Development of simple and specific DNA diagnostic method of Malaria in Malaria endemic areas

我们建立了一种不经酚抽提DNA的比色法--“微量滴定板杂交法”，即用聚合酶链反应（PCR）扩增的靶DNA序列（18 S小亚基核糖体RNA基因）与固定在微量滴定孔上的种特异性探针杂交。结合在孔上的PCR产物通过生物素-链霉亲和素系统和随后的显色反应以类似于酶免疫测定的方式可视化。该测定系统使我们能够检测和鉴定四种人类疟疾寄生虫（恶性疟原虫、间日疟原虫）。卵圆疟原虫和三日疟原虫）和卵圆疟原虫的一种变体，在日本对每种寄生虫特异性微量滴定板进行输入性疟疾检测。我们从327名无症状献血者中通过手指穿刺获得血液样本，他们几乎都在东京大学医学科学研究所进行了医学检查。327例标本中，恶性疟原虫阳性118例（36.1%），间日疟原虫阳性83例（25.4%），卵圆疟原虫阳性16例（4.9%），疟疾疟原虫阳性3例（0.9%），阴性101例。该方法具有快速、灵敏的特点，可用于疟疾流行区的大规模筛查和疟疾防治效果的评估，并建立了适用于疟疾流行区的人疟原虫快速诊断系统--环介导等温扩增（LAMP）方法，该方法操作简单，不需要PCR设备，检测时间约为30 min。我们正在开发用于在疟疾流行地区检测疟疾寄生虫的LAMP。

We have developed a colorimetric assay, "microtiter plate-hybridization", for the detection of malaria parasites in human blood without phenol extraction of DNA, in which the target DNA sequences (18S small subunit ribosomal RNA gene) amplified by polymerase chain reaction (PCR) are hybridized with the species-specific probes immobilized on a microtiter well. The PCR products bound on a well are visualized by the biotin-streptavidin system and the following chromogenic reaction in a manner similar to that of an enzyme immunoassay. This assay system allowed us to detect and identify the four species of human malaria parasites (Plasmodium falciparum, P. vivax. P. ovale and P. malariae) and one variant of P. ovale, each parasite-specific microtiter plates were tested for imported malaria in Japan. We obtained blood samples by finger puncture from 327 asymptomatic donors, almost they had medical examination at the Institute of Medical Science, The University of Tokyo. Among the 327 samples, 118 (36.1%) were P. falciparum-positive, 83 (25.4%) were P. vivax-positive, 16 (4.9%) were P. ovale-positive, 3 (0.9%) were P. malariae-positive and negative were 101 cases. Our method provides a rapid and sensitive assay, and thus may be useful for large scale screening or assessment of malaria control programs in endemic areas.Furthermore, we developed of quick diagnostic system of human malaria parasites for the use of malaria endemic areas, loop-mediated isothermal amplification (LAMP), this method is simple, PCR equipment is not required and detection time is about 30 minutes. We are developing the LAMP for detection of malaria parasites in endemic areas.