Li Tianming, Chen Shiying, Chen Xiaoying, Yang Shuang, Huang Haiqian, Xu Jingsong, Liu Yu, Zhang Junheng, Cao Liou, Kang Zhihua, Li Min, Wang Hua
Department of Laboratory Medicine, Renji Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai, China.
Jiading District Central Hospital Affiliated Shanghai University of Medicine & Health Sciences, Shanghai, China.
Anal Chim Acta. 2025 Oct 8;1370:344390. doi: 10.1016/j.aca.2025.344390. Epub 2025 Jul 8.
Group B Streptococcus (GBS) is a major cause of perinatal infections. Prenatal screening is critical to prevent maternal and neonatal GBS infections, reduce adverse outcomes, and guide clinical interventions. While bacterial culture is the gold standard, it is time-consuming and delays decision-making. Rapid molecular tests like PCR are sensitive and specific but require expensive equipment and skilled personnel. Point-of-care tests (e.g., Xpert GBS LB) offer speed and sensitivity but remain costly and underutilized. This study aims to develop a portable CRISPR/Cas13a-based device for rapid, on-site GBS detection.
A palm-sized CRISPR detection platform, PalmCS, was developed. PalmCS includes two key components: an integrated tube for nucleic acid extraction, gene amplification, and CRISPR-based reaction, and a multifunctional device for thermal regulation, fluorescence detection, and automatic result interpretation. The sealed plug needle valve controls fluid flow for sample input and result output. By optimizing crRNA selection, Cas13a/crRNA ribonucleoprotein (RNP) complex concentrations, and isothermal recombinase polymerase amplification primers, we established a one-pot CRISPR/Cas13a-RPA method for GBS detection. Results showed that 3 M guanidinium salt combined with 5 % Tween 20 achieved the highest extraction efficiency. The system extracted nucleic acids from samples in 5 min at room temperature, demonstrating its potential for rapid diagnostics. PalmCS was validated using 40 clinical samples, achieving a limit of detection (LOD) of 20 copies/reaction, 97.5 % sensitivity, and 100 % specificity.
This study used a one-step nucleic acid extraction combined with a closed one-pot RPA-CRISPR reaction for rapid, sensitive, and specific GBS detection. This provides a new platform for prenatal screening in pregnant women. In resource-limited settings with emergency deliveries, PalmCS enables on-site GBS testing, allowing antibiotic administration during delivery for positive cases to significantly reduce neonatal infection risk.
B族链球菌（GBS）是围产期感染的主要原因。产前筛查对于预防孕产妇和新生儿GBS感染、减少不良结局以及指导临床干预至关重要。虽然细菌培养是金标准，但它耗时且会延迟决策。像PCR这样的快速分子检测灵敏且特异，但需要昂贵的设备和技术人员。即时检测（例如Xpert GBS LB）速度快且灵敏，但成本仍然很高且未得到充分利用。本研究旨在开发一种基于CRISPR/Cas13a的便携式设备，用于快速、现场GBS检测。
开发了一种手掌大小的CRISPR检测平台PalmCS。PalmCS包括两个关键组件：一个用于核酸提取、基因扩增和基于CRISPR反应的集成管，以及一个用于温度调节、荧光检测和自动结果解读的多功能设备。密封的插塞针阀控制流体流动以进行样品输入和结果输出。通过优化crRNA选择、Cas13a/crRNA核糖核蛋白（RNP）复合物浓度和等温重组酶聚合酶扩增引物，我们建立了一种用于GBS检测的一锅法CRISPR/Cas13a-RPA方法。结果表明，3M胍盐与5%吐温20结合可实现最高提取效率。该系统在室温下5分钟内从样品中提取核酸，证明了其在快速诊断方面的潜力。使用40份临床样品对PalmCS进行了验证，检测限（LOD）为20拷贝/反应，灵敏度为97.5%，特异性为100%。
本研究采用一步核酸提取结合封闭的一锅法RPA-CRISPR反应进行快速、灵敏和特异的GBS检测。这为孕妇产前筛查提供了一个新平台。在有紧急分娩的资源有限环境中，PalmCS能够进行现场GBS检测，使阳性病例在分娩期间能够使用抗生素，从而显著降低新生儿感染风险。
