Cas12a/Guide RNA-Based Platform for Rapidly and Accurately Detecting bla_KPC Gene in Carbapenem-Resistant Enterobacterales

Abstract

Purpose

Accurate detection and identification of pathogens and their associated resistance mechanisms are essential prerequisites for implementing precision medicine in the management of Carbapenem-resistant Enterobacterales (CRE). Among the various resistance mechanisms, the production of KPC carbapenemase is the most prevalent worldwide. Consequently, this study aims to develop a convenient and precise nucleic acid detection platform specifically for the bla_KPC gene.

Methods

The initial phase of our research methodology involved developing a CRISPR/Cas12a detection framework, which was achieved by designing highly specific single-guide RNAs (sgRNAs) targeting the bla_KPC gene. To enhance the sensitivity of this system, we incorporated three distinct amplification techniques—polymerase chain reaction (PCR), loop-mediated isothermal amplification (LAMP), and recombinase polymerase amplification (RPA)—into the CRISPR/Cas12a framework. Subsequently, we conducted a comparative analysis of the sensitivity and specificity of these three amplification methods when used in combination with the CRISPR/Cas12a system. Additionally, we assessed the clinical applicability of the methodologies by evaluating fluorescence readouts from 80 different clinical isolates. Furthermore, we employed lateral flow assay technology to provide a visual representation of the results, facilitating point-of-care testing.

Results

Following a comparative analysis of the sensitivity and specificity of the three methods, we identified the RPA-Cas12a approach as the optimal detection technique. Our findings demonstrated that the limit of detection (LoD) of the RPA-Cas12a platform was 1 aM (~1 copy/µL) for plasmid DNA and 5 × 10³ fg/µL for genomic DNA. Furthermore, both the sensitivity and specificity of the platform achieved 100% upon validation with 80 clinical isolates.

Conclusion

These findings suggest that the developed RPA-Cas12a platform represents a promising tool for the cost-effective, convenient, and accurate detection of the bla_KPC gene.

Keywords:

• Klebsiella pneumoniae carbapenemase
• CRISPR-Cas12a
• isothermal amplification
• polymerase chain reaction
• loop-mediated isothermal amplification
• recombinase polymerase amplification
• lateral flow strips

Acknowledgments

This study was supported by the Gansu Provincial Hospital Science and Technology Innovation Platform Fund Project (No. ZX-62000001-2021-251) and the Hospital Fund of Gansu Provincial Clinical Research Center for Laboratory Medicine (No. 21JR7RA676). The funders had no role in study design, data collection, and analysis, the decision to publish, or the preparation of the manuscript. We thank the team of Huang Shifeng of the First Affiliated Hospital of Chongqing Medical University for the gift of bacterial strains and their whole genome sequencing results.

Author Contributions

All authors made a significant contribution to the work reported, whether that is in the conception, study design, execution, acquisition of data, analysis, and interpretation, or all these areas; took part in drafting, revising, or critically reviewing the article; gave final approval of the version to be published; have agreed on the journal to which the article has been submitted; and agree to be accountable for all aspects of the work.

Disclosure

The authors report no conflicts of interest in this work, including employment, consultancies, stock ownership, honoraria, paid expert testimony, patent applications/registrations, and grants or other funding.