Mussa Ebthag A M, Al-Maleki Anis Rageh, Aljaberi Musheer A, Alsalahi Abdulsamad, Desa Mohd Nasir Mohd, Jasni Azmiza Syawani, Ramanoon Siti Zubaidah, Abdallah Atiyeh M, Hamat Rukman Awang
Department of Medical Microbiology, Faculty of Medicine and Health Sciences, Universiti Putra Malaysia, Serdang 43400, Selangor, Malaysia.
Department of Medical Microbiology, Faculty of Medicine, Universiti Malaya, Kuala Lumpur 50603, Malaysia.
Diagnostics (Basel). 2025 May 12;15(10):1213. doi: 10.3390/diagnostics15101213.
The increasing prevalence of antimicrobial-resistant (AMR) bacteria in humans, animals, and the environment underscores the necessity for a rapid, sensitive, and specific method to identify resistance genes. : This study aims to develop a reliable detection tool for identifying the tetracycline-resistant gene (M) in species using a real-time loop-mediated isothermal amplification (RT-LAMP) assay. Real-time visualization through a turbidimeter enabled precise estimation of time-to-positivity for gene detection. : Six primers were designed using PrimerExplorer v.5, and the assay was optimized across different temperatures and incubation times. Validation was conducted by testing 52 (M)-positive clinical enterococci isolates and spiking urine samples from a healthy volunteer and a cow with (M)-positive species. The (M) gene was detected as early as 33 min, with optimal amplification occurring within 60 min at 60 °C. The assay demonstrated 100% specificity with the established primers. The sigmoidal graphs were corroborated with visual confirmation methods, including a green color change (visible to the naked eye), green fluorescence (under UV light), and a 200 bp PCR product observed via agarose gel electrophoresis. Notably, the (M) RT-LAMP assay exhibited a detection limit of 0.001 pg/μL, significantly surpassing conventional PCR, which had a detection limit of 0.1 pg/μL. : This rapid, cost-effective, highly sensitive, and specific (M) RT-LAMP assay holds significant promise as a surveillance tool for antimicrobial resistance monitoring within a One Health framework, particularly in low-resource countries.
人类、动物和环境中耐抗菌药物（AMR）细菌的日益流行凸显了采用快速、灵敏且特异的方法来鉴定耐药基因的必要性。本研究旨在开发一种可靠的检测工具，利用实时环介导等温扩增（RT-LAMP）分析法鉴定某物种中的四环素耐药基因（M）。通过浊度仪进行实时可视化能够精确估计基因检测的阳性时间。使用PrimerExplorer v.5设计了六种引物，并在不同温度和孵育时间下对该分析法进行了优化。通过检测52株（M）阳性临床肠球菌分离株以及向来自一名健康志愿者和一头带有（M）阳性某物种的奶牛的尿液样本中加样进行验证。最早在33分钟时检测到（M）基因，在60℃下60分钟内出现最佳扩增。该分析法使用既定引物显示出100%的特异性。S形曲线通过视觉确认方法得到了证实，包括颜色变为绿色（肉眼可见）、绿色荧光（在紫外光下）以及通过琼脂糖凝胶电泳观察到200 bp的PCR产物。值得注意的是，（M）RT-LAMP分析法的检测限为0.001 pg/μL，显著超过传统PCR的检测限0.1 pg/μL。这种快速、经济高效、高度灵敏且特异的（M）RT-LAMP分析法作为一种在“同一健康”框架内监测抗菌药物耐药性的监测工具具有巨大潜力，特别是在资源匮乏的国家。
