An improved polymerase chain reaction assay to detect Trypanosoma cruzi in blood.
Amplification by the polymerase chain reaction of Trypanosoma 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. Nineteen femtograms of parasite DNA that was amplified in the presence of 15 micrograms 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.
Diaz
C
C
Department of Pathology, New York University School of Medicine, New York.
Nussenzweig
V
V
Gonzalez
A
A
D000818:Animals; D001483:Base Sequence; D014355:Chagas Disease / Q000175:diagnosis*; D002908:Chronic Disease; D016054:DNA, Protozoan / Q000097:blood* / Q000737:chemistry; D004276:DNA, Satellite / Q000097:blood* / Q000737:chemistry; D004587:Electrophoresis, Agar Gel; D005260:Female; D005784:Gene Amplification; D006801:Humans; D051379:Mice; D008807:Mice, Inbred BALB C; D008810:Mice, Inbred C57BL; D008969:Molecular Sequence Data; D009693:Nucleic Acid Hybridization; D015345:Oligonucleotide Probes / Q000737:chemistry; D016133:Polymerase Chain Reaction; D012091:Repetitive Sequences, Nucleic Acid; D012680:Sensitivity and Specificity; D014349:Trypanosoma cruzi / Q000235:genetics / Q000302:isolation & purification*
D016054:DNA, Protozoan; D004276:DNA, Satellite; D015345:Oligonucleotide Probes