Single primer polymerase chain reaction fingerprinting for Pasteurella multocida isolates from laboratory rabbits
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1Department of Anatomy, Pathology and Pharmacology, College of Veterinary Medicine, Oklahoma State University, Stillwater 74078-2007, USA.
Objective:
To evaluate a rapid polymerase chain reaction (PCR) fingerprinting technique for discriminating among Pasteurella multocida isolates from laboratory rabbits.
Sample Population:
33 P multocida isolates from rabbits with clinical pasteurellosis.
Procedure:
PCR assays were conducted with 2 minisatellites (core sequence and modified core sequence of phage M13) and 2 microsatellites ([GTG]5 and [GACA]4). Each bacterium was assigned to a PCR type for each of the primers used. Boiled bacterial extracts and purified genomic DNA were compared by use of PCR assays for phage M13 and (GACA)4. Plasmids were isolated from each bacterium, and their influence on PCR fingerprint was determined, using boiled extracts as a DNA source.
Results:
M13 core sequence and M13 modified core sequence yielded 5 and 8 PCR types, respectively. The microsatellites (GTG)5 and (GACA)4 yielded 4 and 9 PCR fingerprint types, respectively. Fingerprint patterns obtained by use of isolated DNA differed from those obtained by use of boiled extracts, although discrimination among P multocida isolates was similar. The presence or absence of plasmids did not affect PCR fingerprints.
Conclusion:
Single primer PCR fingerprinting with minisatellite and microsatellite primers is an efficient and reproducible method for the discrimination of P multocida isolates from rabbits and can be performed directly, using boiled bacterial extracts as a source of template, although more bands were obtained from pure genomic DNA.
Insights
Rapid polymerase chain reaction (PCR) fingerprinting effectively differentiates Pasteurella multocida isolates from rabbits. This method using minisatellite and microsatellite primers is efficient and reproducible, even with boiled bacterial extracts.
Area of Science:
- Microbiology
- Molecular Biology
- Veterinary Science
Background:
- Pasteurella multocida is a significant pathogen in laboratory rabbits, causing pasteurellosis.
- Accurate and rapid identification of P. multocida strains is crucial for disease control and research.
- Existing methods for strain discrimination may be time-consuming or complex.
Purpose of the Study:
- To evaluate a rapid polymerase chain reaction (PCR) fingerprinting technique for discriminating among Pasteurella multocida isolates.
- To assess the efficiency and reproducibility of PCR fingerprinting using different types of primers.
- To determine the optimal sample preparation method for PCR analysis of P. multocida.
Main Methods:
- Utilized 33 P. multocida isolates from rabbits with clinical pasteurellosis.
- Employed PCR assays with minisatellite (M13 core, M13 modified core) and microsatellite ([GTG]5, [GACA]4) primers.
- Compared PCR fingerprint patterns generated from boiled bacterial extracts and purified genomic DNA, and assessed the impact of plasmids.
Main Results:
- Minisatellite and microsatellite primers generated distinct PCR fingerprint types, with [GACA]4 yielding the highest number of types (9).
- PCR fingerprint patterns from isolated DNA differed from boiled extracts, but discrimination efficacy was similar.
- The presence or absence of plasmids did not influence the PCR fingerprint results.
Conclusions:
- Single-primer PCR fingerprinting with minisatellite and microsatellite primers is an efficient and reproducible method for discriminating P. multocida isolates from rabbits.
- The technique can be performed directly using boiled bacterial extracts as a template source.
- While purified genomic DNA yielded more bands, boiled extracts provide a practical and effective alternative for strain differentiation.
