TY - JOUR
T1 - Improved DNA extraction technique from clot for the diagnosis of Chagas disease
AU - Chagas Working Group in Perú and Bolivia
AU - Mayta, Holger
AU - Romero, Yomara K.
AU - Pando, Alejandra
AU - Verastegui, Manuela
AU - Tinajeros, Freddy
AU - Bozo, Ricardo
AU - Henderson-Frost, Josephine
AU - Colanzi, Rony
AU - Flores, Jorge
AU - Lerner, Richard
AU - Bern, Caryn
AU - Gilman, Robert H.
N1 - Funding Information:
This study was funded by the National Institute of Health 5R01AI087776-04 and D43TW010074. The funder had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. To Janeth P. Castillo-Galindo for the initial review and MC Camila for her technical assistance. To Dr. Charles R. Sterling for the review and comments that greatly improved the manuscript. To Dr. Alejandro G. Schijman for the training at his laboratory. The members of the Chagas Working Group in Perú and Bolivia are: Lidabel Rios, Karen Cortez, Edith S. Malaga-Machaca, Leny Sanchez, Angela G. Vidal-Riva, Edward Valencia, Victoria R. Rendell, Malasa Jois, Vishal Shah, Edith Hinojosa, Clariza Chavez, Jean Karla Velarde, Carla Chavarria, Victoria Serrudo, Roberto Araya, Alcides Button and Rita Mendieta.
Publisher Copyright:
© 2019 Mayta et al.
PY - 2019
Y1 - 2019
N2 - 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.
AB - 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.
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U2 - 10.1371/journal.pntd.0007024
DO - 10.1371/journal.pntd.0007024
M3 - Article
C2 - 30633743
AN - SCOPUS:85059906860
SN - 1935-2727
VL - 13
JO - PLoS neglected tropical diseases
JF - PLoS neglected tropical diseases
IS - 1
M1 - e0007024
ER -