CRISPR/Cas12a-Based Diagnostic Platform Accurately Detects Nocardia farcinica Targeting a Novel Species-Specific Gene

Abstract

Under the COVID-19 pandemic background, nucleic acid detection has become the gold standard to rapidly diagnose the infectious disease. A rapid, low cost, reliable nucleic acid detection platform will be the key to control next potential pandemic. In this study, a nucleic acid detection platform, which combined CRISPR/Cas12a-based detection with loop-mediated isothermal amplification (LAMP), was developed and termed CRISPR-CLA. In the CRISPR-CLA system, LAMP preamplification was employed, and CRISPR/Cas12a-based detection was used to monitor the preamplicons. The forward inner primer (FIP) was engineered with a protospacer adjacent motif (PAM) site TTTA of Cas12a effector at the linker region; thus, the CRISPR-CLA platform can detect any sequence as long as the primer design meets the requirement of LAMP. To demonstrate the validity of the CRISPR-CLA system, it was applied for the molecular diagnosis of nocardiosis caused by Nocardia farcinica (N. farcinica). A highly conserved and species-specific gene pbr1 of N. farcinica, which was first reported in this study, was used as the target of detection. A set of LAMP primers targeting a fragment of pbr1 of the N. farcinica reference strain IFM 10152 was designed according to the principle of CRISPR-CLA. Three CRISPR RNAs (crRNAs) with different lengths were designed, and the most efficient crRNA was screened out. Additionally, three single-strand DNA (ssDNA) probes were tested to further optimize the detection system. As a result, the N. farcinica CRISPR-CLA assay was established, and the whole detection process, including DNA extraction (20 min), LAMP preamplification (70°C, 40 min), and CRISPR/Cas12a-mediated detection (37°C, 8 min), can be completed within 70 min. A fluorescence reader (for fluorescence CRISPR-CLA) or a lateral flow biosensor (for lateral-flow CRISPR-CLA) can be the media of the result readout. Up to 132 strains were used to examine the specificity of N. farcinica CRISPR-CLA assay, and no cross-reaction was observed with non-N. farcinica templates. The limit of detection (LoD) of the N. farcinica CRISPR-CLA assay was 100 fg double-strand DNA per reaction. N. farcinica was detected accurately in 41 sputum specimens using the N. farcinica CRISPR-CLA assay, which showed higher specificity than a real-time qPCR method. Hence, the N. farcinica CRISPR-CLA assay is a rapid, economic and accurate method to diagnose N. farcinica infection.

Keywords: CRISPR; CRISPR-CLA; Cas12a; Nocardia farcinica; accurate diagnosis; nucleic acid detection; pbr1.

Figure 1

Schematic illustration of the workflow and the reaction principle of the CRISPR-CLA assay. (A) CRISPR-CLA workflow. The whole reaction of the CRISPR-CLA assay can be completed within 70 min. (B) Primers and crRNA design of the N. farcinica CRISPR-CLA assay. The pbr1 gene of N. farcinica IFM 10152 was used to design LAMP primers. The nucleotide sequence of the sense strand of part of the pbr1 gene (from 904 to 1182 bp) is shown. Right-pointing arrows and left-pointing arrows indicate sense and complementary sequences that were used, respectively. The inserted PAM site (TTTA) is highlighted in red and on a yellow background. The crRNA was boxed. (C) Principle of the PAM site inserted by LAMP preamplification. The amplification products are indicated by the corresponding green arrows. (D) Principle of the CRISPR-CLA system. Step one: the PAM site was introduced into the target amplicons by the modified FIP, with the target amplicons exponentially increased by LAMP. Step two: the Cas12a/crRNA complex was stably formed. Step three: Cas12a was activated after the target amplicons recognized and bound with the Cas12a/crRNA complex, and the single-strand DNA (ssDNA) reporter molecules were cleaved by activated Cas12a.

Figure 2

Principle of results readout of CRISPR-CLA assay by LFB. (A) Construction of LFB. (B) Principle of LFB for visualization of CRISPR-CLA products. (C) Practical application of LFB. Both the control line and the test line showed red bands in the positive result, and only the control line showed a red band in the negative result.

Figure 3

Optimal crRNA screening for the N. farcinica CRISPR-CLA assay. The crRNA3 was screened out, because of the highest fluorescence value. PC, positive control; NC, negative control; BC, blank control. (n = 3 technical replicates, two-tailed Student t-test; ns, no significance; ****p < 0.0001; bars represent mean ± s.e.m.).

Figure 4

Optimal ssDNA probe and work concentration screening for the N. farcinica CRISPR-CLA assay. The 11 nt ssDNA probe with 250 nM work concentration was screened out, because of the highest fluorescence value. nt, nucleotide; NC, negative control; BC, blank control. (n = 3 technical replicates; bars represent mean ± s.e.m.).

Figure 5

Sensitivity of the N. farcinica CRISPR-CLA assay. (A) Sensitivity of the fluorescence N. farcinica CRISPR-CLA assay. One hundred fg dsDNA was determined as the LoD of the fluorescence N. farcinica CRISPR-CLA assay. (n = 3 technical replicates, two-tailed Student t-test; ns, no significance; ***p < 0.001; ****p < 0.0001; bars represent mean ± s.e.m.) (B) Sensitivity of the lateral-flow N. farcinica CRISPR-CLA assay. One hundred fg dsDNA was determined as the LoD of the lateral-flow N. farcinica CRISPR-CLA assay. NC, negative control. BC, blank control.

Figure 6

Feasibility of the N. farcinica CRISPR-CLA assay using clinical samples. (A) The fluorescence N. farcinica CRISPR-CLA assay results. Sample S1 to S21 showed positive results and sample N1 to N20 showed negative. (n = 3 technical replicates; bars represent mean ± s.e.m.) (B) The lateral-flow N. farcinica CRISPR-CLA assay results. Sample S1 to S21 showed positive results and sample N1 to N20 showed negative. S1-S20, simulated positive samples; S21, positive sample; N1-N20, negative samples; PC, positive control; NC, negative control; BC, blank control.