Sensitive detection and schizodeme classification of Trypanosoma cruzi cells by amplification of kinetoplast minicircle DNA sequences: use in diagnosis of Chagas' disease.
Amplification of DNA sequences from the kinetoplast minicircle DNA was employed as a method for the detection and classification of small numbers of Trypanosoma cruzi cells. Two overlapping fragments from the conserved 120 bp minirepeat regions of the minicircle DNA and one fragment covering the adjacent variable regions were amplified. The minimal amount of minicircle DNA required to detect a product by hybridization with an oligonucleotide probe was 0.015 fg, which represents approximately 10 molecules or 0.1% of the minicircle DNA component of a single cell. The amplification worked equally well with kDNA from several strains of T. cruzi and did not occur with kDNA from several other kinetoplastids. kDNA recovered from less than 10 trypanosomes in whole blood could be used as a template for amplification; the presence of a several billion fold excess of human DNA had no effect on the amplification process. Schizodeme analysis by hybridization with specific oligonucleotides or by direct restriction enzyme digestion could be performed on the amplified fragments representing the minicircle conserved region or variable regions. This method should prove useful as a rapid, specific and sensitive assay for Chagas' disease in chronic patients as well as for epidemiological studies of infected animals and insects.
Sturm
N R
NR
Department of Biology, University of California, Los Angeles 90024.
Degrave
W
W
Morel
C
C
Simpson
L
L
D000818:Animals; D001483:Base Sequence; D014355:Chagas Disease / Q000175:diagnosis*; D015342:DNA Probes; D004270:DNA, Circular / Q000032:analysis* / Q000235:genetics; D018105:DNA, Kinetoplast; D004587:Electrophoresis, Agar Gel; D004591:Electrophoresis, Polyacrylamide Gel; D005784:Gene Amplification; D006801:Humans; D008969:Molecular Sequence Data; D009693:Nucleic Acid Hybridization; D012150:Polymorphism, Restriction Fragment Length; D012091:Repetitive Sequences, Nucleic Acid; D013045:Species Specificity; D013698:Templates, Genetic; D014349:Trypanosoma cruzi / Q000145:classification / Q000235:genetics* / Q000302:isolation & purification
D015342:DNA Probes; D004270:DNA, Circular; D018105:DNA, Kinetoplast