A CRISPR-Cas12a-based platform for ultrasensitive rapid highly specific detection of Mycobacterium tuberculosis in clinical application

Figure 1. Schematic illustration of the principle of the MTB-MCDA-CRISPR assay. (A) Location and sequences of primers and gRNA used in this study. The MCDA primers are positioned with arrows, and the gRNA is in the box. The right and left arrows represent the meaning and complementary sequences used in this study, respectively. (B) Schematic diagram of MCDA reaction with the modified primer. Primer CP1 is modified by introducing a PAM site (TT) at the linker region. After amplification, the CRISPR-Cas12a recognition site was constructed in the target amplicons. (C) Schematic diagram of CRISPR-Cas12a detection system. When the target DNA is identified, the CRISPR-Cas12a-gRNA complex cuts the single-stranded DNA reporter molecule and releases a fluorescent signal. (D) The entire process of the MTB-MCDA-CRISPR detection system. The MTB-MCDA-CRISPR assay is performed by three closely linked steps: DNA extraction (step 1), MCDA pre-amplification (step 2), CRISPR-Cas12a cleavage, and data reporting (step 3), which can be completed within 60 minutes.

Figure 2. Validation of the MTB-MCDA-CRISPR assay for detection of MTB. The results of the MTB-MCDA-CRISPR assay was assessed by real-time turbidity detection (A), real-time fluorescence detection (B) and visual detection by naked eye under blue light (C). Genomic DNA of MTB strain H37Rv was used as positive control, that of the Mycobaterium cosmeticum was as negative control and distilled water was as blank control.

Figure 3. Specificity verification of the MTB-MCDA-CRISPR detection system. A total of 8 MTB strains (signals/tubes 1-8) and 24 non-MTB strains (signals/tubes 9-32) were tested by the MTB-MCDA-CRISPR assay for specificity evaluation. The produced fluorescent signals were document in a real-time manner by the real-time PCR system (A) and visually interpreted by naked eye under blue light (B).

Figure 4. Sensitivity assessment of the MTB-MCDA-CRISPR detection system. The genomic DNA of MTB strain H37Rv was ten-fold serially diluted from 4 ng to 4 fg per microlitre and used as templates for sensitivity analysis, with distilled water (DW) as blank control. All the detection results were recorded by measuring the real-time turbidity (A), real-time fluorescence intensity (B) and visually interpreted by naked eye under blue light (C). Signals/tubes 1-7 represent the results of the MTB genomic DNA of 4 ng, 400 pg, 40 pg, 4 pg, 400 fg, 40 fg and 4 fg, respectively, and signal/tube 8 represent the result of bank control.

Figure 5. Performance of the MTB-MCDA-CRISPR detection system in clinical samples. A total of 96 samples were tested by the MTB-MCDA-CRISPR assay to validate the clinical feasibility. The generated fluorescence signals were recorded by the real-time PCR platform (A) and visually interpreted by naked eye under blue light (B). Signals/tubes 1-19 present the results of the 19 samples from non-TB patients, and Signals/tubes 20-96 represent the results of the 77 samples from TB patients.

Figure 6. Comparison of the MTB-MCDA-CRISPR assay, Xpert assays and sputum smear microscope test among the clinical sample for both TB and non-TB patients.