title: 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.
  
  
  RESULTS
  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.
authors: Departamento de Bioquímica e Imunologia, Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais, Belo Horizonte, Minas Gerais, Brazil.
authors: Marcela
authors: M
authors: Segatto
authors: Claudiney Melquíades
authors: CM
authors: Rodrigues
authors: Carlos Renato
authors: CR
authors: Machado
authors: Glória Regina
authors: GR
authors: Franco
authors: Sérgio Danilo Junho
authors: SD
authors: Pena
authors: Andréa Mara
authors: AM
authors: Macedo
mesh_terms: D000818:Animals; D001483:Base Sequence; D017931:DNA Primers; D018105:DNA, Kinetoplast / Q000235:genetics*; D016133:Polymerase Chain Reaction / Q000379:methods*; D014349:Trypanosoma cruzi / Q000235:genetics*
chemical_list: D017931:DNA Primers; D018105:DNA, Kinetoplast
references: Exp Parasitol. 2007 Sep;117(1):35-42
references: 17517390
references: Mol Biochem Parasitol. 1996 Dec 20;83(2):141-52
references: 9027747
references: Int J Parasitol. 2008 Jun;38(7):839-50
references: 18154957
references: Lancet. 1996 Sep 28;348(9031):891-2
references: 8826826
references: Trans R Soc Trop Med Hyg. 1977;71(3):217-25
references: 407674
references: PLoS Negl Trop Dis. 2010 Oct 12;4(10):e846
references: 20967289
references: EXS. 1993;67:339-45
references: 8400704
references: Mem Inst Oswaldo Cruz. 1999;94 Suppl 1:65-70
references: 10677692
references: Acta Virol. 2009;53(1):53-6
references: 19301952
references: Vet Microbiol. 2006 Dec 20;118(3-4):278-88
references: 16962259
references: Mol Cell Probes. 1995 Feb;9(1):45-8
references: 7760859
references: Biomedica. 2005 Mar;25(1):76-86
references: 15962904
references: Proc Natl Acad Sci U S A. 1979 Oct;76(10):5269-73
references: 291943
references: Diagn Microbiol Infect Dis. 2010 Apr;66(4):361-5
references: 20226327
references: Nature. 1978 Apr 27;272(5656):819-21
references: 417267
references: Exp Parasitol. 1998 May;89(1):30-9
references: 9603486
references: Am J Trop Med Hyg. 2009 Sep;81(3):396-403
references: 19706903
references: Clin Infect Dis. 2002 Jan 1;34(1):118-23
references: 11731955
references: Clin Chem Lab Med. 2001 Oct;39(10):948-52
references: 11758608
references: Proc Natl Acad Sci U S A. 1993 Feb 15;90(4):1335-9
references: 8433991
references: Curr Mol Med. 2004 Sep;4(6):549-62
references: 15357207
references: Am J Hum Genet. 1996 Mar;58(3):609-16
references: 8644720
references: Comput Appl Biosci. 1994 Sep;10(5):569-70
references: 7828077
references: Parasitology. 1997 Jun;114 ( Pt 6):517-20
references: 9172422
references: EXS. 1993;67:261-5
references: 8400696
references: Proc Natl Acad Sci U S A. 1994 Mar 1;91(5):1946-9
references: 8127912
references: PLoS One. 2009;4(4):e5113
references: 19337367
references: Exp Parasitol. 2009 Apr;121(4):317-22
references: 19135053
references: Parasitol Res. 2009 Jan;104(2):399-410
references: 18850114
references: Mol Biochem Parasitol. 1989 Mar 15;33(3):205-14
references: 2565018
references: Mol Biochem Parasitol. 1993 Jul;60(1):71-9
references: 8366896
references: Exp Parasitol. 2002 Apr;100(4):269-75
references: 12128054
references: Mol Biochem Parasitol. 1993 Nov;62(1):45-52
references: 8114825
references: Mol Biochem Parasitol. 1999 May 25;100(2):163-72
references: 10391378
references: J Clin Microbiol. 2006 Jun;44(6):2167-71
references: 16757616
references: Parasitology. 2009 Oct;136(12):1509-28
references: 19691868
references: Am J Trop Med Hyg. 1980 Sep;29(5 Suppl):1070-4
references: 6254386
references: Am J Pathol. 2000 May;156(5):1805-9
references: 10793092
references: Acta Trop. 2006 Nov;100(1-2):110-8
references: 17101108
references: Proc Natl Acad Sci U S A. 1998 Mar 31;95(7):3776-80
references: 9520443
references: Rev Inst Med Trop Sao Paulo. 2000 May-Jun;42(3):157-61
references: 10887376
references: Parasitol Res. 2008 Oct;103(5):1111-7
references: 18633644
references: Am J Trop Med Hyg. 2005 Dec;73(6):1016-8
references: 16354804
references: Biomedica. 2007 Jan;27 Suppl 1:64-74
references: 18154246
references: Am J Pathol. 1996 Dec;149(6):2153-9
references: 8952547