Sequence optimized diagnostic assay for Ebola virus detection

Rapid pathogen identification is a critical first step in patient isolation, treatment, and controlling an outbreak. Real-time PCR is a highly sensitive and specific approach commonly used for infectious disease diagnostics. However, mismatches in the primer or probe sequence and the target organism...

Descripción completa

Detalles Bibliográficos
Autores principales: Koehler, Jeffrey W., Stefan, Christopher P., Hall, Adrienne T., Delp, Korey L., O’Hearn, Aileen E., Taylor-Howell, Cheryl L., Wauquier, Nadia, Schoepp, Randal J., Minogue, Timothy D.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Nature Publishing Group UK 2023
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10620139/
https://www.ncbi.nlm.nih.gov/pubmed/37914767
http://dx.doi.org/10.1038/s41598-023-29390-6
_version_ 1785130141759832064
author Koehler, Jeffrey W.
Stefan, Christopher P.
Hall, Adrienne T.
Delp, Korey L.
O’Hearn, Aileen E.
Taylor-Howell, Cheryl L.
Wauquier, Nadia
Schoepp, Randal J.
Minogue, Timothy D.
author_facet Koehler, Jeffrey W.
Stefan, Christopher P.
Hall, Adrienne T.
Delp, Korey L.
O’Hearn, Aileen E.
Taylor-Howell, Cheryl L.
Wauquier, Nadia
Schoepp, Randal J.
Minogue, Timothy D.
author_sort Koehler, Jeffrey W.
collection PubMed
description Rapid pathogen identification is a critical first step in patient isolation, treatment, and controlling an outbreak. Real-time PCR is a highly sensitive and specific approach commonly used for infectious disease diagnostics. However, mismatches in the primer or probe sequence and the target organism can cause decreased sensitivity, assay failure, and false negative results. Limited genomic sequences for rare pathogens such as Ebola virus (EBOV) can negatively impact assay performance due to undiscovered genetic diversity. We previously developed and validated several EBOV assays prior to the 2013–2016 EBOV outbreak in West Africa, and sequencing EBOV Makona identified sequence variants that could impact assay performance. Here, we assessed the impact sequence mismatches have on EBOV assay performance, finding one or two primer or probe mismatches resulted in a range of impact from minimal to almost two log sensitivity reduction. Redesigning this assay improved detection of all EBOV variants tested. Comparing the performance of the new assay with the previous assays across a panel of human EBOV samples confirmed increased assay sensitivity as reflected in decreased Cq values with detection of three positive that tested negative with the original assay.
format Online
Article
Text
id pubmed-10620139
institution National Center for Biotechnology Information
language English
publishDate 2023
publisher Nature Publishing Group UK
record_format MEDLINE/PubMed
spelling pubmed-106201392023-11-03 Sequence optimized diagnostic assay for Ebola virus detection Koehler, Jeffrey W. Stefan, Christopher P. Hall, Adrienne T. Delp, Korey L. O’Hearn, Aileen E. Taylor-Howell, Cheryl L. Wauquier, Nadia Schoepp, Randal J. Minogue, Timothy D. Sci Rep Article Rapid pathogen identification is a critical first step in patient isolation, treatment, and controlling an outbreak. Real-time PCR is a highly sensitive and specific approach commonly used for infectious disease diagnostics. However, mismatches in the primer or probe sequence and the target organism can cause decreased sensitivity, assay failure, and false negative results. Limited genomic sequences for rare pathogens such as Ebola virus (EBOV) can negatively impact assay performance due to undiscovered genetic diversity. We previously developed and validated several EBOV assays prior to the 2013–2016 EBOV outbreak in West Africa, and sequencing EBOV Makona identified sequence variants that could impact assay performance. Here, we assessed the impact sequence mismatches have on EBOV assay performance, finding one or two primer or probe mismatches resulted in a range of impact from minimal to almost two log sensitivity reduction. Redesigning this assay improved detection of all EBOV variants tested. Comparing the performance of the new assay with the previous assays across a panel of human EBOV samples confirmed increased assay sensitivity as reflected in decreased Cq values with detection of three positive that tested negative with the original assay. Nature Publishing Group UK 2023-11-01 /pmc/articles/PMC10620139/ /pubmed/37914767 http://dx.doi.org/10.1038/s41598-023-29390-6 Text en © This is a U.S. Government work and not under copyright protection in the US; foreign copyright protection may apply 2023 https://creativecommons.org/licenses/by/4.0/Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/ (https://creativecommons.org/licenses/by/4.0/) .
spellingShingle Article
Koehler, Jeffrey W.
Stefan, Christopher P.
Hall, Adrienne T.
Delp, Korey L.
O’Hearn, Aileen E.
Taylor-Howell, Cheryl L.
Wauquier, Nadia
Schoepp, Randal J.
Minogue, Timothy D.
Sequence optimized diagnostic assay for Ebola virus detection
title Sequence optimized diagnostic assay for Ebola virus detection
title_full Sequence optimized diagnostic assay for Ebola virus detection
title_fullStr Sequence optimized diagnostic assay for Ebola virus detection
title_full_unstemmed Sequence optimized diagnostic assay for Ebola virus detection
title_short Sequence optimized diagnostic assay for Ebola virus detection
title_sort sequence optimized diagnostic assay for ebola virus detection
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10620139/
https://www.ncbi.nlm.nih.gov/pubmed/37914767
http://dx.doi.org/10.1038/s41598-023-29390-6
work_keys_str_mv AT koehlerjeffreyw sequenceoptimizeddiagnosticassayforebolavirusdetection
AT stefanchristopherp sequenceoptimizeddiagnosticassayforebolavirusdetection
AT halladriennet sequenceoptimizeddiagnosticassayforebolavirusdetection
AT delpkoreyl sequenceoptimizeddiagnosticassayforebolavirusdetection
AT ohearnaileene sequenceoptimizeddiagnosticassayforebolavirusdetection
AT taylorhowellcheryll sequenceoptimizeddiagnosticassayforebolavirusdetection
AT wauquiernadia sequenceoptimizeddiagnosticassayforebolavirusdetection
AT schoepprandalj sequenceoptimizeddiagnosticassayforebolavirusdetection
AT minoguetimothyd sequenceoptimizeddiagnosticassayforebolavirusdetection