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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...

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Autores principales: Flannery, John, Rajko-Nenow, Paulina, Hicks, Hayley, Hill, Holly, Gubbins, Simon, Batten, Carrie
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
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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.
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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
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