Ultrasensitive CRISPR/Cas12a-Based System for Detection of BlaOXA-1 Gene in Antibiotic-Resistant Microorganisms

Abstract

The blaOXA-1 gene encodes an oxacillin-hydrolyzing beta-lactamase of extended-spectrum beta-lactamase (ESBL)-producing microorganisms. The blaOXA-1 gene is found in the resistomes of some Enterobacteriaceae, Morganellaceae, Pasteurellaceae, Moraxellaceae, Aeromonadaceae, Pseudomonadaceae, Yersiniaceae, and Vibrionaceae. Most ESBL detection methods, including those to detect OXA-1-producing microorganisms, are time-consuming, and require specialized equipment and qualified personnel. Here, we report a new CRISPR(Clustered Regularly Interspaced Short Palindromic Repeats)/Cas12a-based detection assay coupled with polymerase chain reaction (PCR) to sensitively detect OXA-1-bearing microorganisms. The PCR-coupled CRISPR/Cas12a-based fluorescence assay includes (i) a pre-amplification step and (ii) a nucleic acid detection step. The pre-amplification step is based on a commonly used PCR, and the detection step is based on the CRISPR/Cas12a property to nonspecifically hydrolyze single-stranded DNA fluorescent reporter molecules. The pre-amplification step takes 65 min, and the detection step is shortened and takes only 5 min. The developed assay can easily detect single (1.25) copies of the blaOXA-1 gene in a reaction and is efficient not only in the detection of a blaOXA-1 model matrix but also in the detection of blaOXA-1-positive microorganisms. We hope that our assay has the potential to improve the monitoring of OXA-1-producing microorganisms and therefore contribute to mitigating the deadly global threat of antibiotic-resistant microorganisms.