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

我们开发了一种比色法，“微滴板杂交”，用于检测人类血液中的疟疾寄生虫，无需苯酚提取DNA，其中通过聚合酶链反应（PCR）扩增的目标DNA序列（18S小亚基核糖体RNA基因）与固定在微滴孔上的物种特异性探针杂交。结合在孔上的PCR产物通过生物素-链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。该检测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）。对日本输入性疟疾的每个寄生虫特异性微滴板进行了检测。我们对327名无症状献血者进行了手指穿刺采血，他们几乎都在东京大学医学研究所接受了医学检查。327份样本中，恶性疟原虫阳性118例（36.1%），间日疟原虫阳性83例（25.4%），卵形疟原虫阳性16例（4.9%），疟疾疟原虫阳性3例（0.9%），阴性101例。本方法提供了一种快速、灵敏的检测方法，可用于疟疾流行地区疟疾控制方案的大规模筛选或评估。此外，我们开发了用于疟疾流行地区的人疟原虫快速诊断系统——环介导等温扩增（LAMP），该方法操作简单，不需要PCR设备，检测时间约为30分钟。我们正在开发LAMP，用于在流行地区检测疟疾寄生虫。

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.