Molecular cloning and sequencing of the aroA gene from Actinobacillus pleuropneumoniae and its use in a PCR assay for rapid identification

The gene (aroA) of Actinobacillus pleuropneumoniae, serotype 2, encoding 5- enolpyruvylshikimate-3-phosphate synthase was cloned by complementation of the apoA mutation in Escherichia coli K-12 strain AB2829, and the nucleotid e sequence was determined, ii pair of primers from the 5' and 3' termini we re selected to be the basis for development of a specific PCR assay. ii DNA fragment of 1,025 bp was amplified from used A. pleuropneumoniae serotypes I to 12 of biovar 1 or from isolated DNA. No PCR products were detected wh en chromosomal DNAs from other genera were used as target DNAs; however, a 1,025-bp DNA fragment was amplified when Actinobacillus equuli chromosomal DIVA was used as a target, which could be easily differentiated by its NAD independence. The PCR assay developed was ver sensitive, with lower detecti on limits of 12 CFU with A. pleuropneumoniae cells and 0.8 pg with extracte d DNA. Specificity and sensitivity make this PCR assay a useful method for the rapid identification and diagnosis of A. pleuropneumoniae infections.