Use of multiple displacement amplification to increase the detection and genotyping of trypanosoma species samples immobilized on FTA filters.
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.
Morrison
Liam J
LJ
Wellcome Centre for Molecular Parasitology, University of Glasgow, Glasgow, United Kingdom. lm78y@udcf.gla.ac.uk
McCormack
Gillian
G
Sweeney
Lindsay
L
Likeufack
Anne C L
AC
Truc
Philippe
P
Turner
C Michael
CM
Tait
Andy
A
MacLeod
Annette
A
D000818:Animals; D016054:DNA, Protozoan / Q000737:chemistry / Q000235:genetics; D021903:DNA, Ribosomal Spacer / Q000737:chemistry / Q000235:genetics; D018503:Genome, Protozoan / Q000235:genetics*; D005838:Genotype; D006801:Humans; D018895:Microsatellite Repeats / Q000235:genetics; D016133:Polymerase Chain Reaction / Q000379:methods*; D014346:Trypanosoma brucei brucei / Q000235:genetics* / Q000302:isolation & purification; D014353:Trypanosomiasis, African / Q000469:parasitology*
D016054:DNA, Protozoan; D021903:DNA, Ribosomal Spacer