Detection assay of polymyxin resistance coding gene based on CRISPR/Cas13a system.

Introduction: Polymyxins are reserved as an ultimate defense against multidrug-resistant bacteria. The emergence of the polymyxin resistance gene poses a potential risk for the treatment of severe infections caused by Gram-negative bacteria. Timely detection and monitoring the gene are essential for guiding anti-infective therapy and controlling the spread of polymyxin resistance. Quantitative real-time PCR (qPCR) is one of the common methods for detecting resistance genes. However, qPCR has equipment dependency, and is not feasible in primary healthcare settings. Currently, there remains a lack of a highly sensitive and portable method for detecting the gene.

Methods: We established and optimized detection assays of the gene based on CRISPR/Cas13a system and lateral flow strips. The detection method was preliminarily evaluated using clinical isolates from , compared with qPCR.

Results: The method for detecting the gene based on the CRISPR/Cas13a system and lateral flow strips was established, with a detection limit of 100 copies/mL. This method demonstrated high analytical specificity, with no cross-reactivity detected in non- and non-resistant strains. Among 36 clinical isolates, the method identified 31 strains as positive for the gene, and had a 100% concordance rate with the results of qPCR.

Conclusions: We established a detection method for the polymyxin resistance gene based on the CRISPR/Cas13a system. This method enables visual readouts without instruments, making it potentially applicable to primary healthcare settings and field surveillance.