Use of multiple displacement amplification to increase the detection and genotyping of trypanosoma species samples immobilized on FTA filters
Morrison, L.J.; McCormack, G.; Sweeney, L.; Likeufack, A.C.L.; Truc, P.; Turner, C.Michael.; Tait, A.; MacLeod, A.
American Journal of Tropical Medicine and Hygiene 76(6): 1132-1137
2007
ISSN/ISBN: 0002-9637 PMID: 17556624 Document Number: 610103
Whole genome amplification methods are a recently developed tool for amplifying DNA from limited template. We report its application in trypanosome infections, characterized by low parasitemias. Multiple displacement amplification (MDA) amplifies DNA with a simple in vitro step and was evaluated on mouse blood samples on FTA filter cards with known numbers of Trypanosoma brucei parasites. The data showed a 20-fold increase in the number of PCRs possible per sample, using primers diagnostic for the multicopy ribosomal ITS region or 177-bp repeats, and a 20-fold increase in sensitivity over nested PCR against a single-copy microsatellite. Using MDA for microsatellite genotyping caused allele dropout at low DNA concentrations, which was overcome by pooling multiple MDA reactions. The validity of using MDA was established with samples from Human African Trypanosomiasis patients. The use of MDA allows maximal use of finite DNA samples and may prove a valuable tool in studies where multiple reactions are necessary, such as population genetic analyses.