Aonuma Hiroka, Suzuki Moemi, Iseki Hiroshi, Perera Namal, Nelson Bryce, Igarashi Ikuo, Yagi Takeshi, Kanuka Hirotaka, Fukumoto Shinya
National Research Center for Protozoan Diseases, Obihiro University of Agriculture and Veterinary Medicine, Inada-cho, Obihiro, Hokkaido 080-8555, Japan.
Biochem Biophys Res Commun. 2008 Nov 28;376(4):671-6. doi: 10.1016/j.bbrc.2008.09.061. Epub 2008 Sep 20.
With an aim to develop a quick and simple method to survey pathogen-transmitting vectors, LAMP (loop-mediated isothermal amplification) was applied to the identification of Plasmodium-carrying mosquitoes, specifically a Plasmodium-transmitting experimental model using rodent malaria parasite (Plasmodium berghei) and anopheline mosquitoes (Anopheles stephensi). The detection sensitivity limit of the LAMP reaction amplifying the SPECT2 gene was determined to be 1 x 10(2) purified Plasmodium parasites, estimated to be sufficient for reliable identification of infectious mosquitoes. The robustness of the LAMP reaction was revealed by its ability to detect both Plasmodium oocysts and sporozoites from an "all-in-one" template using whole mosquito bodies. Moreover, LAMP successfully identified an infectious mosquito carrying just a single oocyst in its midgut, a level that can be easily overlooked in conventional microscopic analysis. These observations suggest that LAMP is more reliable and useful for routine diagnosis of vector mosquitoes in regions where vector-borne diseases such as malaria are endemic.
为了开发一种快速简便的方法来调查病原体传播媒介，环介导等温扩增技术（LAMP）被应用于鉴定携带疟原虫的蚊子，特别是使用啮齿动物疟原虫（伯氏疟原虫）和按蚊（斯氏按蚊）的疟原虫传播实验模型。LAMP反应扩增SPECT2基因的检测灵敏度极限被确定为1×10²个纯化的疟原虫，估计这足以可靠地鉴定感染性蚊子。LAMP反应的稳健性通过其使用整个蚊体从“一体化”模板中检测疟原虫卵囊和子孢子的能力得以体现。此外，LAMP成功鉴定出一只中肠仅携带单个卵囊的感染性蚊子，这一水平在传统显微镜分析中很容易被忽视。这些观察结果表明，在疟疾等媒介传播疾病流行的地区，LAMP对于媒介蚊子的常规诊断更可靠且有用。
