Comparing two protocols of DNA extraction of Trypanosoma cruzi cultured in axenic medium

• Published in: Revista peruana de medicina experimental y salud pública Vol. 31; no. 2; pp. 222 - 227
• Main Authors: López, Mariela, Rivera, María G, Viettri, Mercedes, Lares, María, Morocoima, Antonio, Herrera, Leidi, Ferrer, Elizabeth
• Format: Journal Article
• Language: Spanish
• Published: Peru Instituto Nacional de Salud 01-04-2014
• Subjects: adn; Axenic Culture; diagnóstico; DNA, Kinetoplast - isolation & purification; enfermedad de chagas; Parasitology - methods; Polymerase Chain Reaction - methods; reacción en cadena de la polimerasa; trypanosoma cruzi; Trypanosoma cruzi - genetics
• ISSN: 1726-4634; 1726-4642
• DOI: 10.17843/rpmesp.2014.312.38

To compare two extraction protocols of Trypanosoma cruzi DNA for use in DNA amplification of kinetoplast minicircles (kDNA) through the technique of Polymerase Chain Reaction (PCR).
Epimastigotes of T. cruzi were cultured in axenic conditions and masses from 1.5 to 100 x 106 parasites were obtained. DNA extraction was performed using two protocols: extraction with organic solvents (phenol/chloroform), and with resin (Chelex100), from different parasitic sediments. Concentration and purity of DNA was determined by spectrophotometry, and integrity was assessed by agarose gel electrophoresis. Analysis of variance and comparisons of means were performed through Tukey's test, using the Statistix 8.0 software.
Ten DNA extractions were done of each one of the different amounts of parasitic sediments. In the DNA extraction with Chelex100 resin, a higher performance was obtained but a lower purity and integrity compared to the extraction with organic solvents. However, it allowed a product amplification of 330 bp of T. cruzi kDNA.
Although the technique of Chelex100 provided less purity and integrity of DNA, it allowed a successful amplification of kDNA by PCR, avoiding the use of laborious techniques and toxic organic solvents.