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Direct PCR analysis for toxigenic Pasteurella multocida
C A Lichtensteiger1, S M Steenbergen, R M Lee
1Department of Veterinary Pathobiology, College of Veterinary Medicine, University of Illinois, Urbana 61801, USA. clichten@uiuc.edu
Abstract:
A more rapid, accurate method to detect toxigenic Pasteurella multocida is needed for improved clinical diagnosis, farm biosecurity, and epidemiological studies. Toxigenic and nontoxigenic P. multocida isolates cannot be differentiated by morphology or standard biochemical reactions. The feasibility of using PCR for accurate, rapid detection of toxigenic P. multocida from swabs was investigated. A PCR protocol which results in amplification of an 846-nucleotide segment of the toxA gene was developed. The PCR amplification protocol is specific for toxigenic P. multocida and can detect fewer than 100 bacteria. There was concordance of PCR results with (i) detection of toxA gene with colony blot hybridization, (ii) detection of ToxA protein with colony immunoblot analysis, and (iii) lethal toxicity of sonicate in mice in a test set of 40 swine diagnostic isolates. Results of an enzyme-linked immunosorbent assay for ToxA agreed with the other assays except for a negative reaction in one of the 19 isolates that the other assays identified as toxigenic. In addition to accuracy, as required for a rapid direct specimen assay, toxigenic P. multocida was recovered efficiently from inoculated swabs without inhibition of the PCR. The results show that PCR detection of toxigenic P. multocida directly from clinical swab specimens should be feasible.
Insights
A new PCR method accurately detects toxigenic Pasteurella multocida (toxA gene) in swine. This rapid test improves diagnosis, farm biosecurity, and disease tracking.
Area of Science:
- Veterinary Microbiology
- Molecular Diagnostics
- Bacterial Pathogenesis
Background:
- Accurate detection of toxigenic Pasteurella multocida is crucial for animal health and disease control.
- Conventional methods struggle to differentiate toxigenic from nontoxigenic strains of P. multocida.
- There is a need for rapid, reliable diagnostic tools for clinical and epidemiological purposes.
Purpose of the Study:
- To investigate the feasibility of using Polymerase Chain Reaction (PCR) for rapid and accurate detection of toxigenic P. multocida directly from clinical samples.
- To develop and validate a specific PCR assay targeting the toxA gene.
Main Methods:
- Development of a PCR protocol to amplify an 846-nucleotide segment of the toxA gene.
- Testing the PCR assay on 40 swine diagnostic isolates.
- Comparison of PCR results with colony blot hybridization, colony immunoblot analysis, and mouse toxicity assays.
- Evaluation of PCR performance on inoculated swabs to assess direct specimen detection.
Main Results:
- The developed PCR protocol specifically amplifies the toxA gene in toxigenic P. multocida.
- The assay is highly sensitive, detecting fewer than 100 bacterial cells.
- PCR results showed strong concordance with gene detection, protein detection, and toxicity assays.
- The PCR method demonstrated efficient recovery of bacteria from swabs without inhibition.
Conclusions:
- PCR offers a rapid, accurate, and sensitive method for detecting toxigenic P. multocida.
- This molecular technique is feasible for direct detection from clinical swab specimens.
- The PCR assay can significantly enhance clinical diagnosis, farm biosecurity, and epidemiological surveillance of P. multocida infections.