Detection of Trypanosoma congolense and Trypanosoma brucei subspecies by DNA amplification using the polymerase chain reaction.
The nuclear DNA of Trypanosoma congolense contains a family of highly conserved 369 base pair (bp) repeats. The sequences of three cloned copies of these repeats were determined. An unrelated family of 177 bp repeats has previously been shown to occur in the nuclear DNA of Trypanosoma brucei brucei (Sloof et al. 1983a). Oligonucleotides were synthesized which prime the specific amplification of each of these repetitive DNAs by the polymerase chain reaction (PCR). Amplification of 10% of the DNA in a single parasite of T. congolense or T. brucei spp. produced sufficient amplified product to be visible as a band in an agarose gel stained with ethidium bromide. This level of detection, which does not depend on the use of radioactivity, is about 100 times more sensitive than previous detection methods based on radioactive DNA probes. The oligonucleotides did not prime the amplification of DNA sequences in other trypanosome species nor in Leishmania, mouse or human DNAs. Amplification of DNA from the blood of animals infected with T. congolense and/or T. brucei spp. permitted the identification of parasite levels far below that detectable by microscopic inspection. Since PCR amplification can be conducted on a large number of samples simultaneously, it is ideally suited for large-scale studies on the prevalence of African trypanosomes in both mammalian blood and insect vectors.
Moser
D R
DR
Department of Biochemistry, University of Iowa, Iowa City 52242.
Cook
G A
GA
Ochs
D E
DE
Bailey
C P
CP
McKane
M R
MR
Donelson
J E
JE
D000818:Animals; D001483:Base Sequence; D004247:DNA / Q000235:genetics; D004259:DNA-Directed DNA Polymerase; D004587:Electrophoresis, Agar Gel; D005784:Gene Amplification; D051379:Mice; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D051381:Rats; D012091:Repetitive Sequences, Nucleic Acid; D014346:Trypanosoma brucei brucei / Q000235:genetics* / Q000302:isolation & purification; D014348:Trypanosoma congolense / Q000235:genetics* / Q000302:isolation & purification; D014353:Trypanosomiasis, African / Q000175:diagnosis / Q000469:parasitology*
D004247:DNA; D004259:DNA-Directed DNA Polymerase