Improved DNA extraction technique from clot for the diagnosis of Chagas disease

by Holger Mayta, Yomara K. Romero, Alejandra Pando, Manuela Verastegui, Freddy Tinajeros, Ricardo Bozo, Josephine Henderson-Frost, Rony Colanzi, Jorge Flores, Richard Lerner, Caryn Bern, Robert H. Gilman, for the Chagas Working Group in Perú and Bolivia

Background

The detection of Trypanosoma cruzi genetic material in clinical samples is considered an important diagnostic tool for Chagas disease. We have previously demonstrated that PCR using clot samples yields greater sensitivity than either buffy coat or whole blood samples. However, phenol-chloroform DNA extraction from clot samples is difficult and toxic. The objective of the present study was to improve and develop a more sensitive method to recover parasite DNA from clot samples for the diagnosis of Chagas disease.

Methodology/Principal findings

A total of 265 match pair samples of whole blood–guanidine (GEB) and clot samples were analyzed; 150 were from Chagas seropositive subjects. DNA was extracted from both whole blood-guanidine samples, using a previously standardized methodology, and from clot samples, using a newly developed methodology based on a combination of the FastPrep technique and the standard method for GEB extraction. A qPCR targeting the nuclear satellite sequences was used to compare the sample source and the extraction method. Of the 150 samples from Chagas positive individuals by serology, 47 samples tested positive by qPCR with DNA extracted by both GEB and clot, but an additional 13 samples tested positive only in DNA extracted from clot. No serology-negative samples resulted positive when tested by qPCR.

Conclusions

The new methodology for DNA extraction from clot samples improves the molecular diagnosis of Chagas disease.

Tratto da: www.plos.org
Note sul Copyright: Articles and accompanying materials published by PLOS on the PLOS Sites, unless otherwise indicated, are licensed by the respective authors of such articles for use and distribution by you subject to citation of the original source in accordance with the Creative Commons Attribution (CC BY) license.

Lascia un commento

Il tuo indirizzo email non sarà pubblicato. I campi obbligatori sono contrassegnati *