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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Public Library of Science (PLoS)
2014-01-01
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| Series: | PLoS ONE |
| Online Access: | https://journals.plos.org/plosone/article/file?id=10.1371/journal.pone.0089921&type=printable |
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| author | Stephen A Clark Jay Lucidarme Lynne S Newbold Ray Borrow |
| author_facet | Stephen A Clark Jay Lucidarme Lynne S Newbold Ray Borrow |
| author_sort | Stephen A Clark |
| collection | DOAJ |
| 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 | Article |
| id | doaj-art-32bd460f8cb84a15b63a051047ff55f8 |
| institution | DOAJ |
| issn | 1932-6203 |
| language | English |
| publishDate | 2014-01-01 |
| publisher | Public Library of Science (PLoS) |
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| series | PLoS ONE |
| spelling | doaj-art-32bd460f8cb84a15b63a051047ff55f82025-08-20T03:11:58ZengPublic Library of Science (PLoS)PLoS ONE1932-62032014-01-0192e8992110.1371/journal.pone.0089921Genotypic analysis of meningococcal factor h-binding protein from non-culture clinical specimens.Stephen A ClarkJay LucidarmeLynne S NewboldRay BorrowFactor 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.https://journals.plos.org/plosone/article/file?id=10.1371/journal.pone.0089921&type=printable |
| spellingShingle | Stephen A Clark Jay Lucidarme Lynne S Newbold Ray Borrow Genotypic analysis of meningococcal factor h-binding protein from non-culture clinical specimens. PLoS ONE |
| 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 |
| url | https://journals.plos.org/plosone/article/file?id=10.1371/journal.pone.0089921&type=printable |
| work_keys_str_mv | AT stephenaclark genotypicanalysisofmeningococcalfactorhbindingproteinfromnoncultureclinicalspecimens AT jaylucidarme genotypicanalysisofmeningococcalfactorhbindingproteinfromnoncultureclinicalspecimens AT lynnesnewbold genotypicanalysisofmeningococcalfactorhbindingproteinfromnoncultureclinicalspecimens AT rayborrow genotypicanalysisofmeningococcalfactorhbindingproteinfromnoncultureclinicalspecimens |