Chen Hua-Wei, Weissenberger Giulia, Ching Wei-Mei
Naval Medical Research Center, 503 Robert Grant Avenue, Silver Spring, MD 20910.
Mil Med. 2016 May;181(5 Suppl):227-31. doi: 10.7205/MILMED-D-15-00149.
Leptospirosis is considered to be the most widespread zoonosis. This worldwide emerging infectious disease is caused by the pathogenic species belonging to the genus Leptospira. Polymerase chain reaction (PCR)-based diagnostic assays have been developed for detecting Leptospira DNA in cell cultures and clinical samples. Because PCR requires specialized equipment and extensive end-user training, it is not suitable for routine work in resource-limited areas. We have developed a loop-mediated isothermal amplification (LAMP) assay to detect the presence of Leptospira in patient, animal and environmental samples using lyophilized reagents at a single temperature of around 63°C with a heating block. The sensitivity of this LAMP assay is very similar to the PCR method. The amplified DNA products can be visualized with the naked eyes using hydroxy naphthol blue or detected by the fluorescence signal of SYBR green dye in the reaction when an ultraviolet lamp or compact fluorescence tube scanner is used. This LAMP assay is simple, rapid, and can be performed with a water bath or heating block. The lyophilized LAMP reagents are stable for 3 months when stored at 4°C and 1 month when stored at 25°C, respectively. It is ideal for resource-limited settings where leptospirosis is endemic.
钩端螺旋体病被认为是最广泛传播的人畜共患病。这种全球范围内新出现的传染病由钩端螺旋体属的致病菌种引起。基于聚合酶链反应（PCR）的诊断检测方法已被开发用于检测细胞培养物和临床样本中的钩端螺旋体DNA。由于PCR需要专门的设备和大量的终端用户培训，因此它不适合资源有限地区的常规工作。我们开发了一种环介导等温扩增（LAMP）检测方法，使用冻干试剂在约63°C的单一温度下通过加热块检测患者、动物和环境样本中钩端螺旋体的存在。这种LAMP检测方法的灵敏度与PCR方法非常相似。当使用紫外灯或紧凑型荧光管扫描仪时，扩增的DNA产物可以用羟基萘酚蓝肉眼观察或通过反应中SYBR绿染料的荧光信号检测。这种LAMP检测方法简单、快速，可以用水浴或加热块进行。冻干的LAMP试剂分别在4°C储存时稳定3个月，在25°C储存时稳定1个月。它非常适合钩端螺旋体病流行的资源有限地区。
