A MIQE-compliant real-time PCR assay for Aspergillus detection
The polymerase chain reaction (PCR) is widely used as a diagnostic tool in clinical laboratories and is particularly effective for detecting and identifying infectious agents for which routine culture and microscopy methods are inadequate. Invasive fungal disease (IFD) is a major cause of morbidity...
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| description | The polymerase chain reaction (PCR) is widely used as a diagnostic tool in clinical laboratories and is particularly effective for detecting and identifying infectious agents for which routine culture and microscopy methods are inadequate. Invasive fungal disease (IFD) is a major cause of morbidity and mortality in immunosuppressed patients, and optimal diagnostic criteria are contentious. Although PCR-based methods have long been used for the diagnosis of invasive aspergillosis (IA), variable performance in clinical practice has limited their value. This shortcoming is a consequence of differing sample selection, collection and preparation protocols coupled with a lack of standardisation of the PCR itself. Furthermore, it has become clear that the performance of PCR-based assays in general is compromised by the inadequacy of experimental controls, insufficient optimisation of assay performance as well as lack of transparency in reporting experimental details. The recently published "Minimum Information for the publication of real-time Quantitative PCR Experiments" (MIQE) guidelines provide a blueprint for good PCR assay design and unambiguous reporting of experimental detail and results. We report the first real-time quantitative PCR (qPCR) assay targeting Aspergillus species that has been designed, optimised and validated in strict compliance with the MIQE guidelines. The hydrolysis probe-based assay, designed to target the 18S rRNA DNA sequence of Aspergillus species, has an efficiency of 100% (range 95-107%), a dynamic range of at least six orders of magnitude and limits of quantification and detection of 6 and 0.6 Aspergillus fumigatus genomes, respectively. It does not amplify Candida, Scedosporium, Fusarium or Rhizopus species and its clinical sensitivity is demonstrated in histological material from proven IA cases, as well as concordant PCR and galactomannan data in matched broncho-alveolar lavage and blood samples. The robustness, specificity and sensitivity of this assay make it an ideal molecular diagnostic tool for clinical use. |
| doi_str_mv | 10.1371/journal.pone.0040022 |
| format | Article |
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Invasive fungal disease (IFD) is a major cause of morbidity and mortality in immunosuppressed patients, and optimal diagnostic criteria are contentious. Although PCR-based methods have long been used for the diagnosis of invasive aspergillosis (IA), variable performance in clinical practice has limited their value. This shortcoming is a consequence of differing sample selection, collection and preparation protocols coupled with a lack of standardisation of the PCR itself. Furthermore, it has become clear that the performance of PCR-based assays in general is compromised by the inadequacy of experimental controls, insufficient optimisation of assay performance as well as lack of transparency in reporting experimental details. The recently published "Minimum Information for the publication of real-time Quantitative PCR Experiments" (MIQE) guidelines provide a blueprint for good PCR assay design and unambiguous reporting of experimental detail and results. We report the first real-time quantitative PCR (qPCR) assay targeting Aspergillus species that has been designed, optimised and validated in strict compliance with the MIQE guidelines. The hydrolysis probe-based assay, designed to target the 18S rRNA DNA sequence of Aspergillus species, has an efficiency of 100% (range 95-107%), a dynamic range of at least six orders of magnitude and limits of quantification and detection of 6 and 0.6 Aspergillus fumigatus genomes, respectively. It does not amplify Candida, Scedosporium, Fusarium or Rhizopus species and its clinical sensitivity is demonstrated in histological material from proven IA cases, as well as concordant PCR and galactomannan data in matched broncho-alveolar lavage and blood samples. The robustness, specificity and sensitivity of this assay make it an ideal molecular diagnostic tool for clinical use.</description><identifier>ISSN: 1932-6203</identifier><identifier>EISSN: 1932-6203</identifier><identifier>DOI: 10.1371/journal.pone.0040022</identifier><identifier>PMID: 22808087</identifier><language>eng</language><publisher>United States: Public Library of Science</publisher><subject>Alveoli ; Annealing ; Aspergillosis ; Aspergillus ; Aspergillus fumigatus ; Aspergillus fumigatus - genetics ; Aspergillus fumigatus - isolation & purification ; Aspergillus nidulans ; Assaying ; Base Sequence ; Biology ; Biomedical laboratories ; Bronchoalveolar Lavage Fluid - chemistry ; Calibration ; Chemotherapy ; Deoxyribonucleic acid ; Design ; Diagnostic software ; Diagnostic systems ; DNA ; DNA Primers - chemistry ; DNA Primers - genetics ; DNA sequencing ; DNA, Fungal - blood ; Fungal diseases ; Fungal infections ; Genetic testing ; Genome, Fungal ; Genomes ; Genomics ; Guidelines ; Health aspects ; Hematology ; Humans ; Identification methods ; Infectious diseases ; Invasive Pulmonary Aspergillosis - blood ; Invasive Pulmonary Aspergillosis - diagnosis ; Invasive Pulmonary Aspergillosis - microbiology ; Laboratories ; Limit of Detection ; Medicine ; Microscopy ; Molecular Sequence Data ; Morbidity ; Mortality ; Mycoses ; Nucleotide sequence ; Polymerase chain reaction ; Practice Guidelines as Topic ; Real time ; Real-Time Polymerase Chain Reaction - methods ; Real-Time Polymerase Chain Reaction - standards ; Reproducibility of Results ; Ribosomal DNA ; RNA ; RNA, Ribosomal, 18S - blood ; RNA, Ribosomal, 18S - genetics ; rRNA 18S ; Sensitivity ; Species ; Stem cells ; Transparency</subject><ispartof>PloS one, 2012-07, Vol.7 (7), p.e40022-e40022</ispartof><rights>COPYRIGHT 2012 Public Library of Science</rights><rights>2012 Johnson et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License: https://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. 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Invasive fungal disease (IFD) is a major cause of morbidity and mortality in immunosuppressed patients, and optimal diagnostic criteria are contentious. Although PCR-based methods have long been used for the diagnosis of invasive aspergillosis (IA), variable performance in clinical practice has limited their value. This shortcoming is a consequence of differing sample selection, collection and preparation protocols coupled with a lack of standardisation of the PCR itself. Furthermore, it has become clear that the performance of PCR-based assays in general is compromised by the inadequacy of experimental controls, insufficient optimisation of assay performance as well as lack of transparency in reporting experimental details. The recently published "Minimum Information for the publication of real-time Quantitative PCR Experiments" (MIQE) guidelines provide a blueprint for good PCR assay design and unambiguous reporting of experimental detail and results. We report the first real-time quantitative PCR (qPCR) assay targeting Aspergillus species that has been designed, optimised and validated in strict compliance with the MIQE guidelines. The hydrolysis probe-based assay, designed to target the 18S rRNA DNA sequence of Aspergillus species, has an efficiency of 100% (range 95-107%), a dynamic range of at least six orders of magnitude and limits of quantification and detection of 6 and 0.6 Aspergillus fumigatus genomes, respectively. It does not amplify Candida, Scedosporium, Fusarium or Rhizopus species and its clinical sensitivity is demonstrated in histological material from proven IA cases, as well as concordant PCR and galactomannan data in matched broncho-alveolar lavage and blood samples. The robustness, specificity and sensitivity of this assay make it an ideal molecular diagnostic tool for clinical use.</description><subject>Alveoli</subject><subject>Annealing</subject><subject>Aspergillosis</subject><subject>Aspergillus</subject><subject>Aspergillus fumigatus</subject><subject>Aspergillus fumigatus - genetics</subject><subject>Aspergillus fumigatus - isolation & purification</subject><subject>Aspergillus nidulans</subject><subject>Assaying</subject><subject>Base Sequence</subject><subject>Biology</subject><subject>Biomedical laboratories</subject><subject>Bronchoalveolar Lavage Fluid - chemistry</subject><subject>Calibration</subject><subject>Chemotherapy</subject><subject>Deoxyribonucleic acid</subject><subject>Design</subject><subject>Diagnostic software</subject><subject>Diagnostic systems</subject><subject>DNA</subject><subject>DNA Primers - chemistry</subject><subject>DNA Primers - genetics</subject><subject>DNA sequencing</subject><subject>DNA, Fungal - blood</subject><subject>Fungal diseases</subject><subject>Fungal infections</subject><subject>Genetic testing</subject><subject>Genome, Fungal</subject><subject>Genomes</subject><subject>Genomics</subject><subject>Guidelines</subject><subject>Health aspects</subject><subject>Hematology</subject><subject>Humans</subject><subject>Identification methods</subject><subject>Infectious diseases</subject><subject>Invasive Pulmonary Aspergillosis - blood</subject><subject>Invasive Pulmonary Aspergillosis - diagnosis</subject><subject>Invasive Pulmonary Aspergillosis - microbiology</subject><subject>Laboratories</subject><subject>Limit of Detection</subject><subject>Medicine</subject><subject>Microscopy</subject><subject>Molecular Sequence Data</subject><subject>Morbidity</subject><subject>Mortality</subject><subject>Mycoses</subject><subject>Nucleotide sequence</subject><subject>Polymerase chain reaction</subject><subject>Practice Guidelines as Topic</subject><subject>Real time</subject><subject>Real-Time Polymerase Chain Reaction - methods</subject><subject>Real-Time Polymerase Chain Reaction - standards</subject><subject>Reproducibility of Results</subject><subject>Ribosomal DNA</subject><subject>RNA</subject><subject>RNA, Ribosomal, 18S - blood</subject><subject>RNA, Ribosomal, 18S - genetics</subject><subject>rRNA 18S</subject><subject>Sensitivity</subject><subject>Species</subject><subject>Stem cells</subject><subject>Transparency</subject><issn>1932-6203</issn><issn>1932-6203</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2012</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><sourceid>ABUWG</sourceid><sourceid>AFKRA</sourceid><sourceid>AZQEC</sourceid><sourceid>BENPR</sourceid><sourceid>CCPQU</sourceid><sourceid>DWQXO</sourceid><sourceid>GNUQQ</sourceid><sourceid>DOA</sourceid><recordid>eNqNkl2L1DAUhoso7jr6D0QLguhFx3w1aW8WhmHVgZXV9eM2pGkykyVtxiQV99-bOt1lKnshISQkz3nPOcmbZc8hWELM4LtrN_he2OXe9WoJAAEAoQfZKawxKigC-OHR_iR7EsI1ACWuKH2cnSBUgTTYaXa2yj9tvpwX0nV7a0Qfc6-ELaLpVP55fZWLEMRNrp3PV2Gv_NZYO4S8VVHJaFz_NHukhQ3q2bQusu_vz7-tPxYXlx8269VFIWmNYtEiRIgSDENVlZIBIFiFGkRbgtOFEEyWomlhAxtN27bWAAJEGq2hpgpKDfAie3nQ3VsX-NR64BCjEtcEUJaIzYFonbjme2864W-4E4b_PXB-y4WPRlrFK0hrBURNMaoIlWVNUCoJY1oyQmE5ap1N2YamU61UffTCzkTnN73Z8a37xTGuMUtzkb2ZBLz7OagQeWeCVNaKXrkh1Q1SRgIZHNFX_6D3dzdRW5EaML12Ka8cRfmKMAYhJtVILe-h0mhVZ2TyiTbpfBbwdhaQmKh-x60YQuCbr1f_z17-mLOvj9hdclTcBWeH0TJhDpIDKL0LwSt998gQ8NHmt6_BR5vzyeYp7MXxB90F3foa_wHPNPPo</recordid><startdate>20120710</startdate><enddate>20120710</enddate><creator>Johnson, Gemma L</creator><creator>Bibby, David F</creator><creator>Wong, Stephenie</creator><creator>Agrawal, Samir G</creator><creator>Bustin, Stephen A</creator><general>Public Library of Science</general><general>Public Library of Science (PLoS)</general><scope>CGR</scope><scope>CUY</scope><scope>CVF</scope><scope>ECM</scope><scope>EIF</scope><scope>NPM</scope><scope>AAYXX</scope><scope>CITATION</scope><scope>IOV</scope><scope>ISR</scope><scope>3V.</scope><scope>7QG</scope><scope>7QL</scope><scope>7QO</scope><scope>7RV</scope><scope>7SN</scope><scope>7SS</scope><scope>7T5</scope><scope>7TG</scope><scope>7TM</scope><scope>7U9</scope><scope>7X2</scope><scope>7X7</scope><scope>7XB</scope><scope>88E</scope><scope>8AO</scope><scope>8C1</scope><scope>8FD</scope><scope>8FE</scope><scope>8FG</scope><scope>8FH</scope><scope>8FI</scope><scope>8FJ</scope><scope>8FK</scope><scope>ABJCF</scope><scope>ABUWG</scope><scope>AEUYN</scope><scope>AFKRA</scope><scope>ARAPS</scope><scope>ATCPS</scope><scope>AZQEC</scope><scope>BBNVY</scope><scope>BENPR</scope><scope>BGLVJ</scope><scope>BHPHI</scope><scope>C1K</scope><scope>CCPQU</scope><scope>D1I</scope><scope>DWQXO</scope><scope>FR3</scope><scope>FYUFA</scope><scope>GHDGH</scope><scope>GNUQQ</scope><scope>H94</scope><scope>HCIFZ</scope><scope>K9.</scope><scope>KB.</scope><scope>KB0</scope><scope>KL.</scope><scope>L6V</scope><scope>LK8</scope><scope>M0K</scope><scope>M0S</scope><scope>M1P</scope><scope>M7N</scope><scope>M7P</scope><scope>M7S</scope><scope>NAPCQ</scope><scope>P5Z</scope><scope>P62</scope><scope>P64</scope><scope>PATMY</scope><scope>PDBOC</scope><scope>PIMPY</scope><scope>PQEST</scope><scope>PQQKQ</scope><scope>PQUKI</scope><scope>PRINS</scope><scope>PTHSS</scope><scope>PYCSY</scope><scope>RC3</scope><scope>7X8</scope><scope>5PM</scope><scope>DOA</scope></search><sort><creationdate>20120710</creationdate><title>A MIQE-compliant real-time PCR assay for Aspergillus detection</title><author>Johnson, Gemma L ; Bibby, David F ; Wong, Stephenie ; Agrawal, Samir G ; Bustin, Stephen A</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c692t-d2244ea731e85c700a782b26d43244aa7c5abd1b1bf6dd9f01024bff1f6e1cf03</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2012</creationdate><topic>Alveoli</topic><topic>Annealing</topic><topic>Aspergillosis</topic><topic>Aspergillus</topic><topic>Aspergillus fumigatus</topic><topic>Aspergillus fumigatus - genetics</topic><topic>Aspergillus fumigatus - isolation & purification</topic><topic>Aspergillus nidulans</topic><topic>Assaying</topic><topic>Base Sequence</topic><topic>Biology</topic><topic>Biomedical laboratories</topic><topic>Bronchoalveolar Lavage Fluid - chemistry</topic><topic>Calibration</topic><topic>Chemotherapy</topic><topic>Deoxyribonucleic acid</topic><topic>Design</topic><topic>Diagnostic software</topic><topic>Diagnostic systems</topic><topic>DNA</topic><topic>DNA Primers - chemistry</topic><topic>DNA Primers - genetics</topic><topic>DNA sequencing</topic><topic>DNA, Fungal - blood</topic><topic>Fungal diseases</topic><topic>Fungal infections</topic><topic>Genetic testing</topic><topic>Genome, Fungal</topic><topic>Genomes</topic><topic>Genomics</topic><topic>Guidelines</topic><topic>Health aspects</topic><topic>Hematology</topic><topic>Humans</topic><topic>Identification methods</topic><topic>Infectious diseases</topic><topic>Invasive Pulmonary Aspergillosis - blood</topic><topic>Invasive Pulmonary Aspergillosis - 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Academic</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>Johnson, Gemma L</au><au>Bibby, David F</au><au>Wong, Stephenie</au><au>Agrawal, Samir G</au><au>Bustin, Stephen A</au><au>Munro, Carol A.</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>A MIQE-compliant real-time PCR assay for Aspergillus detection</atitle><jtitle>PloS one</jtitle><addtitle>PLoS One</addtitle><date>2012-07-10</date><risdate>2012</risdate><volume>7</volume><issue>7</issue><spage>e40022</spage><epage>e40022</epage><pages>e40022-e40022</pages><issn>1932-6203</issn><eissn>1932-6203</eissn><abstract>The polymerase chain reaction (PCR) is widely used as a diagnostic tool in clinical laboratories and is particularly effective for detecting and identifying infectious agents for which routine culture and microscopy methods are inadequate. Invasive fungal disease (IFD) is a major cause of morbidity and mortality in immunosuppressed patients, and optimal diagnostic criteria are contentious. Although PCR-based methods have long been used for the diagnosis of invasive aspergillosis (IA), variable performance in clinical practice has limited their value. This shortcoming is a consequence of differing sample selection, collection and preparation protocols coupled with a lack of standardisation of the PCR itself. Furthermore, it has become clear that the performance of PCR-based assays in general is compromised by the inadequacy of experimental controls, insufficient optimisation of assay performance as well as lack of transparency in reporting experimental details. The recently published "Minimum Information for the publication of real-time Quantitative PCR Experiments" (MIQE) guidelines provide a blueprint for good PCR assay design and unambiguous reporting of experimental detail and results. We report the first real-time quantitative PCR (qPCR) assay targeting Aspergillus species that has been designed, optimised and validated in strict compliance with the MIQE guidelines. The hydrolysis probe-based assay, designed to target the 18S rRNA DNA sequence of Aspergillus species, has an efficiency of 100% (range 95-107%), a dynamic range of at least six orders of magnitude and limits of quantification and detection of 6 and 0.6 Aspergillus fumigatus genomes, respectively. It does not amplify Candida, Scedosporium, Fusarium or Rhizopus species and its clinical sensitivity is demonstrated in histological material from proven IA cases, as well as concordant PCR and galactomannan data in matched broncho-alveolar lavage and blood samples. The robustness, specificity and sensitivity of this assay make it an ideal molecular diagnostic tool for clinical use.</abstract><cop>United States</cop><pub>Public Library of Science</pub><pmid>22808087</pmid><doi>10.1371/journal.pone.0040022</doi><oa>free_for_read</oa></addata></record> |
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| recordid | cdi_plos_journals_1325394067 |
| source | MEDLINE; DOAJ Directory of Open Access Journals; Public Library of Science (PLoS); EZB-FREE-00999 freely available EZB journals; PubMed Central; Free Full-Text Journals in Chemistry |
| subjects | Alveoli Annealing Aspergillosis Aspergillus Aspergillus fumigatus Aspergillus fumigatus - genetics Aspergillus fumigatus - isolation & purification Aspergillus nidulans Assaying Base Sequence Biology Biomedical laboratories Bronchoalveolar Lavage Fluid - chemistry Calibration Chemotherapy Deoxyribonucleic acid Design Diagnostic software Diagnostic systems DNA DNA Primers - chemistry DNA Primers - genetics DNA sequencing DNA, Fungal - blood Fungal diseases Fungal infections Genetic testing Genome, Fungal Genomes Genomics Guidelines Health aspects Hematology Humans Identification methods Infectious diseases Invasive Pulmonary Aspergillosis - blood Invasive Pulmonary Aspergillosis - diagnosis Invasive Pulmonary Aspergillosis - microbiology Laboratories Limit of Detection Medicine Microscopy Molecular Sequence Data Morbidity Mortality Mycoses Nucleotide sequence Polymerase chain reaction Practice Guidelines as Topic Real time Real-Time Polymerase Chain Reaction - methods Real-Time Polymerase Chain Reaction - standards Reproducibility of Results Ribosomal DNA RNA RNA, Ribosomal, 18S - blood RNA, Ribosomal, 18S - genetics rRNA 18S Sensitivity Species Stem cells Transparency |
| title | A MIQE-compliant real-time PCR assay for Aspergillus detection |
| url | https://sfx.bib-bvb.de/sfx_tum?ctx_ver=Z39.88-2004&ctx_enc=info:ofi/enc:UTF-8&ctx_tim=2025-01-12T13%3A14%3A57IST&url_ver=Z39.88-2004&url_ctx_fmt=infofi/fmt:kev:mtx:ctx&rfr_id=info:sid/primo.exlibrisgroup.com:primo3-Article-gale_plos_&rft_val_fmt=info:ofi/fmt:kev:mtx:journal&rft.genre=article&rft.atitle=A%20MIQE-compliant%20real-time%20PCR%20assay%20for%20Aspergillus%20detection&rft.jtitle=PloS%20one&rft.au=Johnson,%20Gemma%20L&rft.date=2012-07-10&rft.volume=7&rft.issue=7&rft.spage=e40022&rft.epage=e40022&rft.pages=e40022-e40022&rft.issn=1932-6203&rft.eissn=1932-6203&rft_id=info:doi/10.1371/journal.pone.0040022&rft_dat=%3Cgale_plos_%3EA477113487%3C/gale_plos_%3E%3Curl%3E%3C/url%3E&disable_directlink=true&sfx.directlink=off&sfx.report_link=0&rft_id=info:oai/&rft_pqid=1325394067&rft_id=info:pmid/22808087&rft_galeid=A477113487&rft_doaj_id=oai_doaj_org_article_8169e0a9632846c59427003365746157&rfr_iscdi=true |