Zhu Xiaofan, Jiao Jingran, Ni Qun, Yao Li, Zhang Yule, Liu Kaiyong, Huang Lin, Bo Qingli, Qin Panzhu
Department of Nutrition and Food Hygiene, School of Public Health, Anhui Medical University, Hefei, 230032, Anhui, PR China.
School of Food and Biological Engineering, Hefei University of Technology, Hefei, 230009, Anhui, PR China.
Biosens Bioelectron. 2025 Jun 20;287:117718. doi: 10.1016/j.bios.2025.117718.
The integration of recombinase polymerase amplification (RPA) with CRISPR/Cas12a holds great promise for point-of-care assay of bacterial infections, but still confronts challenges such as the input of unstable RNA, dependence on PAM, and multi-step operations. To address these challenges, we here present a tagged primer-based one-pot CRISPR/Cas12a assay, called TOP-CRISPR. Strategically, TOP-CRISPR utilized target gene-induced amplicons to generate crRNA sequences with the help of T7 RNA polymerase, thus eliminating the need for additional crRNA. The resulting Cas12a/crRNA complex were activated by the tag sequences at the end of specific primers rather than by the target amplicons, avoiding dependence on the PAM and disruption of the target amplicon by CRISPR/Cas12a. The ingenious design of tagged primer enables perfect compatibility between RPA and CRISPR/Cas12a system in a closed single tube, which is expected to overcome aerosol contamination caused by multi-step and open-cap operations. Under optimal conditions, the method was able to detect target bacteria down to ∼1 CFU/mL and the entire process took only less than 50 min, which is significantly better than the traditional two-step or one-step methods. By grafting the tag sequence onto different specific primers, bacteria such as Staphylococcus aureus, Escherichia coli O157:H7 and Pseudomonas aeruginosa has been detected, respectively. The practicability and robustness of the method was further validated by real samples. We hope that the method can be developed into a point-of-care detection tool for bacterial infections.
重组酶聚合酶扩增（RPA）与CRISPR/Cas12a的整合在细菌感染的即时检测方面具有巨大潜力，但仍面临诸如不稳定RNA输入、对原间隔相邻基序（PAM）的依赖以及多步骤操作等挑战。为应对这些挑战，我们在此展示一种基于标记引物的单管CRISPR/Cas12a检测方法，称为TOP-CRISPR。从策略上讲，TOP-CRISPR利用靶基因诱导的扩增子在T7 RNA聚合酶的帮助下生成crRNA序列，从而无需额外的crRNA。产生的Cas12a/crRNA复合物由特异性引物末端的标记序列激活，而非靶扩增子，避免了对PAM的依赖以及CRISPR/Cas12a对靶扩增子的破坏。标记引物的巧妙设计使RPA与CRISPR/Cas12a系统在封闭的单管中实现完美兼容，有望克服多步骤和开盖操作导致的气溶胶污染。在最佳条件下，该方法能够检测低至约1 CFU/mL的靶细菌，整个过程仅需不到50分钟，明显优于传统的两步法或一步法。通过将标记序列嫁接到不同的特异性引物上，分别检测了金黄色葡萄球菌、大肠杆菌O157:H7和铜绿假单胞菌等细菌。实际样本进一步验证了该方法的实用性和稳健性。我们希望该方法能够发展成为细菌感染的即时检测工具。
