To establish a method for specific detection and typing of A. pleuropneumoniae, we investigated the feasibility of PCR and restriction fragment length polymorphism (RFLP). Primers for PCR were selected from a conserved region of the outer membrane lipoprotein gene (omlA) of A. pleuropneumoniae serotypes 1 and 5a. A DNA fragment of 970 bp was amplified from the genomic DNA of all 12 serotypes of A. pleuropneumoniae. The amplification was specific to the A. pleuropneumoniae DNA but not to those of other bacteria tested, some of them are found as normal flora in upper respiratory tract of swine. The amplified DNA from all 12 serotypes specifically hybridized to the cloned omlA5a gene of A. pleuropneumoniae. This PCR system could detect as little as 20 pg of purified chromosomal DNA. Furthermore, digestion of the amplified DNA with the enzyme VspI yielded serotype specific polymorphic patterns, allowing discrimination of all serotypes into five distinct groups.
(Lab. of Molecular Bacteriology, Department of Bacteriology and Parasitology)
