Journal: Frontiers in Microbiology

Article Title: One-pot MCDA-CRISPR-Cas-based detection platform for point-of-care testing of severe acute respiratory syndrome coronavirus 2

doi: 10.3389/fmicb.2024.1503356

Figure Lengend Snippet: The primers, reporter molecule and gRNAs used in this study.

Article Snippet: Plasmids containing the target sequences of ORF1ab and N genes from each virus genome (SARS-CoV-2, SARS-CoV, and MERS-CoV) were synthesized by Sangon Biotech Co., Ltd. (Shanghai, China).

Techniques:

Journal: Frontiers in Microbiology

Article Title: One-pot MCDA-CRISPR-Cas-based detection platform for point-of-care testing of severe acute respiratory syndrome coronavirus 2

doi: 10.3389/fmicb.2024.1503356

Figure Lengend Snippet: Optimal reaction conditions of the COVID-19 MCTOP assay. Error bars represent the means ± standard error of means (SEM) from three replicates. (A) Optimal reaction temperature for ORF1ab gene detection. The reactions were performed under temperatures ranging from 62°C to 66°C at 1°C intervals. (B) Optimal primer concentration for ORF1ab gene detection. Serial volumes of MCDA primer premixture (2.2 μL and 1.1 to 2.3 μL with 0.2 μL intervals) were used to prepare the reaction mixtures. Primer concentrations for serial volumes were as follows. 1.1 μL (0.2 μM each of F1 and F2, 0.4 μM each of C1, C2, R1, R2, D1 and D2, 0.8 μM each of CP1 and CP2), 1.3 μL (0.24 μM each of F1 and F2, 0.47 μM each of C1, C2, R1, R2, D1 and D2, 0.95 μM each of CP1 and CP2), 1.5 μL (0.27 μM each of F1 and F2, 0.55 μM each of C1, C2, R1, R2, D1 and D2, 1.09 μM each of CP1 and CP2), 1.7 μL (0.31 μM each of F1 and F2, 0.62 μM each of C1, C2, R1, R2, D1 and D2, 1.24 μM each of CP1 and CP2), 1.9 μL (0.35 μM each of F1 and F2, 0.69 μM each of C1, C2, R1, R2, D1 and D2, 1.38 μM each of CP1 and CP2), 2.1 μL (0.38 μM each of F1 and F2, 0.76 μM each of C1, C2, R1, R2, D1 and D2, 1.53 μM each of CP1 and CP2), 2.2 μL (0.4 μM each of F1 and F2, 0.8 μM each of C1, C2, R1, R2, D1 and D2, 1.6 μM each of CP1 and CP2), 2.3 μL (0.42 μM each of F1 and F2, 0.84 μM each of C1, C2, R1, R2, D1 and D2, 1.67 μM each of CP1 and CP2). (C) Optimal reaction temperature for N gene detection. The reactions were performed under temperatures ranging from 62°C to 66°C at 1°C intervals. (D) Optimal primer concentration for N gene detection. Serial volumes of MCDA primer premixture (2.0 μL and 1.1 to 2.1 μL with 0.2 μL intervals) were used to prepare the reaction mixtures. Primer concentrations for serial volumes were as follows. 1.1 μL (0.22 μM each of F1 and F2, 0.44 μM each of C1, C2, R1, R2, D1 and D2, 0.88 μM each of CP1 and CP2), 1.3 μL (0.26 μM each of F1 and F2, 0.52 μM each of C1, C2, R1, R2, D1 and D2, 1.04 μM each of CP1 and CP2), 1.5 μL (0.3 μM each of F1 and F2, 0.6 μM each of C1, C2, R1, R2, D1 and D2, 1.2 μM each of CP1 and CP2), 1.7 μL (0.34 μM each of F1 and F2, 0.68 μM each of C1, C2, R1, R2, D1 and D2, 1.36 μM each of CP1 and CP2), 1.9 μL (0.38 μM each of F1 and F2, 0.76 μM each of C1, C2, R1, R2, D1 and D2, 1.52 μM each of CP1 and CP2), 2.0 μL (0.4 μM each of F1 and F2, 0.8 μM each of C1, C2, R1, R2, D1 and D2, 1.6 μM each of CP1 and CP2), 2.1 μL (0.42 μM each of F1 and F2, 0.84 μM each of C1, C2, R1, R2, D1 and D2, 1.68 μM each of CP1 and CP2). Ultrapure water served as negative control (NC).

Article Snippet: Plasmids containing the target sequences of ORF1ab and N genes from each virus genome (SARS-CoV-2, SARS-CoV, and MERS-CoV) were synthesized by Sangon Biotech Co., Ltd. (Shanghai, China).

Techniques: Concentration Assay, Negative Control

Journal: Frontiers in Microbiology

Article Title: One-pot MCDA-CRISPR-Cas-based detection platform for point-of-care testing of severe acute respiratory syndrome coronavirus 2

doi: 10.3389/fmicb.2024.1503356

Figure Lengend Snippet: Sensitivity and specificity analysis of COVID-19 MCTOP assay. (A,C) COVID-19 MCTOP results were reported by real-time fluorescence (A) for ORF1ab-MCTOP result, and (C) for N-MCTOP result. Error bars represent the means ± standard error of means (SEM) from three replicates. (B,D) COVID-19 results were reported by lateral flow biosensor (B) for ORF1ab-MCTOP result, and (D) for NP-MCTOP result. Signals (A) /Biosensors (B) 1–8 represented the results of corresponding ORF1ab-plasmid levels of SARS-CoV-2 from 1 × 10 4 to 1 × 10 0 copies, and 1 × 10 4 copies ORF1ab-plasmid of SARS-CoV and MERS-CoV, respectively, and ultrapure water served as negative control (NC). Signals (C) /Biosensors (D) 1–8 represented the results of corresponding N-plasmid levels of SARS-CoV-2 from 1 × 10 4 to 1 × 10 0 copies, and 1 × 10 4 copies N-plasmid of SARS-CoV and MERS-CoV respectively, and ultrapure water served as negative control (NC). Control line (CL), Test line (TL).

Article Snippet: Plasmids containing the target sequences of ORF1ab and N genes from each virus genome (SARS-CoV-2, SARS-CoV, and MERS-CoV) were synthesized by Sangon Biotech Co., Ltd. (Shanghai, China).

Techniques: Fluorescence, Plasmid Preparation, Negative Control, Control

Journal: Frontiers in Microbiology

Article Title: One-pot MCDA-CRISPR-Cas-based detection platform for point-of-care testing of severe acute respiratory syndrome coronavirus 2

doi: 10.3389/fmicb.2024.1503356

Figure Lengend Snippet: The effects of reaction inhibitors on COVID-19 MCTOP assay. 1 × 10 4 copies of SARS-CoV-2 plasmid were used in the tolerance test. (A) For ORF1ab-MCTOP result, (B) for N-MCTOP result. Ultrapure water served as negative control (NC). Error bars represent the means ± standard error of means (SEM) from at three replicates.

Article Snippet: Plasmids containing the target sequences of ORF1ab and N genes from each virus genome (SARS-CoV-2, SARS-CoV, and MERS-CoV) were synthesized by Sangon Biotech Co., Ltd. (Shanghai, China).

Techniques: Plasmid Preparation, Negative Control