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Subspecific kDNA probes for major clones of Trypanosoma cruzi






Acta Tropica 48(1): 79-82

Subspecific kDNA probes for major clones of Trypanosoma cruzi

The authors describe the generation of 2 zymodeme-specific cloned probes for natural populations of Trypanosoma cruzi, utilizing the polymerase chain reaction (PCR) methodology. Oligonucleotide primers 20-23 bases in length derived from the highly conserved regions of kDNA minicircles have been used to generate these probes based on the variable or hypervariable regions of the kDNA in T. cruzi. By use of this protocol 250 base-pair fragments were amplified from the total DNA of 26 genetically highly diversified T. cruzi stocks to generate specific probes SO 34 and SC43 c11. The first, probe SO 34, is specific for clone or zymodeme 20; the second, SC43 c11, is specific for clonal zymodeme 39. These clone numbers refer to those described by Tibayrenc and Ayala, (Evolution, 1988, 42, 277). These 2 probes were then hybridized to total DNA from 24 stocks of T. cruzi. Each of the cloned probes hybridized specifically to its own stocks and all other clones to which it is related. This protocol therefore describes a simpler approach to the generation of zymodeme-specific probes utilizing PCR of the highly variable regions of kDNA in T. cruzi. The authors of this paper suggest that the success of this approach supports their hypothesis of the clonality of T. cruzi. Perhaps, more importantly, these probes should prove useful to assess easily the clinical and epidemiological studies of T. cruzi below the species level. Finally, these probes should allow the detection and analysis of mixed infection by several clones of this parasite within the same host parasite.J. Crampton Investigations concerning DNA probes specific for 2 of the major clones of T. cruzi (stock 5034, clone 20 and stock SC43 c11, clone 39) are presented. The polymerase chain reaction was carried out in order to generate selectively important amounts of high variable regions (HVRm) from the kDNA minicircles of T. cruzi. Oligonucleotide sequences (20-23) were selected from high constant regions of the minicircle in order to anneal sites flanking the variable region. An artificial restriction site was introduced close to the 3'-ends of each oligoprimer, allowing a fast and easy purification of HVRm (250 bp band), ready to use as probe.

Accession: 001959470

PMID: 1980806

DOI: 10.1016/0001-706x(90)90067-a

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