1992 Fiscal Year Final Research Report Summary
DNA Diagnosis of Malaria Using PCR Techniques.
| Project/Area Number |
03557020
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| Research Category |
Grant-in-Aid for Developmental Scientific Research (B)
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| Allocation Type | Single-year Grants |
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| Research Field |
寄生虫学(含医用動物学）
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| Research Institution | Okayama University |
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Principal Investigator |
WATAYA Yusuke Okayama. Univ., Faculty it Pharm. Sci., 薬学部, 助教授 (90127598)
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| Co-Investigator(Kenkyū-buntansha) |
YAMANE Akio WAKunaga. Phdlm. Co. Bio Res. Inst. Chef Researcher., バイオ研究所, 主任研究員
OTA Nobuo Okayama Univ. School of Med. Aso. Prof., 医学部, 助教授 (10143611)
NEGISHI Kazuo Okayama Univ. Gene Res. Center. Aso. Prof, 遺伝子実験施設, 助教授 (70116490)
ISHII Akira NIH of Japan. Dept of Parasitology. Directs, 寄生動物部, 部長 (40012752)
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| Project Period (FY) |
1991 – 1992
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| Keywords | Malaria / DNA Diagnosis / PCR / Plasmodium / Vivax / falciparum / Solomon Islands / Gadalcanal |
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| Research Abstract |
We have developed three DNA diagnosis systems for detection of malaria parasites in human blood using the polymerase chain reaction (PCR) techniques. Our systems are simple and require neither DNA extraction nor radioisotopes. The double PCR is highly sensitive and specific detection system of Plasmodium falciparum parasites using two-step PCR. The target DNA sequence used in the double PCR was that encoded by the dihydrofolate reductase-thymidylate synthase (DHFR-TS) gene of P. falciparum. As little as 10 parasites in 10 muIOTA of blood gave a positive band of 226 base pairs (bp) by agarose gel electrophoresis of the PCR products. In the ED-PCR, enzymatic detection of PCR products, we have chosen a part of 18S ribosomal RNA gene of P. falciparum and P. vivax parasites as the target DNA. The PCR products labeled with dinitrophenyl(DNP) were detected using enzyme-labeled anti-DNP antibody. Both P. falciparum and P. vivax parasites were detected without distinction by the ED-PCR. The microplate hybridization is a diagnosis system which can detect P. falciparum and P. vivax distinctively, using the P. falciparum-specific probe and the P. vivax-specific probe to detect the PCR products. In this method, the part of 18S rRNA gene was also used as the target DNA for the PCR amplification.
We studied these three DNA diagnosis systems in the Guadalcanal of the Solomon lslands, where is a highly endemic area of malaria. 101 blood samples from 98 people were examined by the DNA diagnosis systems and microscopy. The results of our systems well accorded with that of microscopic examination. Our systems were also proved to be useful for a judgment of the effects of treatments.
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