Implementation and Evaluation of a Fully Automated Multiplex Real-Time PCR Assay on the BD Max Platform to Detect and Differentiate Herpesviridae from Cerebrospinal Fluids
A fully automated multiplex real-time PCR assay--including a sample process control and a plasmid based positive control--for the detection and differentiation of herpes simplex virus 1 (HSV1), herpes simplex virus 2 (HSV2) and varicella-zoster virus (VZV) from cerebrospinal fluids (CSF) was develop...
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| Veröffentlicht in: | PloS one 2016-04, Vol.11 (4), p.e0153991-e0153991 |
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| creator | Köller, Thomas Kurze, Daniel Lange, Mirjam Scherdin, Martin Podbielski, Andreas Warnke, Philipp |
| description | A fully automated multiplex real-time PCR assay--including a sample process control and a plasmid based positive control--for the detection and differentiation of herpes simplex virus 1 (HSV1), herpes simplex virus 2 (HSV2) and varicella-zoster virus (VZV) from cerebrospinal fluids (CSF) was developed on the BD Max platform. Performance was compared to an established accredited multiplex real time PCR protocol utilizing the easyMAG and the LightCycler 480/II, both very common devices in viral molecular diagnostics. For clinical validation, 123 CSF specimens and 40 reference samples from national interlaboratory comparisons were examined with both methods, resulting in 97.6% and 100% concordance for CSF and reference samples, respectively. Utilizing the BD Max platform revealed sensitivities of 173 (CI 95%, 88-258) copies/ml for HSV1, 171 (CI 95%, 148-194) copies/ml for HSV2 and 84 (CI 95%, 5-163) copies/ml for VZV. Cross reactivity could be excluded by checking 25 common viral, bacterial and fungal human pathogens. Workflow analyses displayed shorter test duration as well as remarkable fewer and easier preparation steps with the potential to reduce error rates occurring when manually assessing patient samples. This protocol allows for a fully automated PCR assay on the BD Max platform for the simultaneously detection of herpesviridae from CSF specimens. Singular or multiple infections due to HSV1, HSV2 and VZV can reliably be differentiated with good sensitivities. Control parameters are included within the assay, thereby rendering its suitability for current quality management requirements. |
| doi_str_mv | 10.1371/journal.pone.0153991 |
| format | Article |
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Performance was compared to an established accredited multiplex real time PCR protocol utilizing the easyMAG and the LightCycler 480/II, both very common devices in viral molecular diagnostics. For clinical validation, 123 CSF specimens and 40 reference samples from national interlaboratory comparisons were examined with both methods, resulting in 97.6% and 100% concordance for CSF and reference samples, respectively. Utilizing the BD Max platform revealed sensitivities of 173 (CI 95%, 88-258) copies/ml for HSV1, 171 (CI 95%, 148-194) copies/ml for HSV2 and 84 (CI 95%, 5-163) copies/ml for VZV. Cross reactivity could be excluded by checking 25 common viral, bacterial and fungal human pathogens. Workflow analyses displayed shorter test duration as well as remarkable fewer and easier preparation steps with the potential to reduce error rates occurring when manually assessing patient samples. This protocol allows for a fully automated PCR assay on the BD Max platform for the simultaneously detection of herpesviridae from CSF specimens. Singular or multiple infections due to HSV1, HSV2 and VZV can reliably be differentiated with good sensitivities. 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Performance was compared to an established accredited multiplex real time PCR protocol utilizing the easyMAG and the LightCycler 480/II, both very common devices in viral molecular diagnostics. For clinical validation, 123 CSF specimens and 40 reference samples from national interlaboratory comparisons were examined with both methods, resulting in 97.6% and 100% concordance for CSF and reference samples, respectively. Utilizing the BD Max platform revealed sensitivities of 173 (CI 95%, 88-258) copies/ml for HSV1, 171 (CI 95%, 148-194) copies/ml for HSV2 and 84 (CI 95%, 5-163) copies/ml for VZV. Cross reactivity could be excluded by checking 25 common viral, bacterial and fungal human pathogens. Workflow analyses displayed shorter test duration as well as remarkable fewer and easier preparation steps with the potential to reduce error rates occurring when manually assessing patient samples. This protocol allows for a fully automated PCR assay on the BD Max platform for the simultaneously detection of herpesviridae from CSF specimens. Singular or multiple infections due to HSV1, HSV2 and VZV can reliably be differentiated with good sensitivities. Control parameters are included within the assay, thereby rendering its suitability for current quality management requirements.</abstract><cop>United States</cop><pub>Public Library of Science</pub><pmid>27092772</pmid><doi>10.1371/journal.pone.0153991</doi><oa>free_for_read</oa></addata></record> |
| fulltext | fulltext |
| identifier | ISSN: 1932-6203 |
| ispartof | PloS one, 2016-04, Vol.11 (4), p.e0153991-e0153991 |
| issn | 1932-6203 1932-6203 |
| language | eng |
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| source | Public Library of Science (PLoS) Journals Open Access; MEDLINE; DOAJ Directory of Open Access Journals; EZB-FREE-00999 freely available EZB journals; PubMed Central; Free Full-Text Journals in Chemistry |
| subjects | Accreditation Analysis Assaying Automation Bacteria Biological Assay - methods Biology and Life Sciences Care and treatment Cerebrospinal fluid Cerebrospinal Fluid - virology Chicken pox Comparative analysis Deoxyribonucleic acid DNA DNA, Viral - genetics Encephalitis Fluids Health aspects Herpes simplex Herpes viruses Herpesviridae - genetics Herpesviruses Humans Hygiene Medicine and Health Sciences Meningitis Multiplex Polymerase Chain Reaction - methods Multiplexing Nervous system Parameter sensitivity Patients Polymerase chain reaction Process control Process controls Quality management Real time Research and Analysis Methods Risk factors Sensitivity and Specificity Technology application Varicella Virology Viruses Workflow |
| title | Implementation and Evaluation of a Fully Automated Multiplex Real-Time PCR Assay on the BD Max Platform to Detect and Differentiate Herpesviridae from Cerebrospinal Fluids |
| url | https://sfx.bib-bvb.de/sfx_tum?ctx_ver=Z39.88-2004&ctx_enc=info:ofi/enc:UTF-8&ctx_tim=2025-01-28T20%3A15%3A38IST&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=Implementation%20and%20Evaluation%20of%20a%20Fully%20Automated%20Multiplex%20Real-Time%20PCR%20Assay%20on%20the%20BD%20Max%20Platform%20to%20Detect%20and%20Differentiate%20Herpesviridae%20from%20Cerebrospinal%20Fluids&rft.jtitle=PloS%20one&rft.au=K%C3%B6ller,%20Thomas&rft.date=2016-04-19&rft.volume=11&rft.issue=4&rft.spage=e0153991&rft.epage=e0153991&rft.pages=e0153991-e0153991&rft.issn=1932-6203&rft.eissn=1932-6203&rft_id=info:doi/10.1371/journal.pone.0153991&rft_dat=%3Cgale_plos_%3EA453447981%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=1782205359&rft_id=info:pmid/27092772&rft_galeid=A453447981&rft_doaj_id=oai_doaj_org_article_75ce4e9e36344deeab7973cff42d0af3&rfr_iscdi=true |