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.