Fig. 1: Implementation of CARMEN using a microfluidic system improves sensitivity and speed. | Nature Medicine

Fig. 1: Implementation of CARMEN using a microfluidic system improves sensitivity and speed.

From: Multiplexed CRISPR-based microfluidic platform for clinical testing of respiratory viruses and identification of SARS-CoV-2 variants

Fig. 1

a, Schematic of CARMEN v.1 (top) and mCARMEN (bottom) workflows. b, Heatmap showing mCARMEN fluorescent data across 21 human respiratory viruses (synthetic DNA fragments and corresponding viral Cas13 crRNAs) at 1 h post-reaction initiation, which were serially diluted from 103 to 101 copies μl−1 and amplified using 2 separate primer pools. All samples were background-subtracted from the no target control (NTC)-noMg negative control. c, Concordance between CARMEN v.1 and mCARMEN from b. Blue: targets at 103 copies μl−1; green: targets at 102 copies μl−1; red: targets at 101 copies μl−1. d, Fluorescence kinetics of amplified SARS-CoV-2 DNA gene fragments from 104–101 copies μl−1 at 0, 1 and 3 h post-reaction initiation. Blue: mCARMEN; red: CARMEN v.1. e, A 21-human respiratory virus panel was tested on clinical specimens from 6 SARS-CoV-2-positive, 4 SARS-CoV-2-negative nasopharyngeal (NP) swabs and 8 FLUAV-positive specimens, collected before December 2019, and 5 NTCs. The heatmap shows fluorescent signals from SARS-CoV-2 crRNA, FLUAV crRNA and no crRNA control. Blue: mCARMEN at 1 h post-reaction initiation; red: CARMEN v.1 at 3 h post-reaction initiation. f, Concordance of mCARMEN and NGS on 58 suspected respiratory virus-infected patient specimens collected before December 2019 shown as a bar graph; overall concordance is shown as a confusion matrix. Black: detected by both mCARMEN and NGS; blue: detected by mCARMEN only; green: detected by NGS only. mCARMEN values are shown as the normalized fluorescence signal (FAM/ROX) (FAM signal divided by the signal for the passive reference dye, ROX, 1 h). CARMEN v.1 values are shown as the raw fluorescence signal (FAM). NTC-extract: no target control taken through extraction, cDNA synthesis, amplification, and detection; NTC-cDNA: no target control taken through cDNA synthesis, amplification and detection; NTC-amp: no target control taken through amplification and detection; NTC-det: no target control taken through detection; NTC-noMg: no target control expected to have no fluorescent signal due to lack on Mg2+ needed to activate Cas13.

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