Department of Medical Microbiology, Faculty of Medicine and Health Sciences, Universiti Putra Malaysia, Serdang, Selangor, Malaysia.
Department of Biological Sciences and Biotechnology, Faculty of Science and Technology, Universiti Kebangsaan Malaysia, Bangi, Selangor, Malaysia.
PeerJ. 2022 Mar 10;10:e12850. doi: 10.7717/peerj.12850. eCollection 2022.
Leptospirosis is a zoonotic disease caused by bacteria of the genus that affects both humans and animals worldwide. Early detection of the pathogen in humans is crucial for early intervention and control of the progression of the disease to a severe state. It is also vitally important to be able to detect the presence of the pathogen in carrier animals to control the spread of the disease from the environment. Here we developed a simple and rapid loop-mediated isothermal amplification (LAMP) assay targeting the leptospiral gene.
Several reaction conditions of the LAMP reaction were optimized to ensure efficient amplification of the target DNA. The sensitivity of the developed LAMP assay obtained using a pure culture was 2 × 10 copies of genomic DNA per reaction (equivalent to 0.1 ng) for a 40-minute reaction time. No cross-reactions were observed in the LAMP reaction against a series of non-leptospiral bacteria, indicating a specific reaction. The applicability of the LAMP assay was demonstrated on human blood and urine specimens collected from suspected leptospirosis patients and rat kidney specimens collected from suspected leptospirosis outbreak areas and high-risk areas. The developed LAMP assay demonstrated a higher detection rate for leptospiral DNA compared with the polymerase chain reaction (PCR) assay, possibly due to the presence of inhibitory substances, especially in rat kidney specimens, to which the PCR method is more susceptible. The present findings also highlight the importance of urine sample collection from patients for routine monitoring of the disease.
In short, the developed LAMP assay can serve as a feasible alternative tool for the diagnosis of leptospirosis and be used for epidemiological and environmental surveillance of the disease, considering its robustness, rapidity, sensitivity, and specificity, as demonstrated in this study.
钩端螺旋体病是一种由属细菌引起的人畜共患疾病，在全球范围内影响人类和动物。早期发现人类病原体对于早期干预和控制疾病进展至关重要。能够在携带病原体的动物中检测到病原体，以控制疾病从环境中的传播也至关重要。在这里，我们开发了一种针对钩端螺旋体的简单快速环介导等温扩增（LAMP）检测方法。
优化了 LAMP 反应的几种反应条件，以确保目标 DNA 的有效扩增。使用纯培养物获得的开发的 LAMP 检测的灵敏度为每个反应 2×10 拷贝基因组 DNA（相当于 0.1ng），反应时间为 40 分钟。在 LAMP 反应中，未观察到与一系列非钩端螺旋体细菌的交叉反应，表明反应具有特异性。该 LAMP 检测方法在疑似钩端螺旋体病患者的人血和尿液标本以及疑似钩端螺旋体病暴发地区和高风险地区的大鼠肾脏标本中的适用性得到了证明。与聚合酶链反应（PCR）检测相比，开发的 LAMP 检测对钩端螺旋体 DNA 的检测率更高，这可能是由于存在抑制物质，特别是在大鼠肾脏标本中，PCR 方法更易受到抑制。目前的研究结果还强调了从患者中收集尿液样本进行常规疾病监测的重要性。
总之，考虑到该方法的稳健性、快速性、敏感性和特异性，如本研究所示，开发的 LAMP 检测可作为诊断钩端螺旋体病的可行替代工具，并可用于疾病的流行病学和环境监测。
