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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...
Autores principales: | , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Public Library of Science
2014
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3933679/ https://www.ncbi.nlm.nih.gov/pubmed/24587125 http://dx.doi.org/10.1371/journal.pone.0089921 |
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author | Clark, Stephen A. Lucidarme, Jay Newbold, Lynne S. Borrow, Ray |
author_facet | Clark, Stephen A. Lucidarme, Jay Newbold, Lynne S. Borrow, Ray |
author_sort | Clark, Stephen A. |
collection | PubMed |
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. |
format | Online Article Text |
id | pubmed-3933679 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2014 |
publisher | Public Library of Science |
record_format | MEDLINE/PubMed |
spelling | pubmed-39336792014-02-25 Genotypic Analysis of Meningococcal Factor H-Binding Protein from Non-Culture Clinical Specimens Clark, Stephen A. Lucidarme, Jay Newbold, Lynne S. Borrow, Ray PLoS One Research Article 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. Public Library of Science 2014-02-24 /pmc/articles/PMC3933679/ /pubmed/24587125 http://dx.doi.org/10.1371/journal.pone.0089921 Text en © 2014 Clark et al http://creativecommons.org/licenses/by/4.0/ This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited. |
spellingShingle | Research Article Clark, Stephen A. Lucidarme, Jay Newbold, Lynne S. Borrow, Ray Genotypic Analysis of Meningococcal Factor H-Binding Protein from Non-Culture Clinical Specimens |
title | Genotypic Analysis of Meningococcal Factor H-Binding Protein from Non-Culture Clinical Specimens |
title_full | Genotypic Analysis of Meningococcal Factor H-Binding Protein from Non-Culture Clinical Specimens |
title_fullStr | Genotypic Analysis of Meningococcal Factor H-Binding Protein from Non-Culture Clinical Specimens |
title_full_unstemmed | Genotypic Analysis of Meningococcal Factor H-Binding Protein from Non-Culture Clinical Specimens |
title_short | Genotypic Analysis of Meningococcal Factor H-Binding Protein from Non-Culture Clinical Specimens |
title_sort | genotypic analysis of meningococcal factor h-binding protein from non-culture clinical specimens |
topic | Research Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3933679/ https://www.ncbi.nlm.nih.gov/pubmed/24587125 http://dx.doi.org/10.1371/journal.pone.0089921 |
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