Introducing automation to the molecular diagnosis of Trypanosoma cruzi infection: A comparative study of sample treatments, DNA extraction methods and real-time PCR assays
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 commerci...
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| creator | Abras, Alba Ballart, Cristina Llovet, Teresa Roig, Carme Gutiérrez, Cristina Tebar, Silvia Berenguer, Pere Pinazo, María-Jesús Posada, Elizabeth Gascón, Joaquim Schijman, Alejandro G Gállego, Montserrat Muñoz, Carmen |
| description | 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. |
| doi_str_mv | 10.1371/journal.pone.0195738 |
| format | Article |
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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.</description><identifier>ISSN: 1932-6203</identifier><identifier>EISSN: 1932-6203</identifier><identifier>DOI: 10.1371/journal.pone.0195738</identifier><identifier>PMID: 29664973</identifier><language>eng</language><publisher>United States: Public Library of Science</publisher><subject>Assaying ; Automation ; Biology and Life Sciences ; Chagas Disease - diagnosis ; Chagas Disease - parasitology ; Comparative studies ; Deoxyribonucleic acid ; Diagnosis ; Diagnostic software ; Diagnostic systems ; DNA ; DNA, Protozoan ; Ethylenediaminetetraacetic acids ; Guanidine ; Humans ; Infections ; Laboratories ; Life assessment ; Malalties parasitàries ; Medical diagnosis ; Medicine and Health Sciences ; Parasites ; Parasitic diseases ; Physical Sciences ; Polymerase chain reaction ; Protozoa ; Real time ; Real-Time Polymerase Chain Reaction ; Reproducibility of Results ; Research and Analysis Methods ; Satellite DNA ; Sensitivity and Specificity ; Silica ; Silicon dioxide ; Standardization ; Tripanosoma ; Trypanosoma ; Trypanosoma cruzi ; Trypanosoma cruzi - genetics ; Viruses</subject><ispartof>PloS one, 2018-04, Vol.13 (4), p.e0195738-e0195738</ispartof><rights>2018 Abras et al. This is an open access article distributed under the terms of the Creative Commons Attribution License: http://creativecommons.org/licenses/by/4.0/ (the “License”), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Notwithstanding the ProQuest Terms and Conditions, you may use this content in accordance with the terms of the License.</rights><rights>cc-by (c) Abras, Alba et al., 2018 info:eu-repo/semantics/openAccess <a href="http://creativecommons.org/licenses/by/3.0/es">http://creativecommons.org/licenses/by/3.0/es</a></rights><rights>2018 Abras et al 2018 Abras et al</rights><lds50>peer_reviewed</lds50><oa>free_for_read</oa><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c568t-813ac83d95df508e7af9d220f4583fafc5de4ea90ccb15219eaae3392bbc058b3</citedby><cites>FETCH-LOGICAL-c568t-813ac83d95df508e7af9d220f4583fafc5de4ea90ccb15219eaae3392bbc058b3</cites><orcidid>0000-0002-8143-9773</orcidid></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><linktopdf>$$Uhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC5903661/pdf/$$EPDF$$P50$$Gpubmedcentral$$Hfree_for_read</linktopdf><linktohtml>$$Uhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC5903661/$$EHTML$$P50$$Gpubmedcentral$$Hfree_for_read</linktohtml><link.rule.ids>230,314,725,778,782,862,883,2098,2917,23849,26957,27907,27908,53774,53776,79351,79352</link.rule.ids><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/29664973$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><contributor>Melcher, Ulrich</contributor><creatorcontrib>Abras, Alba</creatorcontrib><creatorcontrib>Ballart, Cristina</creatorcontrib><creatorcontrib>Llovet, Teresa</creatorcontrib><creatorcontrib>Roig, Carme</creatorcontrib><creatorcontrib>Gutiérrez, Cristina</creatorcontrib><creatorcontrib>Tebar, Silvia</creatorcontrib><creatorcontrib>Berenguer, Pere</creatorcontrib><creatorcontrib>Pinazo, María-Jesús</creatorcontrib><creatorcontrib>Posada, Elizabeth</creatorcontrib><creatorcontrib>Gascón, Joaquim</creatorcontrib><creatorcontrib>Schijman, Alejandro G</creatorcontrib><creatorcontrib>Gállego, Montserrat</creatorcontrib><creatorcontrib>Muñoz, Carmen</creatorcontrib><title>Introducing automation to the molecular diagnosis of Trypanosoma cruzi infection: A comparative study of sample treatments, DNA extraction methods and real-time PCR assays</title><title>PloS one</title><addtitle>PLoS One</addtitle><description>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). 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Academic</collection><collection>Recercat</collection><collection>PubMed Central (Full Participant titles)</collection><collection>DOAJ Directory of Open Access Journals</collection><jtitle>PloS one</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Abras, Alba</au><au>Ballart, Cristina</au><au>Llovet, Teresa</au><au>Roig, Carme</au><au>Gutiérrez, Cristina</au><au>Tebar, Silvia</au><au>Berenguer, Pere</au><au>Pinazo, María-Jesús</au><au>Posada, Elizabeth</au><au>Gascón, Joaquim</au><au>Schijman, Alejandro G</au><au>Gállego, Montserrat</au><au>Muñoz, Carmen</au><au>Melcher, Ulrich</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Introducing automation to the molecular diagnosis of Trypanosoma cruzi infection: A comparative study of sample treatments, DNA extraction methods and real-time PCR assays</atitle><jtitle>PloS one</jtitle><addtitle>PLoS One</addtitle><date>2018-04-17</date><risdate>2018</risdate><volume>13</volume><issue>4</issue><spage>e0195738</spage><epage>e0195738</epage><pages>e0195738-e0195738</pages><issn>1932-6203</issn><eissn>1932-6203</eissn><abstract>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.</abstract><cop>United States</cop><pub>Public Library of Science</pub><pmid>29664973</pmid><doi>10.1371/journal.pone.0195738</doi><tpages>14</tpages><orcidid>https://orcid.org/0000-0002-8143-9773</orcidid><oa>free_for_read</oa></addata></record> |
| fulltext | fulltext |
| identifier | ISSN: 1932-6203 |
| ispartof | PloS one, 2018-04, Vol.13 (4), p.e0195738-e0195738 |
| issn | 1932-6203 1932-6203 |
| language | eng |
| recordid | cdi_plos_journals_2026390770 |
| source | MEDLINE; DOAJ Directory of Open Access Journals; Elektronische Zeitschriftenbibliothek - Frei zugängliche E-Journals; Public Library of Science (PLoS); Recercat; PubMed Central; Free Full-Text Journals in Chemistry |
| subjects | Assaying Automation Biology and Life Sciences Chagas Disease - diagnosis Chagas Disease - parasitology Comparative studies Deoxyribonucleic acid Diagnosis Diagnostic software Diagnostic systems DNA DNA, Protozoan Ethylenediaminetetraacetic acids Guanidine Humans Infections Laboratories Life assessment Malalties parasitàries Medical diagnosis Medicine and Health Sciences Parasites Parasitic diseases Physical Sciences Polymerase chain reaction Protozoa Real time Real-Time Polymerase Chain Reaction Reproducibility of Results Research and Analysis Methods Satellite DNA Sensitivity and Specificity Silica Silicon dioxide Standardization Tripanosoma Trypanosoma Trypanosoma cruzi Trypanosoma cruzi - genetics Viruses |
| title | Introducing automation to the molecular diagnosis of Trypanosoma cruzi infection: A comparative study of sample treatments, DNA extraction methods and real-time PCR assays |
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