Department of Microbiology, Faculty of Science, Mahidol University, Bangkok 10400, Thailand.
Drug Research Unit for Malaria, Faculty of Tropical Medicine, Mahidol University, Bangkok 10400, Thailand.
Acta Trop. 2020 Dec;212:105651. doi: 10.1016/j.actatropica.2020.105651. Epub 2020 Aug 4.
In Thailand, asymptomatic leishmaniasis cases have continuously increased, especially among patients with HIV at risk to develop further symptoms of cutaneous and visceral leishmaniasis. Thus, earlier diagnosis using a simple, sensitive and reliable diagnostic tool is needed because populations at risk mostly reside in rural communities where only basic laboratory equipment is available for health care services. In this present study, a closed tube loop mediated isothermal amplification (LAMP) was developed using a piece of parafilm placed between the dye and LAMP reaction mixture to form semi-layer that partially secured SYBR green I from spilling during amplification. No post-amplification preparation was required and accidental spill of the dye during LAMP amplification was prevented. The result could be visually interpreted under visible and UV lights after dye spinning down. The semi-layer modification of a closed tube LAMP showed successful amplification of Leishmania DNA with clear interpretation using both color and fluorescence dyes when observing by the naked eye. The sensitivity and specificity were as high as 94.4 and 96.9%, respectively whereas detection limits were 10 parasites/mL being ten fold more sensitive than other related studies. This user-friendly inexpensive approach is affordable and suitable for empowering leishmaniasis surveillance without the need of expensive devices in all levels of hospitals, including health services, as well as fieldwork, especially in low income countries.
在泰国，无症状利什曼病病例持续增加，尤其是在有 HIV 风险的患者中，他们可能会进一步出现皮肤和内脏利什曼病的症状。因此，需要使用简单、敏感和可靠的诊断工具进行早期诊断，因为高危人群大多居住在农村社区，那里的医疗保健服务只能提供基本的实验室设备。在本研究中，使用放置在染料和 LAMP 反应混合物之间的一段 Parafilm 形成半层，开发了一种封闭式管环介导等温扩增（LAMP），从而部分防止 SYBR 绿 I 在扩增过程中溢出。不需要扩增后的准备，并且可以防止在 LAMP 扩增过程中染料意外溢出。染料旋转沉淀后，可在可见光和紫外线下通过肉眼观察来对结果进行直观解释。封闭式管 LAMP 的半层修饰显示可以成功扩增利什曼原虫 DNA，通过肉眼观察到颜色和荧光染料的清晰解释。灵敏度和特异性分别高达 94.4%和 96.9%，而检测限为 10 个寄生虫/mL，比其他相关研究敏感十倍。这种易于使用且价格低廉的方法在各级医院，包括卫生服务以及现场工作中都具有成本效益，并且适合于增强利什曼病监测，而无需昂贵的设备，尤其是在低收入国家。
