Cas12a/Guide RNA-Based Platforms for Rapidly and Accurately Identifying Staphylococcus aureus and Methicillin-Resistant S. aureus

• Published in: Microbiology spectrum 2023-03, Vol.11 (2), p.e0487022-e0487022
• Main Authors: Cao, Xiaoying, Chang, Yanbin, Tao, Chunqing, Chen, Sen, Lin, Qiuxia, Ling, Chao, Huang, Shifeng, Zhang, Hengshu
• Format: Article
• Language: English
• Subjects: Bacteriology; Research Article
• ISSN: 2165-0497; 2165-0497
• DOI: 10.1128/spectrum.04870-22

In order to ensure the prevention and control of methicillin-resistant Staphylococcus aureus (MRSA) infection, rapid and accurate detection of pathogens and their resistance phenotypes is a must. Therefore, this study aimed to develop a fast and precise nucleic acid detection platform for identifying S. aureus and MRSA. We initially constructed a CRISPR-Cas12a detection system by designing single guide RNAs (sgRNAs) specifically targeting the thermonuclease ( ) and genes. To increase the sensitivity of the CRISPR-Cas12a system, we incorporated PCR, loop-mediated isothermal amplification (LAMP), and recombinase polymerase amplification (RPA). Subsequently, we compared the sensitivity and specificity of the three amplification methods paired with the CRISPR-Cas12a system. Finally, the clinical performance of the methods was tested by analyzing the fluorescence readout of 111 clinical isolates. In order to visualize the results, lateral-flow test strip technology, which enables point-of-care testing, was also utilized. After comparing the sensitivity and specificity of three different methods, we determined that the -LAMP-Cas12a and -LAMP-Cas12a methods were the optimal detection methods. The -LAMP-Cas12a platform showed a limit of detection (LOD) of 10 aM (~6 copies μL ), while the -LAMP-Cas12a platform demonstrated a LOD of 1 aM (~1 copy μL ). The LOD of both platforms reached 4 × 10 fg/μL of genomic DNA. Critical evaluation of their efficiencies on 111 clinical bacterial isolates showed that they were 100% specific and 100% sensitive with both the fluorescence readout and the lateral-flow readout. Total detection time for the present assay was approximately 80 min (based on fluorescence readout) or 85 min (based on strip readout). These results indicated that the -LAMP-Cas12a and -LAMP-Cas12a platforms are promising tools for the rapid and accurate identification of S. aureus and MRSA. The spread of methicillin-resistant Staphylococcus aureus (MRSA) poses a major threat to global health. Isothermal amplification combined with the -cleavage activity of Cas12a has been exploited to generate diagnostic platforms for pathogen detection. Here, we describe the design and clinical evaluation of two highly sensitive and specific platforms, -LAMP-Cas12a and -LAMP-Cas12a, for the detection of S. aureus and MRSA in 111 clinical bacterial isolates. With a limit of detection (LOD) of 4 × 10 fg/μL of genomic DNA and a turnaround time of 80 to 85 min, the present assay w