Sensitive detection and strain classification of Trypanosoma cruzi by amplification of a ribosomal RNA sequence.
A sequence of about 100 bp of the 24S alpha ribosomal RNA was investigated for sensitive detection of Trypanosoma cruzi. It was shown that the target sequence is specific for this parasite and no cross-reactivity was observed with different species of pathogenic Leishmania, two strains of Trypanosoma rangeli or human RNA. Amplification of the sequence was obtained by reverse transcription coupled to polymerase chain reaction. Following this procedure the equivalent to 0.1% of the nucleic acid content of a single parasite cell could be detected either by ethidium staining or blot hybridization. The distribution of the target sequence in sixteen strains of T. cruzi was investigated. Positive amplification was obtained for all samples employing the same oligonucleotides as primers. However, amplified fragments of 125 bp were obtained in eight strains, while fragments of 110 bp were detected in the remaining eight isolates. No amplification of both classes of fragments has been detected in any of the strains examined. Dimorphism in the target region was confirmed by hybridization to specific internal probes and sequencing, allowing the division of T. cruzi strains in two groups. It is proposed that sensitive parasite detection could be achieved by rRNA amplification followed by hybridization to two probes derived from the target sequences of both groups of T. cruzi strains. Furthermore, the sequence dimorphism found in this sequence opens the perspective of strain typing simultaneous with parasite detection.
Souto
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RP
Departamento de Bioquímica, Universidade de São Paulo, Brazil.
Zingales
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B
D000818:Animals; D001483:Base Sequence; D014355:Chagas Disease / Q000175:diagnosis; D017931:DNA Primers / Q000235:genetics; D016054:DNA, Protozoan / Q000235:genetics; D005784:Gene Amplification; D017125:Genes, Protozoan; D006801:Humans; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction / Q000706:statistics & numerical data; D016053:RNA, Protozoan / Q000235:genetics*; D012335:RNA, Ribosomal / Q000235:genetics*; D012680:Sensitivity and Specificity; D013045:Species Specificity; D014349:Trypanosoma cruzi / Q000145:classification / Q000235:genetics*
D017931:DNA Primers; D016054:DNA, Protozoan; D016053:RNA, Protozoan; D012335:RNA, Ribosomal