Cargando…
Evaluating the most appropriate pooling ratio for EDTA blood samples to detect Bluetongue virus using real-time RT-PCR
The control of Bluetongue virus (BTV) presents a significant challenge to European Union (EU) member states as trade restrictions are placed on animals imported from BTV-affected countries. BTV surveillance programs are costly to maintain, thus, pooling of EDTA blood samples is used to reduce costs...
Autores principales: | , , , , , |
---|---|
Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Elsevier Scientific Pub. Co
2018
|
Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5904549/ https://www.ncbi.nlm.nih.gov/pubmed/29615257 http://dx.doi.org/10.1016/j.vetmic.2018.03.001 |
_version_ | 1783315127874879488 |
---|---|
author | Flannery, John Rajko-Nenow, Paulina Hicks, Hayley Hill, Holly Gubbins, Simon Batten, Carrie |
author_facet | Flannery, John Rajko-Nenow, Paulina Hicks, Hayley Hill, Holly Gubbins, Simon Batten, Carrie |
author_sort | Flannery, John |
collection | PubMed |
description | The control of Bluetongue virus (BTV) presents a significant challenge to European Union (EU) member states as trade restrictions are placed on animals imported from BTV-affected countries. BTV surveillance programs are costly to maintain, thus, pooling of EDTA blood samples is used to reduce costs and increase throughput. We investigated different pooling ratios (1:2, 1:5, 1:10 and 1:20) for EDTA blood samples to detect a single BTV positive animal. A published real-time RT-PCR assay (Hofmann et al., 2008) and a commercial assay (ThermoFisher VetMax™ BTV NS3 kit) were used to analyse BTV RNA extracted from pooled EDTA blood samples. The detection rate was low for the onset of infection sample (0–2 days post infection (dpi); C(T) 36) irrespective of the pooling ratio. Both assays could reliably detect a single BTV-positive animal at early viraemia (3–6 dpi; C(T) 33) when pooled, however, detection rate diminished with increasing pooling ratio. A statistical model indicated that pooling samples up to 1:20, is suitable to detect a single BTV positive animal at peak viraemia (7–12 dpi) or late infection (13–30 dpi) with a probability of detection of >80% and >94% using the Hofmann et al. (2008) and VetMAX assays, respectively. Using the assays highlighted in our study, pooling at ratios of 1:20 would be technically suitable in BTV-endemic countries for surveillance purposes. As peak viraemia occurs between 7–12 days post infection, a 1:10 pooling ratio is appropriate for post-import testing when animals are sampled within a similar time frame post-import. |
format | Online Article Text |
id | pubmed-5904549 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2018 |
publisher | Elsevier Scientific Pub. Co |
record_format | MEDLINE/PubMed |
spelling | pubmed-59045492018-04-19 Evaluating the most appropriate pooling ratio for EDTA blood samples to detect Bluetongue virus using real-time RT-PCR Flannery, John Rajko-Nenow, Paulina Hicks, Hayley Hill, Holly Gubbins, Simon Batten, Carrie Vet Microbiol Article The control of Bluetongue virus (BTV) presents a significant challenge to European Union (EU) member states as trade restrictions are placed on animals imported from BTV-affected countries. BTV surveillance programs are costly to maintain, thus, pooling of EDTA blood samples is used to reduce costs and increase throughput. We investigated different pooling ratios (1:2, 1:5, 1:10 and 1:20) for EDTA blood samples to detect a single BTV positive animal. A published real-time RT-PCR assay (Hofmann et al., 2008) and a commercial assay (ThermoFisher VetMax™ BTV NS3 kit) were used to analyse BTV RNA extracted from pooled EDTA blood samples. The detection rate was low for the onset of infection sample (0–2 days post infection (dpi); C(T) 36) irrespective of the pooling ratio. Both assays could reliably detect a single BTV-positive animal at early viraemia (3–6 dpi; C(T) 33) when pooled, however, detection rate diminished with increasing pooling ratio. A statistical model indicated that pooling samples up to 1:20, is suitable to detect a single BTV positive animal at peak viraemia (7–12 dpi) or late infection (13–30 dpi) with a probability of detection of >80% and >94% using the Hofmann et al. (2008) and VetMAX assays, respectively. Using the assays highlighted in our study, pooling at ratios of 1:20 would be technically suitable in BTV-endemic countries for surveillance purposes. As peak viraemia occurs between 7–12 days post infection, a 1:10 pooling ratio is appropriate for post-import testing when animals are sampled within a similar time frame post-import. Elsevier Scientific Pub. Co 2018-04 /pmc/articles/PMC5904549/ /pubmed/29615257 http://dx.doi.org/10.1016/j.vetmic.2018.03.001 Text en © 2018 The Authors http://creativecommons.org/licenses/by/4.0/ This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/). |
spellingShingle | Article Flannery, John Rajko-Nenow, Paulina Hicks, Hayley Hill, Holly Gubbins, Simon Batten, Carrie Evaluating the most appropriate pooling ratio for EDTA blood samples to detect Bluetongue virus using real-time RT-PCR |
title | Evaluating the most appropriate pooling ratio for EDTA blood samples to detect Bluetongue virus using real-time RT-PCR |
title_full | Evaluating the most appropriate pooling ratio for EDTA blood samples to detect Bluetongue virus using real-time RT-PCR |
title_fullStr | Evaluating the most appropriate pooling ratio for EDTA blood samples to detect Bluetongue virus using real-time RT-PCR |
title_full_unstemmed | Evaluating the most appropriate pooling ratio for EDTA blood samples to detect Bluetongue virus using real-time RT-PCR |
title_short | Evaluating the most appropriate pooling ratio for EDTA blood samples to detect Bluetongue virus using real-time RT-PCR |
title_sort | evaluating the most appropriate pooling ratio for edta blood samples to detect bluetongue virus using real-time rt-pcr |
topic | Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5904549/ https://www.ncbi.nlm.nih.gov/pubmed/29615257 http://dx.doi.org/10.1016/j.vetmic.2018.03.001 |
work_keys_str_mv | AT flanneryjohn evaluatingthemostappropriatepoolingratioforedtabloodsamplestodetectbluetonguevirususingrealtimertpcr AT rajkonenowpaulina evaluatingthemostappropriatepoolingratioforedtabloodsamplestodetectbluetonguevirususingrealtimertpcr AT hickshayley evaluatingthemostappropriatepoolingratioforedtabloodsamplestodetectbluetonguevirususingrealtimertpcr AT hillholly evaluatingthemostappropriatepoolingratioforedtabloodsamplestodetectbluetonguevirususingrealtimertpcr AT gubbinssimon evaluatingthemostappropriatepoolingratioforedtabloodsamplestodetectbluetonguevirususingrealtimertpcr AT battencarrie evaluatingthemostappropriatepoolingratioforedtabloodsamplestodetectbluetonguevirususingrealtimertpcr |