Development of a non-infectious control for viral hemorrhagic fever PCR assays.
Assay validation is an essential component of disease surveillance testing, but can be problematic in settings where access to positive control material is limited and a safety risk for handlers. Here we describe a single non-infectious synthetic control that can help develop and validate the PCR ba...
Saved in:
| Main Authors: | , , |
|---|---|
| Format: | Article |
| Language: | English |
| Published: |
Public Library of Science (PLoS)
2024-04-01
|
| Series: | PLoS Neglected Tropical Diseases |
| Online Access: | https://doi.org/10.1371/journal.pntd.0011390 |
| Tags: |
Add Tag
No Tags, Be the first to tag this record!
|
| _version_ | 1849427774889000960 |
|---|---|
| author | Matthew A Knox Collette Bromhead David Ts Hayman |
| author_facet | Matthew A Knox Collette Bromhead David Ts Hayman |
| author_sort | Matthew A Knox |
| collection | DOAJ |
| description | Assay validation is an essential component of disease surveillance testing, but can be problematic in settings where access to positive control material is limited and a safety risk for handlers. Here we describe a single non-infectious synthetic control that can help develop and validate the PCR based detection of the viral causes of Crimean-Congo hemorrhagic fever, Ebola virus disease, Lassa fever, Marburg virus disease and Rift Valley fever. We designed non-infectious synthetic DNA oligonucleotide sequences incorporating primer binding sites suitable for five assays, and a T7 promotor site which was used to transcribe the sequence. Transcribed RNA was used as template in a dilution series, extracted and amplified with RT-PCR and RT-qPCR to demonstrate successful recovery and determine limits of detection in a range of laboratory settings. Our results show this approach is adaptable to any diagnostic assay requiring validation of nucleic acid extraction and/or amplification, particularly where sourcing reliable, safe material for positive controls is infeasible. |
| format | Article |
| id | doaj-art-184f355e7c83400a8e0fad84053030db |
| institution | Kabale University |
| issn | 1935-2727 1935-2735 |
| language | English |
| publishDate | 2024-04-01 |
| publisher | Public Library of Science (PLoS) |
| record_format | Article |
| series | PLoS Neglected Tropical Diseases |
| spelling | doaj-art-184f355e7c83400a8e0fad84053030db2025-08-20T03:28:55ZengPublic Library of Science (PLoS)PLoS Neglected Tropical Diseases1935-27271935-27352024-04-01184e001139010.1371/journal.pntd.0011390Development of a non-infectious control for viral hemorrhagic fever PCR assays.Matthew A KnoxCollette BromheadDavid Ts HaymanAssay validation is an essential component of disease surveillance testing, but can be problematic in settings where access to positive control material is limited and a safety risk for handlers. Here we describe a single non-infectious synthetic control that can help develop and validate the PCR based detection of the viral causes of Crimean-Congo hemorrhagic fever, Ebola virus disease, Lassa fever, Marburg virus disease and Rift Valley fever. We designed non-infectious synthetic DNA oligonucleotide sequences incorporating primer binding sites suitable for five assays, and a T7 promotor site which was used to transcribe the sequence. Transcribed RNA was used as template in a dilution series, extracted and amplified with RT-PCR and RT-qPCR to demonstrate successful recovery and determine limits of detection in a range of laboratory settings. Our results show this approach is adaptable to any diagnostic assay requiring validation of nucleic acid extraction and/or amplification, particularly where sourcing reliable, safe material for positive controls is infeasible.https://doi.org/10.1371/journal.pntd.0011390 |
| spellingShingle | Matthew A Knox Collette Bromhead David Ts Hayman Development of a non-infectious control for viral hemorrhagic fever PCR assays. PLoS Neglected Tropical Diseases |
| title | Development of a non-infectious control for viral hemorrhagic fever PCR assays. |
| title_full | Development of a non-infectious control for viral hemorrhagic fever PCR assays. |
| title_fullStr | Development of a non-infectious control for viral hemorrhagic fever PCR assays. |
| title_full_unstemmed | Development of a non-infectious control for viral hemorrhagic fever PCR assays. |
| title_short | Development of a non-infectious control for viral hemorrhagic fever PCR assays. |
| title_sort | development of a non infectious control for viral hemorrhagic fever pcr assays |
| url | https://doi.org/10.1371/journal.pntd.0011390 |
| work_keys_str_mv | AT matthewaknox developmentofanoninfectiouscontrolforviralhemorrhagicfeverpcrassays AT collettebromhead developmentofanoninfectiouscontrolforviralhemorrhagicfeverpcrassays AT davidtshayman developmentofanoninfectiouscontrolforviralhemorrhagicfeverpcrassays |