Deng Zhongliang, Weng Xingyong, Tang Honghua, Zou Tintao, Zhou Xuan, Liu Hangxi, Wen Piaoting, Luo Gemiao, Gan Tian, He Jun
The Affiliated Nanhua Hospital, Department of Clinical Laboratory, Hengyang Medical School, University of South China, Hengyang, Hunan, China.
Department of Public Health Laboratory Sciences, College of Public Health, Hengyang Medical School, University of South China, Hengyang, Hunan, China.
Microbiol Spectr. 2025 Jul;13(7):e0265224. doi: 10.1128/spectrum.02652-24. Epub 2025 Jun 9.
Tuberculosis remains a major global health threat, with existing detection methods often limited by efficiency and resource demands. Our previous PAM-dependent dsDNA Target-activated Cas12f1 Reporter (PDTCTR) fluorescence sensing platform, while effective for PAM-dependent pathogen detection, was constrained by its reliance on specialized fluorescence equipment and lack of visual output, limiting its use in resource-limited settings. To overcome these limitations, we introduce an innovative RPA/CRISPR-Cas12f1_ge4.1 dual-mode system for rapid detection. This system combines engineered Cas12f_ge4.1 with recombinase polymerase amplification (RPA), offering both fluorescent and lateral flow detection. It achieves high sensitivity with detection limits of 10 copies/µL (fluorescence) and 100 copies/µL (lateral flow), alongside 100% specificity. In clinical validation, compared with a commercial qPCR kit, the fluorescent and lateral flow approaches demonstrate sensitivities of 94.52% (69/73, 95% confidence interval [CI]: 85.84%-98.23%) and 90.41% (66/73, 95% CI: 80.67%-95.73%), respectively, while maintaining 100% (40/40, 95% CI: 89.09%-100%) specificity and high concordance (kappa values: 0.924 and 0.878). Detection is completed within 1 h, providing a rapid, sensitive, and specific solution for identification. This dual-mode capability represents a significant advancement in current tuberculosis diagnostics, enabling both sensitive laboratory confirmation and rapid point-of-care screening. Our versatile and efficient method promises to transform tuberculosis diagnostics, particularly in resource-constrained environments.
Tuberculosis (TB) remains a significant global health challenge, demanding rapid and accurate detection for effective management. The innovative RPA/CRISPR-Cas12f1_ge4.1 dual-mode system represents a major advancement in TB diagnostics, offering highly sensitive and specific detection of DNA. This adaptable system, incorporating both fluorescent and lateral flow detection modes, is designed for use in both advanced laboratories and resource-limited settings. Its high performance, rigorously validated through clinical trials, holds the potential to revolutionize TB diagnosis, particularly in high-burden, low-resource areas. By facilitating earlier treatment and enhancing control of TB transmission, this system could significantly contribute to global efforts in combating this persistent public health threat.
结核病仍然是全球主要的健康威胁，现有的检测方法常常受到效率和资源需求的限制。我们之前的基于PAM的双链DNA靶标激活的Cas12f1报告基因（PDTCTR）荧光传感平台，虽然对基于PAM的病原体检测有效，但由于依赖专门的荧光设备且缺乏视觉输出，限制了其在资源有限环境中的应用。为克服这些限制，我们引入了一种创新的RPA/CRISPR-Cas12f1_ge4.1双模式系统用于快速检测。该系统将工程化的Cas12f_ge4.1与重组酶聚合酶扩增（RPA）相结合，提供荧光检测和侧流检测。它实现了高灵敏度，荧光检测限为10拷贝/微升，侧流检测限为100拷贝/微升，特异性为100%。在临床验证中，与商用qPCR试剂盒相比，荧光法和侧流法的灵敏度分别为94.52%（69/73，95%置信区间[CI]：85.84%-98.23%）和90.41%（66/73，95%CI：80.67%-95.73%），同时保持100%（40/40，95%CI：89.09%-100%）的特异性和高一致性（kappa值：0.924和0.878）。检测在1小时内完成，为识别提供了快速、灵敏和特异的解决方案。这种双模式能力代表了当前结核病诊断的重大进展，实现了灵敏的实验室确认和快速的即时检测筛查。我们通用且高效的方法有望改变结核病诊断，特别是在资源受限的环境中。
结核病仍然是一项重大的全球健康挑战，需要快速准确的检测以进行有效管理。创新的RPA/CRISPR-Cas12f1_ge4.1双模式系统代表了结核病诊断的重大进展，提供了对DNA的高度灵敏和特异的检测。这个适应性强的系统结合了荧光和侧流检测模式，设计用于先进实验室和资源有限的环境。其高性能通过临床试验得到严格验证，有潜力彻底改变结核病诊断，特别是在高负担、资源匮乏地区。通过促进早期治疗和加强对结核病传播的控制，该系统可为全球应对这一持续的公共卫生威胁做出重大贡献。
