Abstract
BackgroundPolymerase 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 findingsWe 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/SignificanceThis 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.
Highlights
Chagas disease, a parasitic infection caused by the protozoan Trypanosoma cruzi, is endemic in 21 countries of Latin America, with approximately six million people affected [1]
In endemic settings the parasite is mainly transmitted by blood-sucking triatomine bugs [4,5], whereas in areas without vector-borne exposure the risk of developing T. cruzi infection arises from congenital transmission, blood transfusion, organ transplant, and laboratory accidents [6,7]
The aim of the present study was to assess the usefulness of an automated DNA extraction system based on magnetic particles (EZ1 Virus Mini Kit v2.0, Qiagen, Hilden, Germany) combined with a commercially available Real-Time Polymerase chain reaction (PCR) assay that targets the satellite DNA (SatDNA) of T. cruzi (RealCycler CHAG), a methodology routinely used for T. cruzi-infection diagnosis in the Hospital de la Santa Creu i Sant Pau of Barcelona (Spain)
Summary
A parasitic infection caused by the protozoan Trypanosoma cruzi, is endemic in 21 countries of Latin America, with approximately six million people affected [1]. Migratory flows have expanded Chagas disease worldwide, especially since the beginning of 2000, and the disease has emerged in non-endemic countries of North America, Europe and the Western Pacific Region [2,3]. 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. 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
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