Introducing automation to the molecular diagnosis of Trypanosoma cruzi infection : A comparative study of sample treatments, DNA extraction methods and real-time PCR assays
Abras, Alba (Universitat de Girona. Departament de Biologia)
Ballart, Cristina (Barcelona Institute for Global Health (ISGlobal))
Llovet, Teresa (Universitat Autònoma de Barcelona. Departament de Genètica i de Microbiologia)
Roig, Carme (Institut d'Investigació Biomèdica Sant Pau)
Gutiérrez, Cristina (Institut d'Investigació Biomèdica Sant Pau)
Tebar, Silvia (Barcelona Institute for Global Health (ISGlobal))
Berenguer, Pere (Institut d'Investigació Biomèdica Sant Pau)
Pinazo, María-Jesús (Barcelona Institute for Global Health (ISGlobal))
Posada, Elizabeth (Barcelona Institute for Global Health (ISGlobal))
Gascon, Joaquim (Barcelona Institute for Global Health (ISGlobal))
Schijman, Alejandro G. (Instituto de Investigaciones en Ingeniería Genética y Biología Molecular)
Gállego Culleré, M. (Montserrat) (Barcelona Institute for Global Health (ISGlobal))
Muñoz, Carmen (Universitat Autònoma de Barcelona. Departament de Genètica i de Microbiologia)

Fecha:	2018
Resumen:	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. 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. 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.
Ayudas:	Agència de Gestió d'Ajuts Universitaris i de Recerca 2014/SGR-026 Ministerio de Economía y Competitividad RD12/0015 Ministerio de Economía y Competitividad RD12/0018/0010
Derechos:	Aquest document està subjecte a una llicència d'ús Creative Commons. Es permet la reproducció total o parcial, la distribució, la comunicació pública de l'obra i la creació d'obres derivades, fins i tot amb finalitats comercials, sempre i quan es reconegui l'autoria de l'obra original.
Lengua:	Anglès
Documento:	Article ; recerca ; Versió publicada
Publicado en:	PloS one, Vol. 13, Issue 4 (April 2018) , art. e0195738, ISSN 1932-6203

DOI: 10.1371/journal.pone.0195738
PMID: 29664973

14 p, 1.6 MB

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