Nzelu Chukwunonso O, Gomez Eduardo A, Cáceres Abraham G, Sakurai Tatsuya, Martini-Robles Luiggi, Uezato Hiroshi, Mimori Tatsuyuki, Katakura Ken, Hashiguchi Yoshihisa, Kato Hirotomo
Laboratory of Parasitology, Department of Disease Control, Graduate School of Veterinary Medicine, Hokkaido University, Hokkaido, Japan.
Servicio Nacional de Erradicacion de la Malaria (SNEM), Ministerio de Salud Publica, Guayaquil, Ecuador.
Acta Trop. 2014 Apr;132:1-6. doi: 10.1016/j.actatropica.2013.12.016. Epub 2014 Jan 2.
Entomological monitoring of Leishmania infection in leishmaniasis endemic areas offers epidemiologic advantages for predicting the risk and expansion of the disease, as well as evaluation of the effectiveness of control programs. In this study, we developed a highly sensitive loop-mediated isothermal amplification (LAMP) method for the mass screening of sand flies for Leishmania infection based on the 18S rRNA gene. The LAMP technique could detect 0.01 parasites, which was more sensitive than classical PCR. The method was robust and could amplify the target DNA within 1h from a crude sand fly template without DNA purification. Amplicon detection could be accomplished by the newly developed colorimetric malachite green (MG)--mediated naked eye visualization. Pre-addition of MG to the LAMP reaction solution did not inhibit amplification efficiency. The field applicability of the colorimetric MG-based LAMP assay was demonstrated with 397 field-caught samples from the endemic areas of Ecuador and eight positive sand flies were detected. The robustness, superior sensitivity, and ability to produce better visual discriminatory reaction products than existing LAMP fluorescence and turbidity assays indicated the field potential usefulness of this new method for surveillance and epidemiological studies of leishmaniasis in developing countries.
在利什曼病流行地区对利什曼原虫感染进行昆虫学监测，对于预测疾病风险和传播范围以及评估控制项目的效果具有流行病学优势。在本研究中，我们基于18S rRNA基因开发了一种高灵敏度的环介导等温扩增（LAMP）方法，用于大规模筛查感染利什曼原虫的白蛉。LAMP技术能够检测到0.01个寄生虫，比传统PCR更灵敏。该方法性能稳定，无需对DNA进行纯化，就能在1小时内从白蛉粗模板中扩增出目标DNA。扩增产物检测可通过新开发的比色孔雀石绿（MG）介导的肉眼可视化完成。在LAMP反应溶液中预先添加MG不会抑制扩增效率。利用来自厄瓜多尔流行地区的397份野外捕获样本对基于比色MG的LAMP检测方法的现场适用性进行了验证，检测到8只阳性白蛉。与现有的LAMP荧光和浊度检测相比，该方法性能稳定、灵敏度高且能产生更好的视觉鉴别反应产物，表明这种新方法在发展中国家利什曼病监测和流行病学研究中具有潜在的现场应用价值。
