An improved polymerase chain reaction assay to detect Trypanosoma cruzi in blood
Diaz, C.; Nussenzweig, V.; Gonzalez, A.
American Journal of Tropical Medicine and Hygiene 46(5): 616-623
1992
ISSN/ISBN: 0002-9637 PMID: 1599057 Document Number: 402127
The authors have investigated the detection of Trypanosoma cruzi by amplification of repetitive 195-bp satellite DNA elements which represent about 10% of the total parasite chromosomal DNA. The choice of repetitive elements as targets for the polymerase chain reaction (PCR) enhances the sensitivity of the assay and the authors were able to detect 1 parasite in 10 ml of blood. Hybridization detection of the PCR product with a radiolabelled probe specific for the repeat element is also described and is probably essential for unequivocal detection of the product as the large quantities of host DNA (up to 50 micro g per 50 micro l reaction) tends to mask the PCR product after agarose gel electrophoresis. This could easily be converted to a nonradioactive hybridization system for more widespread use. Inhibition of PCR by blood is well documented and the authors found that purification of cell nuclei by centrifugation after NP40 lysis was necessary to remove the inhibition. DNA was then extracted by SDS/protease digestion, phenol/chloroform extraction and ethanol precipitation. [A more simple sample preparation method would improve this assay.] The authors diluted 10 parasites into 100 ml of blood and after processing 10-ml aliquots noted a Poisson distribution of the densities of the PCR product bands. [This is surprising as PCR is often termed the "Poorly Consistent Reaction" and rarely performs to perfection.] This assay was used to detect T. cruzi in the organs and tissues of chronically infected mice but parasites could not be detected in the blood of these animals. The relevance of this observation to the detection of T. cruzi in the peripheral blood of patients with chronic Chagas's disease remains to be investigated.S.M. Wilson Amplification by the polymerase chain reaction of T. cruzi satellite DNA was used to enhance sensitivity in the detection of the parasite in blood, with the ultimate goal of improving diagnosis of the chronic phase of Chagas' disease. Two contiguous oligonucleotides were synthesized corresponding to the most conserved region of the 195-basepair repeated sequence and used as primers for the amplification reaction. 19 femtograms of parasite DNA that was amplified in the presence of 15 micro g of human or mouse DNA produced a visible band upon electrophoresis in agarose gels and staining with ethidium bromide. In reconstitution experiments, one parasite in 10 ml of blood could be unambiguously determined when the DNA was isolated from nuclei after the blood was treated with NP40 and centrifuged. Polymerase chain reaction assays were carried out to detect T. cruzi in chronically infected mice. Most mice were parasite-positive when organs or tissues were tested, but all were negative when total blood was tested.