LSSP-PCR of Trypanosoma cruzi: how the single primer sequence affects the kDNA signature

BMC Research Notes

Andréa Mara Macedo¹  Sérgio Danilo Junho Pena²  Glória Regina Franco²  Carlos Renato Machado²  Claudiney Melquíades Rodrigues²  Marcela Segatto² 

[1] Laboratório de Genética Bioquímica, Departamento de Bioquímica e Imunologia, Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais, Avenida Antônio Carlos, 6627 - Bloco K4, sala 245 - ICB/UFMG Bairro São Luiz, Belo Horizonte, Minas Gerais, CEP 31270-901, Brazil;Departamento de Bioquímica e Imunologia, Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais, Belo Horizonte, Minas Gerais, Brazil

关键词: Genetic diversity;    kDNA signatures;    Trypanosoma cruzi;    LSSP-PCR;   

Others  :  1142810
DOI  :  10.1186/1756-0500-6-174

 received in 2012-12-27, accepted in 2013-04-25,  发布年份 2013

【 摘 要 】

Background

Low-stringency single specific primer PCR (LSSP-PCR) is a highly sensitive and discriminating technique that has been extensively used to genetically characterize Trypanosoma cruzi populations in the presence of large amounts of host DNA. To ensure high sensitivity, in most T. cruzi studies, the variable regions of the naturally amplified kinetoplast DNA (kDNA) minicircles were targeted, and this method translated the intraspecific polymorphisms of these molecules into specific and reproducible kDNA signatures. Although the LSSP-PCR technique is reproducible under strict assay conditions, the complex banding pattern generated can be significantly altered by even a single-base change in the target DNA. Our survey of the literature identified eight different primers with similar, if not identical, names that have been used for kDNA amplification and LSSP-PCR of T. cruzi. Although different primer sequences were used in these studies, many of the authors cited the same reference report to justify their primer choice. We wondered whether these changes in the primer sequence could affect also the parasite LSSP-PCR profiles.

Findings

To answer this question we compared the kDNA signatures obtained from three different and extensively studied T. cruzi populations with the eight primers found in the literature. Our results clearly demonstrate that even minimal modifications in the oligonucleotide sequences, especially in the 3′ or 5′ end, can significantly change the kDNA signature of a T. cruzi strain.

Conclusions

These results highlight the necessity of careful preservation of primer nomenclature and sequence when reproducing an LSSP-PCR work to avoid confusion and allow comparison of results among different laboratories.

【 授权许可】

   
2013 Segatto et al.; licensee BioMed Central Ltd.

【 图 表 】

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