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.

Accurate, Sensitive, and Rapid Detection of Pseudomonas aeruginosa Based on CRISPR/Cas12b with One Fluid-Handling Step

“…Furthermore, five samples showed positive results by qPCR, whereas they were negative by our CRISPR-based approach and culture. Although we cannot exclude the possibility that the limit of detection (LOD) of qPCR is lower than our method and the five positive qPCR samples corresponded to much lower inoculums, in our previous study ( 13 ) qPCR showed a similar LOD to a CRISPR-based assay but generated some false-positive results.…”

“…The use of guide RNA (gRNA) makes CRISPR/Cas-based detection extremely specific. However, most CRISPR-based detection platforms require two separate fluid-handling steps, namely, preamplification and CRISPR-based detection steps, resulting in multiple operations with the potential for contamination ( 12 – 14 ). More recently, a one-pot, one-step CRISPR-based nucleic acid detection technology, named CRISPR-top, has been successfully devised and applied to the diagnosis of coronavirus disease 2019 ( 15 ).…”

Development of a recombinase-aided amplification assay for rapid detection of the human pathogen Nocardia farcinca

“…To improve the feasibility and practicality of this diagnostic method in the eld, time and temperature were optimized in our preliminary study, and the nal reaction conditions selected were consistent with the results described by Wang et al [26]. Moreover, in terms of sensitivity, we used the results of recombinant plasmid (10 copies/μl) and pure bacterial nucleic acid (10 fg) compared with qPCR, CRISPR-CLA, CRISPR-CPA methods (10 2 copies/μl, 100 fg, 1 pg), respectively, and the results showed that the sensitivity was higher than that of other methods [19,20,24].…”

“…In this study, a novel assay was established for the detection of N. farcinica. The RT-RAA assay was performed at a constant temperature of 39°C for 20 minutes, which provides a signi cantly simpli ed operation and saved turnaround time compared to the cases with existing methods for detecting N. farcinica [19,24]. Furthermore, the assay protected the reaction from contamination (a one-step closed tube reaction) and was not dependent on complex thermal cyclers and skilled personnel, indicating that it can be applied in primary hospitals and communities with portable equipment.…”

“…According to the principles of RT-RAA primer pair and probe design, the lengths of primers and probe were required to be 30 to 35 bp and 46 to 52 bp, respectively [18]. The primers and probe used for the RT-RAA assay were designed to target the conserved region of the pbr1 gene (GenBank accession number: AP006618.1), with encoding a PQQ-binding-like β-propeller repeat protein (GenBank accession number: WP_011210687.1) [19]. The CmSuite (v 8.0) software was used to analyze the potential for primer-dimer and hairpin formation.…”