Detection ofTrypanosoma congolenseandTrypanosoma bruceisubspecies by DNA amplification using the polymerase chain reaction

Abstract

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.

Domestic animals as potential reservoir hosts ofTrypanosoma brucei gambiensein sleeping sickness foci in Cameroon

“…The supernatant containing the DNA was stored at -20°C or used directly for PCR. A first amplification protocol to detect Trypanosoma brucei s.l., was proceeded on the extracts of all the samples using the primers TBR1 (5'-GAATATTAAA CAATGCGCAG-3')/TBR2 (5'-CCATTTATTAGCTTTGTT GC-3') (Moser et al, 1989). This was followed by a second amplification on T. brucei s.l.…”

Usefulness of PCR-based assays to assess drug efficacy in Chagas disease chemotherapy: value and limitations

“…In an effort to develop more sensitive assays, PCR technology has been introduced to specifically detect T. cruzi DNA in blood samples from chagasic patients, opening new possibilities in the diagnosis and follow-up assessment of chemotherapy (Moser et al 1989, Sturm et al 1989, Avila et al 1991, Britto et al 1993, 1995, Wincker et al 1994a, b, Junqueira et al 1996, Gomes et al 1998, Galvão et al 2003. Reconstitution experiments showed that PCR procedures are able to detect the equivalent of a single parasite cell in 10-20 mL of whole blood (Moser et al 1989, Avila et al 1991, Britto et al 1993.…”

“…Reconstitution experiments showed that PCR procedures are able to detect the equivalent of a single parasite cell in 10-20 mL of whole blood (Moser et al 1989, Avila et al 1991, Britto et al 1993. However, there are large genetic differences between distinct T. cruzi strains related to their biological variability, such as virulence or variable susceptibility to the immune response (control of parasitaemia) developed after the acute phase of human infection.…”

Treatment of congenital Chagas' disease diagnosed and followed up by the polymerase chain reaction.

“…17 We have found that the PCR, when used with the T. cruzi-specific primers TCZ-1 and TCZ-2, which anneal to a highly repetitive sequence of T. cruzi nuclear DNA, 18 is far more sensitive than direct microscopic observation for the neonatal diagnosis of T. cruzi infection. 19 In this study, we used the PCR to detect T. cruzi infection in blood samples of newborns for early diagnosis and to monitor treatment.…”