Genetic profiling of Trypanosoma cruzi directly in infected tissues using nested PCR of polymorphic microsatellites
- 作者：Helder Magno Silva Valadares ; Juliana Ramos Pimenta ; Jorge Marcelo de Freitas ; Tomá ; s Duffy ; Daniella C. Bartholomeu ; Riva de Paula Oliveira ; Egler Chiari ; Maria da Consolaç ; ã ; o Vieira Mor
- 关键词：Trypanosoma cruzi ; Chagas disease ; Genome project ; Polymorphic microsatellites ; Full nested PCR
- 刊名：International Journal for Parasitology
- 出版年：2008
- 期刊代码：124_00207519
- 类别：med
- 出版时间：June 2008
- 卷：38
- 期：7
- 页码：839-850
- 文件大小：824 K
摘要
The investigation of the importance of the genetics of Trypanosoma cruzi in determining the clinical course of Chagas disease will depend on precise characterisation of the parasites present in the tissue lesions. This can be adequately accomplished by the use of hypervariable nuclear markers such as microsatellites. However the unilocal nature of these loci and the scarcity of parasites in chronic lesions make it necessary to use high sensitivity PCR with nested primers, whose design depends on the availability of long flanking regions, a feature not hitherto available for any known T. cruzi microsatellites. Herein, making use of the extensive T. cruzi genome sequence now available and using the Tandem Repeats Finder software, it was possible to identify and characterise seven new microsatellite loci – six composed of trinucleotide (TcTAC15, TcTAT20, TcAAT8, TcATT14, TcGAG10 and TcCAA10) and one composed of tetranucleotide (TcAAAT6) motifs. All except the TcCAA10 locus were physically mapped onto distinct intergenic regions of chromosome III of the CL Brener clone contigs. The TcCAA10 locus was localised within a hypothetical protein gene in the T. cruzi genome. All microsatellites were polymorphic and useful for T. cruzi genetic variability studies. Using the TcTAC15 locus it was possible to separate the strains belonging to the T. cruzi I lineage (DTU I) from those belonging to T. cruzi II (DTU IIb), T. cruzi III (DTU IIc) and a hybrid group (DTU IId, IIe). The long flanking regions of these novel microsatellites allowed construction of nested primers and the use of full nested PCR protocols. This strategy enabled us to detect and differentiate T. cruzi strains directly in clinical specimens including heart, blood, CSF and skin tissues from patients in the acute and chronic phases of Chagas disease.