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

PLoS One. 2018 Apr 17;13(4):e0195738. doi: 10.1371/journal.pone.0195738. eCollection 2018.

Abstract

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Chagas Disease / diagnosis*
  • Chagas Disease / parasitology*
  • DNA, Protozoan
  • Humans
  • Real-Time Polymerase Chain Reaction
  • Reproducibility of Results
  • Sensitivity and Specificity
  • Trypanosoma cruzi / genetics*

Substances

  • DNA, Protozoan

Grants and funding

This study was partially supported by Departament d’Universitats, Recerca i Societat de la Informació de la Generalitat de Catalunya (grant 2014SGR026), Institut d’Investigació Biomèdica Sant Pau (IIB Sant Pau) and by Plan Nacional de I+D+i and Instituto de Salud Carlos III, Subdirección General de Redes y Centros de Investigación Cooperativa, Ministerio de Economía y Competividad, Spanish Network for Research in Infectious Diseases (grant REIPI RD12/0015); cofinanced by European Development Regional Fund (ERDF) “A way to achieve Europe”. A.A., C.B., S.T., M-J.P., E.P., J.G., and M.G. belong to RICET, a Tropical Disease Cooperative Research Network in Spain (grant RD12/0018/0010). ISGlobal is a member of the CERCA Programme, Generalitat de Catalunya. The supporters had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.