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Publication Details
AFRICAN RESEARCH NEXUS
SHINING A SPOTLIGHT ON AFRICAN RESEARCH
immunology and microbiology
Development and inter-laboratory validation study of an improved new real-time PCR assay with internal control for detection and laboratory diagnosis of African swine fever virus
Journal of Virological Methods, Volume 178, No. 1-2, Year 2011
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Description
A real-time polymerase chain reaction (PCR) assay for the rapid detection of African swine fever virus (ASFV), multiplexed for simultaneous detection of swine beta-actin as an endogenous control, has been developed and validated by four National Reference Laboratories of the European Union for African swine fever (ASF) including the European Union Reference Laboratory. Primers and a TaqMan ® probe specific for ASFV were selected from conserved regions of the p72 gene. The limit of detection of the new real-time PCR assay is 5.7-57 copies of the ASFV genome. High accuracy, reproducibility and robustness of the PCR assay (CV ranging from 0.7 to 5.4%) were demonstrated both within and between laboratories using different real-time PCR equipments. The specificity of virus detection was validated using a panel of 44 isolates collected over many years in various geographical locations in Europe, Africa and America, including recent isolates from the Caucasus region, Sardinia, East and West Africa. Compared to the OIE-prescribed conventional and real-time PCR assays, the sensitivity of the new assay with internal control was improved, as demonstrated by testing 281 field samples collected in recent outbreaks and surveillance areas in Europe and Africa (170 samples) together with samples obtained through experimental infections (111 samples). This is particularly evident in the early days following experimental infection and during the course of the disease in pigs sub-clinically infected with strains of low virulence (from 35 up to 70dpi). The specificity of the assay was also confirmed on 150 samples from uninfected pigs and wild boar from ASF-free areas. Measured on the total of 431 tested samples, the positive deviation of the new assay reaches 21% or 26% compared to PCR and real-time PCR methods recommended by OIE. This improved and rigorously validated real-time PCR assay with internal control will provide a rapid, sensitive and reliable molecular tool for ASFV detection in pigs in newly infected areas, control in endemic areas and surveillance in ASF-free areas. © 2011 Elsevier B.V.
Authors & Co-Authors
Tignon, Marylène
Belgium, Brussels
Veterinary and Agrochemical Research Centre
Gallardo, Carmina
Spain, Valdeolmos
Csic-inia-cisa - Centro de Investigación en Sanidad Animal
Iscaro, Carmen
Italy, Perugia
Istituto Zooprofilattico Sperimentale Dell'umbria e Delle Marche
Hutet, Evelyne
France, Ploufragan
Agence Nationale de Sécurité Sanitaire de L'alimentation de L'environnement et du Travail Anses
Van der Stede, Yves
Belgium, Brussels
Veterinary and Agrochemical Research Centre
Kolbasov, Denis V.
Russian Federation, Petushki
Federal Research Center for Virology and Microbiology, Russian Academy of Agricultural Sciences
De Mia, Gian Mario
Italy, Perugia
Istituto Zooprofilattico Sperimentale Dell'umbria e Delle Marche
Le Potier, Marie Frédérique L.E.
France, Ploufragan
Agence Nationale de Sécurité Sanitaire de L'alimentation de L'environnement et du Travail Anses
Bishop, Richard Peter
Kenya, Nairobi
International Livestock Research Institute Nairobi
Arias, M.
Spain, Valdeolmos
Csic-inia-cisa - Centro de Investigación en Sanidad Animal
Koenen, Frank
Belgium, Brussels
Veterinary and Agrochemical Research Centre
Statistics
Citations: 118
Authors: 11
Affiliations: 6
Identifiers
Doi:
10.1016/j.jviromet.2011.09.007
ISSN:
01660934
e-ISSN:
18790984
Research Areas
Genetics And Genomics
Study Locations
Multi-countries