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Ready-To-Use qPCR for Detection of DNA from Trypanosoma cruzi or Other Pathogenic Organisms
Published on: January 20, 2022
Instability of Trypanosoma cruzi DNA in blood lysates: importance for PCR DNA-based diagnosis
Ximena Coronado1, Sylvia Ortiz, Olga Lastra
1Cellular and Molecular Biology Program, Biomedical Sciences Institute, Faculty of Medicine, University of Chile, Santiago, Chile.
Insights
Trypanosoma cruzi kinetoplast DNA (kDNA) degrades over time in blood lysates, impacting PCR detection. Storage conditions like buffer and temperature did not significantly alter kDNA stability.
Area of Science:
- Molecular Biology
- Parasitology
- Biochemistry
Background:
- Chagas disease is caused by the parasite Trypanosoma cruzi.
- Accurate detection of Trypanosoma cruzi kinetoplast DNA (kDNA) is crucial for diagnosing Chagas disease.
- Understanding the stability of kDNA under various storage conditions is essential for reliable diagnostic testing.
Purpose of the Study:
- To evaluate the stability of Trypanosoma cruzi kinetoplast DNA (kDNA) in blood samples.
- To determine the impact of different storage buffers and temperatures on kDNA integrity.
- To assess the influence of antioxidants and anticoagulants on kDNA stability.
Main Methods:
- Blood samples from Chagas disease patients were stored in buffers containing guanidine-HCl, EDTA, and/or vitamin E at 4°C and 25°C.
- Phenol extraction was used to isolate T. cruzi kDNA.
- PCR amplification and Southern blot were performed to assess kDNA detection up to 90 days.
- Iron content was measured using atomic absorption spectrophotometry.
Main Results:
- Trypanosoma cruzi kDNA stability was associated with blood storage time.
- The presence of vitamin E or specific anticoagulants (citrate, EDTA) did not significantly affect kDNA stability.
- No statistical difference was observed in PCR detection failure across different storage buffers, temperatures, or iron levels.
Conclusions:
- Blood lysates cause time-dependent damage to T. cruzi kDNA, reducing PCR detectability.
- High concentrations of guanidine-HCl likely lead to non-enzymatic kDNA lysis by denaturing proteins.
- These findings highlight challenges in maintaining kDNA integrity for diagnostic purposes.
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.
