Liang Jiayin, Sui Xintian, Xu Yan, Zheng Xiangqun, Tan Lu
Agro-Environmental Protection Institute, Ministry of Agriculture and Rural Affairs, No. 31 Fukang Road, Nankai District, Tianjin 300191, China.
Key Laboratory of Rural Toilet and Sewage Treatment Technology, Ministry of Agriculture and Rural Affairs, No. 31 Fukang Road, Nankai District, Tianjin 300191, China.
Microorganisms. 2025 May 21;13(5):1166. doi: 10.3390/microorganisms13051166.
Urban sewage, aquaculture wastewater, and medical wastewater are significant reservoirs and transmission sources of . Rapid detection of is crucial for effectively reducing the risk of disease transmission and safeguarding public health. Differentiating viable from inactivated cells presents significant challenges, affecting the accurate assessment of pathogen risks. Moreover, current detection methods face several limitations, including lengthy detection periods, high costs, and limited applicability, underscoring the need for rapid, sensitive, and visual detection diagnostic approaches. In this study, we combined propidium monoazide (PMA) with recombinase polymerase amplification (RPA) and clustered regularly spaced short palindromic repeats (CRISPR)/Cas12a systems to develop a rapid detection system for viable targeting the Y gene. DNA of viable was amplified and visually detected within 60 min and dead cells were effectively excluded. We assessed the specificity and sensitivity of the PMA-RPA-CRISPR/Cas12a assay. The results showed that the assay had a high level of specificity, with no reactions observed with other pathogens. The application of PMA has no effect on the sensitivity of RPA-CRISPR/Cas12a technology and the visibility of the fluorescence reporting system. We successfully detected viable in wastewater with a minimum detection limit of 10 CFU/mL. In summary, the PMA-RPA-CRISPR/Cas12a system developed in this study allows for the rapid and visual detection of viable in wastewater at concentrations as low as 10 CFU/mL. By integrating PMA with the RPA-CRISPR/Cas12a technology, this system offers valuable technical support for the efficient, sensitive, and clear detection of viable in wastewater.
城市污水、水产养殖废水和医疗废水是……的重要储存库和传播源。快速检测……对于有效降低疾病传播风险和保障公众健康至关重要。区分活细胞与死细胞存在重大挑战，影响对病原体风险的准确评估。此外，当前的检测方法面临诸多局限性，包括检测周期长、成本高和适用性有限，这凸显了对快速、灵敏和可视化检测诊断方法的需求。在本研究中，我们将单叠氮碘化丙啶（PMA）与重组酶聚合酶扩增（RPA）以及成簇规律间隔短回文重复序列（CRISPR）/Cas12a系统相结合，开发了一种针对Y基因的活……快速检测系统。活……的DNA在60分钟内被扩增并可视化检测，死细胞被有效排除。我们评估了PMA-RPA-CRISPR/Cas12a检测方法的特异性和灵敏度。结果表明，该检测方法具有高度特异性，与其他病原体未发生反应。PMA的应用对RPA-CRISPR/Cas12a技术的灵敏度和荧光报告系统的可视性没有影响。我们成功检测到废水中的活……，最低检测限为10 CFU/mL。总之，本研究开发的PMA-RPA-CRISPR/Cas12a系统能够快速、可视化地检测废水中低至10 CFU/mL浓度的活……。通过将PMA与RPA-CRISPR/Cas12a技术相结合，该系统为高效、灵敏和清晰地检测废水中的活……提供了有价值的技术支持。
