2003 Fiscal Year Final Research Report Summary
Development of Diagnostic Methods for African Trypanosomes and Their Evaluation Using Field Samples
| Project/Area Number |
13556048
|
|---|
| Research Category |
Grant-in-Aid for Scientific Research (B)
|
| Allocation Type | Single-year Grants |
|---|
| Section | 展開研究 |
|---|
| Research Field |
Applied veterinary science
|
|---|
| Research Institution | Obihiro University of Agriculture and Veterinary Medicine |
|---|
Principal Investigator |
INOUE Noboru Obihiro University of Agriculture and Veterinary Medicine, National Research Center for Protozoan Diseases, Associate Professor, 原虫病研究センター, 助教授 (10271751)
|
|---|
| Co-Investigator(Kenkyū-buntansha) |
XUAN Xuenan Obihiro University of Agriculture and Veterinary Medicine, National Research Center for Protozoan Diseases, Associate Professor, 原虫病研究センター, 助教授 (10292096)
TANAKA Tetsuya Hokkaido University, Graduate School of Agriculture, Instructor, 大学院・農学研究科, 助手 (00322842)
YOKOYAMA Naoaki Obihiro University of Agriculture and Veterinary Medicine, National Research Center for Protozoan Diseases, Associate Professor, 原虫病研究センター, 助教授 (80301802)
NAGASAWA Hideyuki Obihiro University of Agriculture and Veterinary Medicine, National Research Center for Protozoan Diseases, Professor, 原虫病研究センター, 教授 (60172524)
FUJISAKI Kozo Obihiro University of Agriculture and Veterinary Medicine, National Research Center for Protozoan Diseases, Professor, 原虫病研究センター, 教授 (00292095)
|
|---|
| Project Period (FY) |
2001 – 2003
|
| Keywords | African trypanosomes / Diagnosis / ELISA / LAMP |
|---|
| Research Abstract |
Loop-mediated isothermal amplification of DMA (LAMP)
The loop-mediated isothermal amplification (LAMP) reaction is a method that amplifies DNMA with high specificity, efficiency, and rapidity under isothermal conditions with only simple incubators. An added advantage of LAMP over PCR-based methods is that DNA amplification can be monitored spectrophotometrically and/or with the naked eye without the use of dyes. Here we report our conditions for a highly sensitive, specific, and easy diagnostic assay based on LAMP technology for the detection of parasites in the Trypanosoma brucei group (including T.brucei brucei, T.brucei gambiense, T.brucei rhodesiense, and T.evansi) and T.congolense. We show that the sensitivity of the LAMP-based method for detection of trypanosomes in vitro is up to 100 times higher than that of PCR-based methods. In vivo studies in mice infected with human-infective T.brucei gambiense further highlight the potential clinical importance of LAMP as a diagnostic tool for the identification of African trypanosomiasis.
ELISA
The ability to use mitochondrial heat shock protein 70 (MTP) of Trypanosoma congolense as a diagnostic antigen was examined. The B-cell epitope recognized by MAb 10F9 was located within 206 amino acids from the C terminus. Depending on the conditions of protein extraction, MTP was cleaved into smaller polypeptides by endogenous proteases. However, the C-termmal epitope of MTP was preserved with a high degree of antigenicity, even after cleavage. Antibody detection by enzyme-linked immunosorbent assay with the truncated recombinant MTP revealed that anti-MTP antibodies exist in experimentally infected mouse sera. Thus, MTP may be useful as an antigen for the serodiagnosis of primary T.congolense infection.
|
