Genotypic analysis of meningococcal factor h-binding protein from non-culture clinical specimens
Factor H-Binding Protein (fHbp) is an outer membrane protein antigen included in two novel meningococcal group B vaccines and, as such, is an important typing target. Approximately 50% of meningococcal disease cases in England and Wales are confirmed using real-time PCR on non-culture clinical speci...
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| Veröffentlicht in: | PloS one 2014-02, Vol.9 (2), p.e89921-e89921 |
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| description | Factor H-Binding Protein (fHbp) is an outer membrane protein antigen included in two novel meningococcal group B vaccines and, as such, is an important typing target. Approximately 50% of meningococcal disease cases in England and Wales are confirmed using real-time PCR on non-culture clinical specimens only. Protocols for typing fHbp from this subset of cases have not yet been established. Here we present a nested PCR-based assay designed to amplify and sequence fHbp from non-culture clinical specimens. From analytical sensitivity experiments carried out using diluted DNA extracts, an estimated analytical sensitivity limit of 6 fg/µL of DNA ( |
| doi_str_mv | 10.1371/journal.pone.0089921 |
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Approximately 50% of meningococcal disease cases in England and Wales are confirmed using real-time PCR on non-culture clinical specimens only. Protocols for typing fHbp from this subset of cases have not yet been established. Here we present a nested PCR-based assay designed to amplify and sequence fHbp from non-culture clinical specimens. From analytical sensitivity experiments carried out using diluted DNA extracts, an estimated analytical sensitivity limit of 6 fg/µL of DNA (<3 genome copies/µL) was calculated. The sensitivity of the assay was shown to be comparable to the ctrA-directed real-time PCR assay currently used to confirm invasive disease diagnoses from submitted clinical specimens. A panel of 96 diverse, patient-matched clinical specimen/isolate pairs from invasive disease cases was used to illustrate the breadth of strain coverage for the assay. All fHbp alleles sequenced from the isolates matched those derived from previous whole genome analyses. The first-round PCR primer binding sites are highly conserved, however an exceptional second-round PCR primer site mismatch in one validation isolate prevented amplification. In this case, amplification from the corresponding clinical specimen was achieved, suggesting that the use of a nested PCR procedure may compensate for any minor mismatches in round-two primer sites. The assay was successful at typing 91/96 (94.8%) of the non-culture clinical specimens in this study and exhibits sufficient sensitivity to type fHbp from the vast majority of non-culture clinical specimens received by the Meningococcal Reference Unit, Public Health England.</description><identifier>ISSN: 1932-6203</identifier><identifier>EISSN: 1932-6203</identifier><identifier>DOI: 10.1371/journal.pone.0089921</identifier><identifier>PMID: 24587125</identifier><language>eng</language><publisher>United States: Public Library of Science</publisher><subject>Amplification ; Antigens ; Antigens, Bacterial - genetics ; Antigens, Bacterial - metabolism ; Assaying ; Bacterial Proteins - genetics ; Bacterial Proteins - metabolism ; Base Sequence ; Binding sites ; Binding Sites - genetics ; Biology ; Complement Factor H - metabolism ; Culture ; Deoxyribonucleic acid ; Dilution ; DNA ; England ; Factor H-binding protein ; Genomes ; Genomics ; Humans ; Medicine ; Membrane proteins ; Meningitis ; Meningococcal disease ; Meningococcal Infections - diagnosis ; Meningococcal Vaccines - chemistry ; Molecular Sequence Data ; Neisseria ; Neisseria meningitidis ; Neisseria meningitidis - genetics ; Peptides ; Polymerase Chain Reaction ; Protein binding ; Proteins ; Public health ; Real time ; Real-Time Polymerase Chain Reaction ; Sensitivity ; Sensitivity analysis ; Sensitivity and Specificity ; Sequence Analysis, DNA ; Streptococcus infections ; Vaccines ; Wales</subject><ispartof>PloS one, 2014-02, Vol.9 (2), p.e89921-e89921</ispartof><rights>COPYRIGHT 2014 Public Library of Science</rights><rights>2014 Clark 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>2014 Clark et al 2014 Clark et al</rights><lds50>peer_reviewed</lds50><oa>free_for_read</oa><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c692t-423f80f8285926eeb42756daeb38bca654de52f4b44223e9a740dbf3c218f1b83</citedby><cites>FETCH-LOGICAL-c692t-423f80f8285926eeb42756daeb38bca654de52f4b44223e9a740dbf3c218f1b83</cites></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><linktopdf>$$Uhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC3933679/pdf/$$EPDF$$P50$$Gpubmedcentral$$Hfree_for_read</linktopdf><linktohtml>$$Uhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC3933679/$$EHTML$$P50$$Gpubmedcentral$$Hfree_for_read</linktohtml><link.rule.ids>230,315,729,782,786,866,887,2106,2932,23875,27933,27934,53800,53802</link.rule.ids><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/24587125$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><contributor>Lin, Baochuan</contributor><creatorcontrib>Clark, Stephen A</creatorcontrib><creatorcontrib>Lucidarme, Jay</creatorcontrib><creatorcontrib>Newbold, Lynne S</creatorcontrib><creatorcontrib>Borrow, Ray</creatorcontrib><title>Genotypic analysis of meningococcal factor h-binding protein from non-culture clinical specimens</title><title>PloS one</title><addtitle>PLoS One</addtitle><description>Factor H-Binding Protein (fHbp) is an outer membrane protein antigen included in two novel meningococcal group B vaccines and, as such, is an important typing target. Approximately 50% of meningococcal disease cases in England and Wales are confirmed using real-time PCR on non-culture clinical specimens only. Protocols for typing fHbp from this subset of cases have not yet been established. Here we present a nested PCR-based assay designed to amplify and sequence fHbp from non-culture clinical specimens. From analytical sensitivity experiments carried out using diluted DNA extracts, an estimated analytical sensitivity limit of 6 fg/µL of DNA (<3 genome copies/µL) was calculated. The sensitivity of the assay was shown to be comparable to the ctrA-directed real-time PCR assay currently used to confirm invasive disease diagnoses from submitted clinical specimens. A panel of 96 diverse, patient-matched clinical specimen/isolate pairs from invasive disease cases was used to illustrate the breadth of strain coverage for the assay. All fHbp alleles sequenced from the isolates matched those derived from previous whole genome analyses. The first-round PCR primer binding sites are highly conserved, however an exceptional second-round PCR primer site mismatch in one validation isolate prevented amplification. In this case, amplification from the corresponding clinical specimen was achieved, suggesting that the use of a nested PCR procedure may compensate for any minor mismatches in round-two primer sites. The assay was successful at typing 91/96 (94.8%) of the non-culture clinical specimens in this study and exhibits sufficient sensitivity to type fHbp from the vast majority of non-culture clinical specimens received by the Meningococcal Reference Unit, Public Health England.</description><subject>Amplification</subject><subject>Antigens</subject><subject>Antigens, Bacterial - genetics</subject><subject>Antigens, Bacterial - metabolism</subject><subject>Assaying</subject><subject>Bacterial Proteins - genetics</subject><subject>Bacterial Proteins - metabolism</subject><subject>Base Sequence</subject><subject>Binding sites</subject><subject>Binding Sites - genetics</subject><subject>Biology</subject><subject>Complement Factor H - metabolism</subject><subject>Culture</subject><subject>Deoxyribonucleic acid</subject><subject>Dilution</subject><subject>DNA</subject><subject>England</subject><subject>Factor H-binding protein</subject><subject>Genomes</subject><subject>Genomics</subject><subject>Humans</subject><subject>Medicine</subject><subject>Membrane proteins</subject><subject>Meningitis</subject><subject>Meningococcal disease</subject><subject>Meningococcal Infections - 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genetics</topic><topic>Antigens, Bacterial - metabolism</topic><topic>Assaying</topic><topic>Bacterial Proteins - genetics</topic><topic>Bacterial Proteins - metabolism</topic><topic>Base Sequence</topic><topic>Binding sites</topic><topic>Binding Sites - genetics</topic><topic>Biology</topic><topic>Complement Factor H - metabolism</topic><topic>Culture</topic><topic>Deoxyribonucleic acid</topic><topic>Dilution</topic><topic>DNA</topic><topic>England</topic><topic>Factor H-binding protein</topic><topic>Genomes</topic><topic>Genomics</topic><topic>Humans</topic><topic>Medicine</topic><topic>Membrane proteins</topic><topic>Meningitis</topic><topic>Meningococcal disease</topic><topic>Meningococcal Infections - diagnosis</topic><topic>Meningococcal Vaccines - chemistry</topic><topic>Molecular Sequence Data</topic><topic>Neisseria</topic><topic>Neisseria meningitidis</topic><topic>Neisseria meningitidis - genetics</topic><topic>Peptides</topic><topic>Polymerase Chain Reaction</topic><topic>Protein binding</topic><topic>Proteins</topic><topic>Public health</topic><topic>Real time</topic><topic>Real-Time Polymerase Chain Reaction</topic><topic>Sensitivity</topic><topic>Sensitivity analysis</topic><topic>Sensitivity and Specificity</topic><topic>Sequence Analysis, DNA</topic><topic>Streptococcus infections</topic><topic>Vaccines</topic><topic>Wales</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Clark, Stephen A</creatorcontrib><creatorcontrib>Lucidarme, Jay</creatorcontrib><creatorcontrib>Newbold, Lynne S</creatorcontrib><creatorcontrib>Borrow, Ray</creatorcontrib><collection>Medline</collection><collection>MEDLINE</collection><collection>MEDLINE (Ovid)</collection><collection>MEDLINE</collection><collection>MEDLINE</collection><collection>PubMed</collection><collection>CrossRef</collection><collection>Gale In Context: Opposing Viewpoints</collection><collection>Gale In Context: Science</collection><collection>ProQuest Central (Corporate)</collection><collection>Animal Behavior Abstracts</collection><collection>Bacteriology Abstracts (Microbiology B)</collection><collection>Biotechnology Research Abstracts</collection><collection>Nursing & Allied Health Database</collection><collection>Ecology Abstracts</collection><collection>Entomology Abstracts (Full archive)</collection><collection>Immunology Abstracts</collection><collection>Meteorological & Geoastrophysical Abstracts</collection><collection>Nucleic Acids Abstracts</collection><collection>Virology and AIDS Abstracts</collection><collection>Agricultural Science Collection</collection><collection>Health & Medical Collection</collection><collection>ProQuest Central (purchase pre-March 2016)</collection><collection>Medical Database (Alumni Edition)</collection><collection>ProQuest Pharma Collection</collection><collection>Public Health Database</collection><collection>Technology Research Database</collection><collection>ProQuest SciTech Collection</collection><collection>ProQuest Technology Collection</collection><collection>ProQuest Natural Science Collection</collection><collection>Hospital Premium Collection</collection><collection>Hospital Premium Collection (Alumni Edition)</collection><collection>ProQuest Central (Alumni) (purchase pre-March 2016)</collection><collection>Materials Science & Engineering Collection</collection><collection>ProQuest Central (Alumni Edition)</collection><collection>ProQuest Central UK/Ireland</collection><collection>Advanced Technologies & Aerospace Collection</collection><collection>Agricultural & Environmental Science Collection</collection><collection>ProQuest Central Essentials</collection><collection>Biological Science Collection</collection><collection>ProQuest Central</collection><collection>Technology Collection</collection><collection>Natural Science Collection</collection><collection>Environmental Sciences and Pollution Management</collection><collection>ProQuest One Community College</collection><collection>ProQuest Materials Science Collection</collection><collection>ProQuest Central Korea</collection><collection>Engineering Research Database</collection><collection>Health Research Premium Collection</collection><collection>Health Research Premium Collection (Alumni)</collection><collection>ProQuest Central Student</collection><collection>AIDS and Cancer Research Abstracts</collection><collection>SciTech Premium Collection</collection><collection>ProQuest Health & Medical Complete (Alumni)</collection><collection>Materials Science Database</collection><collection>Nursing & Allied Health Database (Alumni Edition)</collection><collection>Meteorological & Geoastrophysical Abstracts - 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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>Clark, Stephen A</au><au>Lucidarme, Jay</au><au>Newbold, Lynne S</au><au>Borrow, Ray</au><au>Lin, Baochuan</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Genotypic analysis of meningococcal factor h-binding protein from non-culture clinical specimens</atitle><jtitle>PloS one</jtitle><addtitle>PLoS One</addtitle><date>2014-02-24</date><risdate>2014</risdate><volume>9</volume><issue>2</issue><spage>e89921</spage><epage>e89921</epage><pages>e89921-e89921</pages><issn>1932-6203</issn><eissn>1932-6203</eissn><abstract>Factor H-Binding Protein (fHbp) is an outer membrane protein antigen included in two novel meningococcal group B vaccines and, as such, is an important typing target. Approximately 50% of meningococcal disease cases in England and Wales are confirmed using real-time PCR on non-culture clinical specimens only. Protocols for typing fHbp from this subset of cases have not yet been established. Here we present a nested PCR-based assay designed to amplify and sequence fHbp from non-culture clinical specimens. From analytical sensitivity experiments carried out using diluted DNA extracts, an estimated analytical sensitivity limit of 6 fg/µL of DNA (<3 genome copies/µL) was calculated. The sensitivity of the assay was shown to be comparable to the ctrA-directed real-time PCR assay currently used to confirm invasive disease diagnoses from submitted clinical specimens. A panel of 96 diverse, patient-matched clinical specimen/isolate pairs from invasive disease cases was used to illustrate the breadth of strain coverage for the assay. All fHbp alleles sequenced from the isolates matched those derived from previous whole genome analyses. The first-round PCR primer binding sites are highly conserved, however an exceptional second-round PCR primer site mismatch in one validation isolate prevented amplification. In this case, amplification from the corresponding clinical specimen was achieved, suggesting that the use of a nested PCR procedure may compensate for any minor mismatches in round-two primer sites. The assay was successful at typing 91/96 (94.8%) of the non-culture clinical specimens in this study and exhibits sufficient sensitivity to type fHbp from the vast majority of non-culture clinical specimens received by the Meningococcal Reference Unit, Public Health England.</abstract><cop>United States</cop><pub>Public Library of Science</pub><pmid>24587125</pmid><doi>10.1371/journal.pone.0089921</doi><tpages>e89921</tpages><oa>free_for_read</oa></addata></record> |
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| subjects | Amplification Antigens Antigens, Bacterial - genetics Antigens, Bacterial - metabolism Assaying Bacterial Proteins - genetics Bacterial Proteins - metabolism Base Sequence Binding sites Binding Sites - genetics Biology Complement Factor H - metabolism Culture Deoxyribonucleic acid Dilution DNA England Factor H-binding protein Genomes Genomics Humans Medicine Membrane proteins Meningitis Meningococcal disease Meningococcal Infections - diagnosis Meningococcal Vaccines - chemistry Molecular Sequence Data Neisseria Neisseria meningitidis Neisseria meningitidis - genetics Peptides Polymerase Chain Reaction Protein binding Proteins Public health Real time Real-Time Polymerase Chain Reaction Sensitivity Sensitivity analysis Sensitivity and Specificity Sequence Analysis, DNA Streptococcus infections Vaccines Wales |
| title | Genotypic analysis of meningococcal factor h-binding protein from non-culture clinical specimens |
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