Rapid Detection of Actinobacillus pleuropneumoniae From Clinical Samples Using Recombinase Polymerase Amplification.
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Authors
Stringer, Oliver W
Li, Yanwen
Forrest, Matthew S
Hernandez-Garcia, Juan
Tucker, Alexander W
Nunes, Tiago
Costa, Francisco
Mortensen, Preben
Velazquez, Eduardo
Penny, Paul
Rodriguez-Manzano, Jesus
Georgiou, Pantelis
Langford, Paul R
Publication Date
2022Journal Title
Front Vet Sci
ISSN
2297-1769
Publisher
Frontiers Media SA
Volume
9
Language
eng
Type
Article
This Version
VoR
Metadata
Show full item recordCitation
Stringer, O. W., Li, Y., Bossé, J. T., Forrest, M. S., Hernandez-Garcia, J., Tucker, A. W., Nunes, T., et al. (2022). Rapid Detection of Actinobacillus pleuropneumoniae From Clinical Samples Using Recombinase Polymerase Amplification.. Front Vet Sci, 9 https://doi.org/10.3389/fvets.2022.805382
Abstract
Actinobacillus pleuropneumoniae (APP) is the causative agent of porcine pleuropneumonia, resulting in high economic impact worldwide. There are currently 19 known serovars of APP, with different ones being predominant in specific geographic regions. Outbreaks of pleuropneumonia, characterized by sudden respiratory difficulties and high mortality, can occur when infected pigs are brought into naïve herds, or by those carrying different serovars. Good biosecurity measures include regular diagnostic testing for surveillance purposes. Current gold standard diagnostic techniques lack sensitivity (bacterial culture), require expensive thermocycling machinery (PCR) and are time consuming (culture and PCR). Here we describe the development of an isothermal point-of-care diagnostic test - utilizing recombinase polymerase amplification (RPA) for the detection of APP, targeting the species-specific apxIVA gene. Our APP-RPA diagnostic test achieved a sensitivity of 10 copies/μL using a strain of APP serovar 8, which is the most prevalent serovar in the UK. Additionally, our APP-RPA assay achieved a clinical sensitivity and specificity of 84.3 and 100%, respectively, across 61 extracted clinical samples obtained from farms located in England and Portugal. Using a small subset (n = 14) of the lung tissue samples, we achieved a clinical sensitivity and specificity of 76.9 and 100%, respectively) using lung imprints made on FTA cards tested directly in the APP-RPA reaction. Our results demonstrate that our APP-RPA assay enables a suitable rapid and sensitive screening tool for this important veterinary pathogen.
Keywords
Point-of-care (Poc), Fta® Card, A. Pleuropneumoniae, Apxiva, Rpa (Recombinase Polymerase Amplification)
Sponsorship
Biotechnology and Biological Sciences Research Council (BB/M011178/1, BB/S005897/1, BB/S002103/1)
Identifiers
35400111, PMC8990124
External DOI: https://doi.org/10.3389/fvets.2022.805382
This record's URL: https://www.repository.cam.ac.uk/handle/1810/337058
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