Jiang Yayun, Chen Zongyao, Liu Xiao, Xin Qi, Wang Dengchao, Ji Caixia, Li Youwei, Mai Gang
Digestive Diseases Center, Deyang People's Hospital, Chengdu University of Traditional Chinese Medicine, Deyang, Sichuan, China.
Department of Clinical Laboratory, Deyang People's Hospital, Chengdu University of Traditional Chinese Medicine, Deyang, Sichuan, China.
Front Microbiol. 2025 Jul 16;16:1592153. doi: 10.3389/fmicb.2025.1592153. eCollection 2025.
Methicillin-resistant (MRSA), a widely drug-resistant bacterium, poses a significant threat to global health. Current culturing and nucleic acid detection methods are time-consuming and require complex instruments, which do not meet the detection needs. Herein, we developed a rapid and visual MRSA detection method (MCFHCR) using ssDNA-functionalized magnetic beads as a trigger chain combined with the trans-cleavage activity of the Cas12a protein and fluorescence signal amplification of the hybridization chain reaction (HCR). MCFHCR is a signal-off platform for the detection of MRSA. In the absence of DNA targets, the trans-cleavage activity of Cas12a is inactivated, allowing HCR to proceed and form long double-stranded DNA, resulting in an increased fluorescent signal. In the presence of the DNA targets, the trans-cleavage activity of CRISPR/Cas12a is activated to cleave the trigger strand, failing HCR and leading to a decrease in the fluorescence signal. Combined with RPA, MCFHCR was completed within 35 min, achieving a limit of detection (LOD) of five copies/μL for DNA and 8 CFU/mL for MRSA. In detecting clinical strains, MCFHCR demonstrated comparable performance to qPCR and drug sensitivity testing. Therefore, with its simple, rapid operation and convenient signal acquisition, MCFHCR shows significant practical applicability in detecting MRSA.
耐甲氧西林金黄色葡萄球菌（MRSA）是一种广泛耐药的细菌，对全球健康构成重大威胁。目前的培养和核酸检测方法耗时且需要复杂仪器，无法满足检测需求。在此，我们开发了一种快速可视化的MRSA检测方法（MCFHCR），该方法使用单链DNA功能化磁珠作为触发链，结合Cas12a蛋白的反式切割活性和杂交链反应（HCR）的荧光信号放大。MCFHCR是一种用于检测MRSA的信号关闭平台。在没有DNA靶标的情况下，Cas12a的反式切割活性失活，使得HCR能够进行并形成长双链DNA，导致荧光信号增强。在存在DNA靶标的情况下，CRISPR/Cas12a的反式切割活性被激活以切割触发链，导致HCR失败并导致荧光信号降低。结合重组酶聚合酶扩增（RPA），MCFHCR在35分钟内完成，DNA的检测限（LOD）达到5拷贝/μL，MRSA的检测限达到8 CFU/mL。在检测临床菌株时，MCFHCR表现出与定量聚合酶链反应（qPCR）和药敏试验相当的性能。因此，凭借其简单、快速的操作和便捷的信号采集，MCFHCR在检测MRSA方面显示出显著的实际应用价值。
