Molecular diagnosis of Trypanosome species

Various parameters for handling samples, extracting nucleic acid and protocols for use of PCR to diagnose trypanosomes have been examined. The use of a commercial kit (Nucleon BACC 2, Amersham) was superior to other extraction methods. After comparisons, a touchdown thermocycling protocol was the procedure used. PCR testing protocols were successfully implemented using Kin primers that bind to an internal transcribed spacer region (ITS1) situated between the 18S and the 5.8S ribosomal subunit genes on nuclear DNA. Their sensitivity was lower than that of satellite DNA primers, particularly for T.vivax. A Kin primer-based PCR could detect and distinguish between a number of Trypanosome species in blood samples. The use of an ITS1 binding primer was 3-5 x cheaper than using classical species-specific primers since the number of PCR reactions per sample is reduced to one. (author)