Construction of a DNA probe and detection of Actinobacillus pleuropneumoniae by using polymerase chain reaction

Abstract

A 1.5-kb Actinobacillus pleuropneumoniae 4074 DNA fragment from a genomic library was found to hybridize to DNA from the 12 A. pleuropneumoniae serotypes by Southern hybridization. No cross-hybridization was detected with DNAs from hemolytic members of the family Pasteurellaceae. From the nucleotide sequence of the putative genomic probe, three primers were synthesized for use in polymerase chain reactions (PCRs), with 31 strains tested by using purified and crude DNA targets. PCR amplification products of 610 and 985 bp were observed in nucleic acids extracted from the 12 known serotypes and a biotype 2 strain. Template DNAs from other gram-negative and gram-positive bacteria, some of them found in the normal flora of swine and the upper respiratory tract, were not amplified by PCR. The only exception was an amplification of a similar 610- or 985-bp sequence in Actinobacillus lignieresii, a species that is closely related to A. pleuropneumoniae but that has never been isolated from swine. Amplification of specific A. pleuropneumoniae sequences by PCR directly from clinical specimens may find applications in the identification of asymptomatic carriers as well as in efforts to eradicate porcine pleuropneumonia.