权益分类	功能权益	普通用户	{{item.name}}会员
{{category.name}}	{{benefitItem.name}}

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

疟疾流行区简便、特异的疟疾DNA诊断方法的建立

基本信息

批准号：
11557183
负责人：
WATAYA Yusuke
金额：
$ 8.06万
依托单位：
OKAYAMA UNIVERSITY
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
1999
资助国家：
日本
起止时间：
1999 至 2001
项目状态：
已结题

项目摘要

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.

我们开发了一种比色测定法“微量滴定板杂交”，用于检测人血液中的疟疾寄生虫，无需苯酚提取 DNA，其中通过聚合酶链式反应 (PCR) 扩增的目标 DNA 序列（18S 小亚基核糖体 RNA 基因）与固定在微量滴定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、卵形疟原虫和三日疟原虫）和卵形疟原虫的一种变种，每种寄生虫特异性微量滴定板均在日本进行了输入性疟疾测试。我们通过手指穿刺采集了327名无症状献血者的血样，他们几乎都是在东京大学医学科学研究所接受过体检的。 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%），三日疟原虫阳性3例（0.9%），阴性101例。我们的方法提供了一种快速、灵敏的检测方法，因此可用于大规模筛查或评估流行地区的疟疾控制方案。此外，我们开发了供疟疾流行地区使用的人类疟原虫快速诊断系统，环介导等温扩增（LAMP），该方法简单，不需要PCR设备，检测时间约为30分钟。我们正在开发用于检测流行地区疟疾寄生虫的 LAMP。