Zhou Zihan, Pan Lele, Luo Shihua, Ma Jiangmei, Ren Baoyan, Liang Lina, Li Xuebin, Wei Guijiang
Center for Medical Laboratory Science, Affiliated Hospital of Youjiang Medical University for Nationalities, Guangxi, Baise, China.
Key Laboratory of Research on Clinical Molecular Diagnosis for High Incidence Diseases in Western Guangxi of Guangxi Higher Education Institutions, Guangxi, Baise, China.
Front Bioeng Biotechnol. 2025 Aug 11;13:1663915. doi: 10.3389/fbioe.2025.1663915. eCollection 2025.
is a significant pathogen associated with nosocomial infections, predominantly affecting immunocompromised patients, and is linked to high mortality rates. To control infection rates, there is an urgent need for a diagnostic method that is cost-effective, rapid, and user-friendly, meeting the current demand for timely diagnosis.
We have developed a one-tube detection method for UV light unlocking based on RPA-CRISPR/Cas12a technology. This method utilizes the photodegradable chemical group NPOM-dt to chemically modify the crRNA base, preventing it from complementary pairing with the base of the target molecule, thereby temporarily silencing the CRISPR system. After RPA preamplification, the caged modification group on the crRNA was removed with brief irradiation with ultraviolet light to restore the activity of the CRISPR/Cas12a. system.
Our results demonstrated that the detection system achieved a limit of detection as low as 10 copies/μL for target fragments, with no cross-reactivity observed with genomic DNA from six clinically common pathogenic bacteria, showcasing excellent sensitivity and specificity. Additionally, clinical validation was performed using 38 sputum samples. The system successfully identified A. baumannii in sputum specimens, with results consistent with those obtained via conventional PCR.
We have successfully developed a light-controlled one-tube RPA-CRISPR/Cas12a detection system. It simplifies the operation and at the same time greatly reduces the risk of laboratory contamination caused by repeated tube opening, providing a new idea for the development of point-of-care testing (POCT).
是一种与医院感染相关的重要病原体，主要影响免疫功能低下的患者，并与高死亡率相关。为了控制感染率，迫切需要一种经济高效、快速且用户友好的诊断方法，以满足当前对及时诊断的需求。
我们基于RPA-CRISPR/Cas12a技术开发了一种用于紫外线解锁的单管检测方法。该方法利用光可降解化学基团NPOM-dt对crRNA碱基进行化学修饰，防止其与靶分子碱基互补配对，从而暂时使CRISPR系统沉默。在RPA预扩增后，用紫外线短暂照射去除crRNA上的笼状修饰基团，以恢复CRISPR/Cas12a系统的活性。
我们的结果表明，该检测系统对靶片段的检测限低至10拷贝/μL，对六种临床常见病原菌的基因组DNA未观察到交叉反应，展现出优异的灵敏度和特异性。此外，使用38份痰液样本进行了临床验证。该系统成功鉴定出痰液标本中的鲍曼不动杆菌，结果与传统PCR法一致。
我们成功开发了一种光控单管RPA-CRISPR/Cas12a检测系统。它简化了操作，同时大大降低了因反复开盖导致实验室污染的风险，为即时检验（POCT）的发展提供了新思路。
