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Rapid Detection of Actinobacillus pleuropneumoniae From Clinical Samples Using Recombinase Polymerase Amplification
Oliver W Stringer1, Yanwen Li1, Janine T Bossé1
1Section of Paediatric Infectious Disease, Department of Infectious Disease, Imperial College London, London, United Kingdom.
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.
Insights
A new rapid diagnostic test for Actinobacillus pleuropneumoniae (APP) was developed using isothermal recombinase polymerase amplification (RPA). This point-of-care assay offers a sensitive and specific tool for detecting APP, improving biosecurity in swine herds.
Area of Science:
- Veterinary Microbiology
- Animal Diagnostics
- Molecular Biology
Background:
- Actinobacillus pleuropneumoniae (APP) causes swine pleuropneumonia, leading to significant global economic losses.
- Current diagnostic methods for APP, such as bacterial culture and PCR, have limitations including low sensitivity, high cost, and time consumption.
- Effective biosecurity and surveillance rely on accurate and timely diagnostic tools for APP detection.
Purpose of the Study:
- To develop and validate a novel isothermal point-of-care diagnostic test for the rapid detection of Actinobacillus pleuropneumoniae.
- To target the species-specific apxIVA gene for enhanced diagnostic accuracy.
- To provide a sensitive and specific screening tool for APP surveillance in swine populations.
Main Methods:
- Development of an isothermal diagnostic assay utilizing recombinase polymerase amplification (RPA).
- Targeting the species-specific apxIVA gene of Actinobacillus pleuropneumoniae.
- Validation of the APP-RPA assay using laboratory strains and clinical samples (lung tissue, FTA cards) from England and Portugal.
Main Results:
- The APP-RPA assay demonstrated a sensitivity of 10 copies/μL for APP serovar 8.
- Clinical sensitivity and specificity were 84.3% and 100%, respectively, across 61 clinical samples.
- Testing lung imprints on FTA cards directly in the APP-RPA reaction yielded 76.9% clinical sensitivity and 100% specificity.
Conclusions:
- The developed APP-RPA assay is a rapid, sensitive, and specific screening tool for Actinobacillus pleuropneumoniae.
- This isothermal point-of-care test can aid in improving biosecurity and surveillance strategies for porcine pleuropneumonia.
- The APP-RPA assay offers a viable alternative to current time-consuming and resource-intensive diagnostic methods for APP.
