Introducing automation to the molecular diagnosis of Trypanosoma cruzi infection: A comparative study of sample treatments, DNA extraction methods and real-time PCR assays

Authors
Abras, Alba; Ballart, Cristina; Llovet, Teresa; Roig, Carme; Gutiérrez, Cristina; Tebar, Silvia; Berenguer, Pere; Pinazo, María-Jesús; Posada, Elizabeth; Gascón, Joaquim; Schijman, Alejandro Gabriel; Gállego, Montserrat; Muñoz, Carmen
Publication Year
2018
Language
English
Format
article
Status
Published version
Description
Background Polymerase chain reaction (PCR) has become a useful tool for the diagnosis of Trypanosoma cruzi infection. The development of automated DNA extraction methodologies and PCR systems is an important step toward the standardization of protocols in routine diagnosis. To date, there are only two commercially available Real-Time PCR assays for the routine laboratory detection of T. cruzi DNA in clinical samples: TCRUZIDNA.CE (Diagnostic Bioprobes Srl) and RealCycler CHAG (Progenie Molecular). Our aim was to evaluate the RealCycler CHAG assay taking into account the whole process. Methodology/Principal findings We assessed the usefulness of an automated DNA extraction system based on magnetic particles (EZ1 Virus Mini Kit v2.0, Qiagen) combined with a commercially available Real-Time PCR assay targeting satellite DNA (SatDNA) of T. cruzi (RealCycler CHAG), a methodology used for routine diagnosis in our hospital. It was compared with a well-known strategy combining a commercial DNA isolation kit based on silica columns (High Pure PCR Template Preparation Kit, Roche Diagnostics) with an in-house Real-Time PCR targeting SatDNA. The results of the two methodologies were in almost perfect agreement, indicating they can be used interchangeably. However, when variations in protocol factors were applied (sample treatment, extraction method and Real-Time PCR), the results were less convincing. A comprehensive fine-tuning of the whole procedure is the key to successful results. Guanidine EDTA-blood (GEB) samples are not suitable for DNA extraction based on magnetic particles due to inhibition, at least when samples are not processed immediately. Conclusions/Significance This is the first study to evaluate the RealCycler CHAG assay taking into account the overall process, including three variables (sample treatment, extraction method and Real-Time PCR). Our findings may contribute to the harmonization of protocols between laboratories and to a wider application of Real-Time PCR in molecular diagnostic laboratories associated with health centers.
Fil: Abras, Alba. Universidad de Barcelona; España. Universidad de Girona; España. Instituto de Salud Global de Barcelona; España
Fil: Ballart, Cristina. Universidad de Barcelona; España. Instituto de Salud Global de Barcelona; España
Fil: Llovet, Teresa. Universitat Autònoma de Barcelona; España. Hospital de la Santa Creu I Sant Pau; España
Fil: Roig, Carme. Hospital de la Santa Creu I Sant Pau; España
Fil: Gutiérrez, Cristina. Hospital de la Santa Creu I Sant Pau; España
Fil: Tebar, Silvia. Universidad de Barcelona; España. Instituto de Salud Global de Barcelona; España
Fil: Berenguer, Pere. Hospital de la Santa Creu I Sant Pau; España
Fil: Pinazo, María-Jesús. Instituto de Salud Global de Barcelona; España
Fil: Posada, Elizabeth. Instituto de Salud Global de Barcelona; España
Fil: Gascón, Joaquim. Instituto de Salud Global de Barcelona; España
Fil: Schijman, Alejandro Gabriel. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; Argentina
Fil: Gállego, Montserrat. Instituto de Salud Global de Barcelona; España. Universidad de Barcelona; España
Fil: Muñoz, Carmen. Hospital de la Santa Creu I Sant Pau; España. Universitat Autònoma de Barcelona; España
Subject
Real time PCR
automatic DNA extraction
molecular diagnosis
Chagas disease
Bioquímica y Biología Molecular
Ciencias Biológicas
CIENCIAS NATURALES Y EXACTAS
Access level
Open access
License
https://creativecommons.org/licenses/by-nc-sa/2.5/ar/
Repository
CONICET Digital (CONICET)
Institution
Consejo Nacional de Investigaciones Científicas y Técnicas
OAI Identifier
oai:ri.conicet.gov.ar:11336/79837