Fu Xupeng, Huang Lintao, Chen Ying, Pi Menglu, Ma Lin, Cai Hu, Wang Xuehao, Chen Zhihao, Shi Hang, Yang Wenhui, Zhang Fulai, Zhang Yang, Jiang Huili, Zhou Zeming, Wang Changhe, Huang Rong, Zhang Juan, Cheng Donghao, Wu Li-An, Qian Airong, Tian Ye
Xi'an Key Laboratory of Special Medicine and Health Engineering, Research Center for Special Medicine and Health Systems Engineering, Lab for Bone Metabolism, Key Lab for Space Biosciences and Biotechnology, NPU-UAB Joint Laboratory for Bone Metabolism, School of Life Sciences, Northwestern Polytechnical University, Xi'an 710072, China.
Shaanxi Eye Hospital, Xian People's Hospital (Xi'an Fourth Hospital), Affiliated People's Hospital of Northwest University, Xi'an 710004, China.
Biomed Opt Express. 2025 Feb 10;16(3):949-964. doi: 10.1364/BOE.549870. eCollection 2025 Mar 1.
Pathogenic microorganisms responsible for infectious diseases pose a significant global threat to human health. Existing detection methods, such as qPCR and ELISA, fail to simultaneously meet the requirements for high sensitivity, high specificity, and rapid detection. This study presents an innovative approach for the rapid, specific, and highly sensitive detection of pathogenic microorganisms, particularly O157:H7 ( O157:H7) and varicella-zoster virus (VZV), by combining recombinase polymerase amplification (RPA) with terahertz time-domain spectroscopy (THz-TDS). The qualitative and quantitative detection method for pathogenic microorganisms was developed and evaluated. The stable and efficient RPA reaction systems were established to specifically amplify the key conserved genes of these pathogens. Then the RPA products were purified, and enriched with MBs. The absorbance spectra were obtained using THz-TDS technology. The linear range of the RPA-THz for detecting O157:H7 was 0.55 to 5.5 × 10 pg/mL, while for VZV, it was 0.75 to 7.5 × 10 pg/mL. The limit of detection (LOD) for bacteria and viruses was 0.226 pg/mL and 0.528 pg/mL, respectively, demonstrating better sensitivity than the qPCR (550 pg/mL and 750 pg/mL, respectively). In addition, the whole amplification and detection process was completed in about 35 minutes. Compared to traditional pathogen detection techniques, the primary advantage of the developed RPA-THz method exhibited high accuracy, good reproducibility, and short detection times, enabling non-ionizing, label-free analysis for rapid detection with high sensitivity and specificity of pathogenic microorganisms. This study provides a theoretical foundation and practical demonstration for the fast and precise detection of pathogenic microorganisms. It establishes a crucial research basis for further development of RPA-THz sensors, advancing technological progress in the field of food safety, medical diagnostics, environmental monitoring, and public health.
导致传染病的致病微生物对全球人类健康构成重大威胁。现有的检测方法，如定量聚合酶链反应（qPCR）和酶联免疫吸附测定（ELISA），无法同时满足高灵敏度、高特异性和快速检测的要求。本研究提出了一种创新方法，通过将重组酶聚合酶扩增（RPA）与太赫兹时域光谱（THz-TDS）相结合，对致病微生物，特别是肠出血性大肠杆菌O157:H7（O157:H7）和水痘带状疱疹病毒（VZV）进行快速、特异性和高灵敏度检测。开发并评估了致病微生物的定性和定量检测方法。建立了稳定高效的RPA反应体系，以特异性扩增这些病原体的关键保守基因。然后对RPA产物进行纯化，并用磁珠进行富集。使用太赫兹时域光谱技术获得吸收光谱。RPA-THz检测O157:H7的线性范围为0.55至5.5×10 pg/mL，而检测VZV的线性范围为0.75至7.5×10 pg/mL。细菌和病毒的检测限分别为0.226 pg/mL和0.528 pg/mL，显示出比qPCR（分别为550 pg/mL和750 pg/mL）更好的灵敏度。此外，整个扩增和检测过程在约35分钟内完成。与传统病原体检测技术相比，所开发的RPA-THz方法的主要优点是具有高准确性、良好的重现性和短检测时间，能够进行非电离无标记分析，以高灵敏度和特异性快速检测致病微生物。本研究为致病微生物的快速精确检测提供了理论基础和实践示范。它为进一步开发RPA-THz传感器建立了关键的研究基础，推动了食品安全、医学诊断、环境监测和公共卫生领域的技术进步。
