|Budget Amount *help
¥2,000,000 (Direct Cost : ¥2,000,000)
Fiscal Year 1993 : ¥500,000 (Direct Cost : ¥500,000)
Fiscal Year 1992 : ¥700,000 (Direct Cost : ¥700,000)
Fiscal Year 1991 : ¥800,000 (Direct Cost : ¥800,000)
Three sets synthesized-oligonucleotide primers have been tested for the polymerase chain reaction (PCR). The first set of the primers amplified only the DNA of L.braziliensis complex (L.braziliensis, L.panamensis and L.guyanensis), and the second one amplified only L.mexicana complex (L.amazonensis, L.pifanoi, L.garnhami) except L.mexicana, and the third one amplified L.mexicana as well as all other Leshimania parasite DNA of WHO reference strains. Three sets of them were applied for already identified-Ecuadorian Leishmania parasites by zymodeme analysis. Of fifty-nine of unidentified-Ecuadorian isolates, fifty were identified as L.braziliensis complex parasites by the first set, two isolates were identified as L.mexicana by the second and third ones, and the other five seemed to be other parasites, Trypanosoma sp. by the first, second and third ones of the primers as well as morphological features. Less than 10 parasites were enough numbers to amplify the specific DNA products by PCR.Southern blot hybridization using non-radioactive digoxigenin-DNA probes supported the identification of Leishmania parasites in molecular level. Lutzomyia ayacuhensis specific DNA probe was developed and constructionof the primers is in progress. These results indicate that the PCR using the three sets of the primers may differentiate L.braziliensis complex and L.mexicana complex and L.mexicana from others, especially Trypanosoma parasites, and apply for epidemiological survey of leishmaniasis in South America.