| Co-Investigator(Kenkyū-buntansha) |
ARAI Meiji Okayama University, Faculty of Pharmaceutical Sciences, Research Associate, 薬学部, 助手 (30294432)
YAMANE Akio Waknaga Pharmaceutical Co., Ltd., Institute for Biotechnology Research, Chief Re, バイオ研究所, 主任研究員
KIMURA Mikio University of Tokyo, Institute of Medical Science, Research Associate, 医科学研究所, 助手 (90114462)
NEGISHI Kazuo Okayama University, Gene Research Center, Associate Professor, 遺伝子実験施設, 助教授 (70116490)
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| Research Abstract |
We have developed a DNA diagnostic method, "microtiter plate-hybridization (MPH)", in which the PCR-amplified products are captured by species-specific probes immobilized on a microtiter well, and visualized by a subsequent chromogenic reaction. This assay system allows us to detect and identify all four species of human malaria parasites, Plasmodium falciparum, P.vivax, P.ovale, and P.malariae, using the DNA sequence of the 18S rRNA gene as the target. We have evaluated the usefulness of the MPH by examining a number of blood samples from patients with clinical malaria. Our study confirmed that the MPH method is valuable for the management of malaria in clinical practice, particularly in the diagnosis of low-grade parasitemia, species differentiation, prediction of recrudescence, and assessment of treatment. We have found a variant type of P.ovale parasite, which was indistinguishable from typical P.ovale by microscopy but which gave negative results by P.ovale-specific MPH.Sequence analysis of the PCR-amplified product revealed that there are one base substitution and two base deletions in the probe region. A newly constructed probe which was designed based on the revealed sequence could detect the P.ovale-variant. We performed a sequence analysis of 2.1 kilobase of 18S rRNA gene of P.ovale-variant and found three types of sequences. They differs about 4% in sequence from typical P.ovale and about 10% from other plasmodial species. We also determined the transcribed RNA sequence in blood stages of P.ovale-variant. In conclusion, the MPH method is valuable not only for clinical practice, but also for searching for a new species or variant of human malaria parasite.
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