Development of Lyophilized Loop-Mediated Isothermal Amplification Reagents for the Detection of Leptospira
Development of Lyophilized Loop-Mediated Isothermal Amplification Reagents for the Detection of Leptospira
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DOI：
10.7205/milmed-d-15-00149
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发表时间：
2016-05-01
影响因子：
1.2
通讯作者：
Ching, Wei-Mei
中科院分区：
文献类型：
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作者：
Chen, Hua-Wei;Weissenberger, Giulia;Ching, Wei-Mei
钩端螺旋体病被认为是最广泛的人畜共患病。这种世界范围内出现的传染病是由属于钩端螺旋体属的致病性物种引起的。已经开发了基于聚合酶链反应（PCR）的诊断测定用于检测细胞培养物和临床样品中的钩端螺旋体DNA。由于PCR需要专门的设备和广泛的最终用户培训，因此不适合在资源有限的地区开展日常工作。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摄氏度的单一温度下用加热块检测患者、动物和环境样品中钩端螺旋体的存在。该LAMP检测的灵敏度与PCR方法非常相似。用羟基萘酚蓝可使扩增的DNA产物肉眼可见，用紫外灯或小型荧光管扫描仪可通过反应中SYBR绿色染料的荧光信号检测扩增产物。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 ℃下储存3个月和在25 ℃下储存1个月时稳定。它是资源有限的地方性钩端螺旋体病的理想选择。
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 degrees 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 degrees C and 1 month when stored at 25 degrees C, respectively. It is ideal for resource-limited settings where leptospirosis is endemic.