Cas12a/Guide RNA-Based Platforms for Rapidly and Accurately Identifying Staphylococcus aureus and Methicillin-Resistant S. aureus
作者全名："Cao, Xiaoying; Chang, Yanbin; Tao, Chunqing; Chen, Sen; Lin, Qiuxia; Ling, Chao; Huang, Shifeng; Zhang, Hengshu"
作者地址："[Cao, Xiaoying; Tao, Chunqing; Chen, Sen; Zhang, Hengshu] Chongqing Med Univ, Dept Plast & Burn Surg, Affiliated Hosp 1, Chongqing, Peoples R China; [Chang, Yanbin] Gansu Prov Hosp, Dept Clin Lab, Lanzhou, Peoples R China; [Lin, Qiuxia; Ling, Chao; Huang, Shifeng] Chongqing Med Univ, Dept Clin Lab, Affiliated Hosp 1, Chongqing, Peoples R China"
通信作者："Zhang, HS (通讯作者)，Chongqing Med Univ, Dept Plast & Burn Surg, Affiliated Hosp 1, Chongqing, Peoples R China.; Huang, SF (通讯作者)，Chongqing Med Univ, Dept Clin Lab, Affiliated Hosp 1, Chongqing, Peoples R China."
文献类型：Article; Early Access
关键词：Staphylococcus aureus; methicillin-resistant Staphylococcus aureus; CRISPR-Cas12a; isothermal amplification; polymerase chain reaction; loop-mediated isothermal amplification; recombinase polymerase amplification; lateral-flow strips
摘要："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 (nuc) and mecA 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 nuc-LAMP-Cas12a and mecA-LAMP-Cas12a methods were the optimal detection methods. The nuc-LAMP-Cas12a platform showed a limit of detection (LOD) of 10 aM (similar to 6 copies mu L-1), while the mecA-LAMP-Cas12a platform demonstrated a LOD of 1 aM (similar to 1 copy mu L-1). The LOD of both platforms reached 4 x 10(3) fg/mu L 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 nuc-LAMP-Cas12a and mecA-LAMP-Cas12a platforms are promising tools for the rapid and accurate identification of S. aureus and MRSA. IMPORTANCE The spread of methicillin-resistant Staphylococcus aureus (MRSA) poses a major threat to global health. Isothermal amplification combined with the trans-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, nuc-LAMP-Cas12a and mecA-LAMP-Cas12a, for the detection of S. aureus and MRSA in 111 clinical bacterial isolates. With a limit of detection (LOD) of 4 x 10(3) fg/mu L of genomic DNA and a turnaround time of 80 to 85 min, the present assay was 100% specific and 100% sensitive using either fluorescence or the lateral-flow readout. The present assay promises clinical application for rapid and accurate identification of S. aureus and MRSA in limited-resource settings or at the point of care. Beyond S. aureus and MRSA, similar CRISPR diagnostic platforms will find widespread use in the detection of various infectious diseases, malignancies, pharmacogenetics, food contamination, and gene mutations."
基金机构："Industry-University-Research Innovation Fund of Universities in China [2021JH049]; Hospital Fund of Gansu Provincial Clinical Research Center for Laboratory Medicine [21GSSYC-41]; Natural Science Foundation of Chongqing, China [cstc2020jcyj-msxmX0519]"
基金资助正文："ACKNOWLEDGMENTS This study was supported by the Industry-University-Research Innovation Fund of Universities in China (no. 2021JH049), the Hospital Fund of Gansu Provincial Clinical Research Center for Laboratory Medicine (no. 21GSSYC-41), and the Natural Science Foundation of Chongqing, China (no. cstc2020jcyj-msxmX0519). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. All authors contributed to data analysis and drafting and revising of the paper and agreed to be accountable for all aspects of the work. We report no conflicts of interest in this work."