR assay had a limit of detection of 1–100 copies/µL for specifically detecting SARS-CoV-2, influenza A and B, and RSV without cross-reaction with other respiratory viruses. The validation of the test in nasopharyngeal specimens showed that it had a 90–100% sensitivity and 100% specificity for the detection of SARS-CoV-2, influenza A and B, and RSV. The CRISPR assay could potentially be used for sensitive detection and specific differentiation of the respiratory viruses.

Analyzing siRNA Concentration, Complexation and Stability in Cationic Dendriplexes by Stem-Loop Reverse Transcription-qPCR

“…Due to slight fluorescence signal increases in NTC reactions in the case of randomized siRNA usage ( Figure 3 B,D), LOD and LOQ values were calculated with different formulas depending on the sample type. For dilution series with negative NTC, the following formulas were used: LOD = 3.3 × (σ/s); LOQ = 10 × (σ/s), where σ is the standard deviation of the linear regression and s is the slope of the calibration curve [ 48 , 49 ]. Increase in NTCs (indicated by threshold line crossing) was considered using the approach by Wolfinger et al [ 50 ], defining the LOQ (and also the LOD) as the lowest measured concentration that shows a C t of at least 3.32 lower than the NTC.…”

“…where σ is the standard deviation of the linear regression and s is the slope of the calibration curve [48,49]. Increase in NTCs (indicated by threshold line crossing) was considered using the approach by Wolfinger et al [50], defining the LOQ (and also the LOD) as the lowest measured concentration that shows a C t of at least 3.32 lower than the NTC.…”

“…All dilution series were performed three times in separate experiments (biological triplicates), each with technical triplicates. Limit of detection and limit of quantification were defined as LOD = 3.3 × (standard deviation of linear regression/slope of the regression line) and LOQ = 10 × (standard deviation of linear regression/slope of the regression line)[48,49]. In case of NTC threshold line crossing, LOQ (and LOD) was calculated according to Wolfinger et al[50].…”

An updated review of SARS‐CoV‐2 detection methods in the context of a novel coronavirus pandemic

“…Zhou et al developed simultaneous detection of SARS‐CoV‐2, influenza, and Respiratory Syncytial Viruses (RSV) based on the CRISPR‐Cas12 system. 110 They demonstrated that CRISPR‐Cas12a with specific gRNAs had an LOD of 1 copy/μl for SARS‐CoV‐2 and 100 copies/μl for influenza A and B and RSV, respectively. The CRISPR‐Cas12a test produced 100.0%, 93.8%, 100.0%, and 90.0% sensitivity for SARS‐CoV‐2, influenza A, influenza B, and RSV, respectively, with a specificity of 100%.…”

Profiling Plasma Cytokines by A CRISPR-ELISA Assay for Early Detection of Lung Cancer

“…We have shown that CRISPR-Cas12a can detect HPV, SARS-CoV-2, influenza, and respiratory syncytial viruses, and druggable genomic DNA mutations with a higher sensitivity compared with PCR and DNA sequencing [ 10 , 11 , 12 , 13 ]. We have also developed a microplate-based CRISPR assay to simultaneously and rapidly detect multiple molecular targets [ 14 ]. Lee et al [ 15 ] and Li et al [ 16 ] recently demonstrated that the Cas12a-based trans-cleavage activity of fluorescently quenched (FQ) reporter substrates could improve the sensitivity of ELISA for protein detection.…”