Infection and Drug Resistance (Jun 2024)
Cas12a/Guide RNA-Based Platform for Rapidly and Accurately Detecting blaKPC Gene in Carbapenem-Resistant Enterobacterales
Abstract
Keke Li,1,* Yaozhou Wu,1,2,* Meng Liu,1 Junwen Yan,1 Lianhua Wei1 1Department of Clinical Laboratory, Gansu Provincial Hospital, Lanzhou, 730000, People’s Republic of China; 2First School of Clinical Medicine, Lanzhou University, Lanzhou, 730000, People’s Republic of China*These authors contributed equally to this workCorrespondence: Lianhua Wei; Junwen Yan, Department of Clinical Laboratory, Gansu Provincial Hospital, No. 204 Donggang West Road, Chengguan District, Lanzhou, 730000, People’s Republic of China, Email [email protected]; [email protected]: 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 blaKPC 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 blaKPC 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 blaKPC gene.Keywords: Klebsiella pneumoniae carbapenemase, CRISPR-Cas12a, isothermal amplification, polymerase chain reaction, loop-mediated isothermal amplification, recombinase polymerase amplification, lateral flow strips
