title: Instability of Trypanosoma cruzi DNA in blood lysates: importance for PCR DNA-based diagnosis.
abstract: OBJECTIVE
  In order to evaluate the stability of Trypanosoma cruzi kinetoplast DNA (kDNA), the blood samples from seven patients with Chagas disease were stored in different buffers and at different temperatures.
  
  
  METHODS
  Three different buffers were used: buffer A, 6 mol/L guanidine-HCl; buffer B, 6M guanidine-HCl and 0.2M EDTA pH 7.5; and buffer C, 6M guanidine-HCl, 0.2M EDTA pH 7.5 and 10 microM dl-alpha-tocopherol (Roche, Basal, Switzerland). Two temperatures were used: 4 degrees C and 25 degrees C. Vitamin E was added to the blood lysates as an antioxidant. T. cruzi kDNA was obtained by phenol extraction, and then PCR amplifications and Southern blot were carried out in each DNA sample up to 90 days of blood storage. The iron content of each sample was determined by atomic absorption spectrophotometry.
  
  
  RESULTS
  Overall, there is an association between T. cruzi kDNA stability and the storage time of blood samples. No significant differences were detected in T. cruzi kDNA stability in the presence or absence of vitamin E or with citrate or EDTA as an anticoagulant. There was no statistical difference in the failure of PCR-based kDNA detection with these different storage buffers, temperatures or iron levels.
  
  
  CONCLUSIONS
  The blood lysates promote T. cruzi kDNA damage in a time-dependent manner that reduces the ability to detect the genomic DNA of an infectious agent by PCR. The high concentration of guanidine-HCl denatured proteins in these storage conditions probably denotes a non-enzymatic kDNA lysis.
authors.affiliation: Cellular and Molecular Biology Program, Biomedical Sciences Institute, Faculty of Medicine, University of Chile, Santiago, Chile.
authors.forename: Ximena
authors.initials: X
authors.lastname: Coronado
authors.forename: Sylvia
authors.initials: S
authors.lastname: Ortiz
authors.forename: Olga
authors.initials: O
authors.lastname: Lastra
authors.forename: Milton
authors.initials: M
authors.lastname: Larrondo
authors.forename: Marlene
authors.initials: M
authors.lastname: Rozas
authors.forename: Aldo
authors.initials: A
authors.lastname: Solari
mesh_terms: D000818:Animals; D000925:Anticoagulants / Q000737:chemistry; D002021:Buffers; D014355:Chagas Disease / Q000175:diagnosis*; D018105:DNA, Kinetoplast / Q000097:blood* / Q000737:chemistry; D004492:Edetic Acid / Q000737:chemistry; D006801:Humans; D016133:Polymerase Chain Reaction; D012680:Sensitivity and Specificity; D013048:Specimen Handling / Q000379:methods*; D014349:Trypanosoma cruzi / Q000235:genetics / Q000302:isolation & purification*; D014810:Vitamin E / Q000737:chemistry
chemical_list: D000925:Anticoagulants; D002021:Buffers; D018105:DNA, Kinetoplast; D014810:Vitamin E; D004492:Edetic Acid