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

Abstract

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.

Figure 1

Amplification of the hypervariable regions of kDNA minicircle molecules of three T. cruzi populations. The PCR assays were performed in triplicate by three independent reactions for each T. cruzi population (CL Brener clone, Col1.7G2 clone or JG strain), as indicated in the figure. NC: negative control (without parasite DNA). MM: molecular size marker (1 Kb Plus DNA ladder - Invitrogen®).

Figure 2

Reproducibility of kDNA signatures obtained with different primers from LSSP-PCR. The amplifications were performed in triplicate by three independent reactions for each T. cruzi population (CL Brener clone, Col1.7G2 clone or JG strain), each strain with a different primer, as indicated in the figure. MM: molecular size marker (1 Kb Plus DNA ladder - Invitrogen®).

Figure 3

Evaluation of the genetic variability among three T. cruzi populations by LSSP-PCR with different primers. The primer used (A to H) is indicated below the profiles. MM: molecular size marker (1 Kb Plus DNA ladder - Invitrogen®).

Figure 4

LSSP-PCR trees. (A) UPGMA tree generated from the distance of the LSSP-PCR profiles produced by each primer based on the sum of present and absent bands for the T. cruzi populations CL Brener, Col1.7G2 and JG. (B) Distance tree constructed from the primer sequences