Ji Caixia, Ru Liqiang, Han Tiao, Mai Gang, Zheng Laibao, Jiang Yayun
Department of Clinical Laboratory, People's Hospital of Deyang City, Chengdu University of Traditional Chinese Medicine, Deyang, Sichuan, China.
Department of Clinical Laboratory, Jincheng People's Hospital, Jincheng, Shanxi, China.
Microbiol Spectr. 2025 Aug 14:e0139525. doi: 10.1128/spectrum.01395-25.
Group B (GBS) is a significant pathogen that causes perinatal infections, seriously threatening the health of pregnant women and newborns. Prophylactic antibiotic treatment for pregnant women who screen positive for GBS can notably reduce the incidence and fatality of neonatal infections. Herein, we developed a isual nucleic acid method for BS that integrates PA and RISPR/Cas12a in a ne-ube setup, termed VGRCOT. The VGRCOT method achieved one-tube detection by adding the appropriate reagents to the bottom and lid of the EP tube, respectively. By rigorous optimization of ssDNA-FQ reporter concentration, crRNA concentration, RPA reaction time, and CRISPR/Cas12a cleavage time, VGRCOT can exhibit fluorescence under ultraviolet light, enabling visual detection. Under optimal conditions, VGRCOT has a satisfactory selectivity, and the detection limit was determined as 10 copies/reaction. Finally, VGRCOT also showed good performance comparable to qPCR in the actual detection of clinical specimens. Due to its ease of operation and convenient signal acquisition, VGRCOT shows promise for point-of-care testing in reproductive health.IMPORTANCEThis study presents a convenient, sensitive, and accurate visual detection method (VGRCOT) for GBS, combining RPA and CRISPR/Cas12a in a single reaction vessel. Through optimization of experimental conditions, VGRCOT enables detection within 60 min, with a minimum detection limit of 10 copies per reaction. VGRCOT offers several advantages by adding the appropriate reagents to the bottom and lid of the EP tube. The one-tube visualization method effectively prevents aerosol contamination, simplifies procedures, and enables visual detection without complex instruments, making it ideal for resource-limited environments. Additionally, its editable crRNA and the use of commonly available laboratory reagents allow for easy reprogramming to detect various pathogens, supporting scalable and low-cost batch production.
B组链球菌（GBS）是一种导致围产期感染的重要病原体，严重威胁孕妇和新生儿的健康。对GBS筛查呈阳性的孕妇进行预防性抗生素治疗可显著降低新生儿感染的发生率和死亡率。在此，我们开发了一种用于GBS的可视化核酸方法，该方法在新的试管设置中整合了重组酶聚合酶扩增（RPA）和CRISPR/Cas12a，称为VGRCOT。VGRCOT方法通过分别向EP管的底部和盖子中添加适当的试剂实现了一管检测。通过严格优化单链DNA-荧光定量（ssDNA-FQ）报告基因浓度、CRISPR RNA（crRNA）浓度、RPA反应时间和CRISPR/Cas12a切割时间，VGRCOT在紫外光下可呈现荧光，实现可视化检测。在最佳条件下，VGRCOT具有令人满意的选择性，检测限确定为10拷贝/反应。最后，在临床标本的实际检测中，VGRCOT也显示出与定量聚合酶链反应（qPCR）相当的良好性能。由于其操作简便且信号采集方便，VGRCOT在生殖健康的即时检测方面显示出前景。重要性本研究提出了一种用于GBS的便捷、灵敏且准确的可视化检测方法（VGRCOT），该方法在单个反应容器中结合了RPA和CRISPR/Cas12a。通过优化实验条件，VGRCOT能够在60分钟内完成检测，最低检测限为每个反应10拷贝。通过向EP管的底部和盖子中添加适当的试剂，VGRCOT具有多个优点。这种一管可视化方法有效防止了气溶胶污染，简化了操作程序，无需复杂仪器即可进行可视化检测，使其成为资源有限环境的理想选择。此外，其可编辑的crRNA以及使用常见的实验室试剂允许轻松重新编程以检测各种病原体，支持可扩展且低成本的批量生产。
