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Single primer polymerase chain reaction fingerprinting for Pasteurella multocida isolates from laboratory rabbits
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.