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Journal of Clinical Microbiology, February 1998, p. 443-448, Vol. 36, No. 2
0095-1137/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.

Improved Diagnostic PCR Assay for Actinobacillus pleuropneumoniae Based on the Nucleotide Sequence of an Outer Membrane Lipoprotein

Trine Gram* and Peter Ahrens

Danish Veterinary Laboratory, DK-1790 Copenhagen V, Denmark

Received 24 June 1997/Accepted 12 November 1997

The gene (omlA) coding for an outer membrane protein of Actinobacillus pleuropneumoniae serotypes 1 and 5 has been described earlier and has formed the basis for development of a specific PCR assay. The corresponding regions of all 12 A. pleuropneumoniae reference strains of biovar 1 were sequenced. Alignment of the sequences revealed conserved terminal and variable middle regions, which divided the reference strains into four distinct groups. Primers were selected from the conserved 5' and 3' termini of the gene. A 950-bp amplicon was obtained from each of 102 tested field isolates of A. pleuropneumoniae obtained from lungs. Their identity was verified by sequencing approximately 500 bp of the amplification product from 50 of the A. pleuropneumoniae isolates, which all showed the expected DNA sequence characteristic of the serotype. To test the specificity of the reaction, 23 other bacterial species related to A. pleuropneumoniae or isolated from pigs were assayed. They were all found negative in the PCR, as were tonsil cultures from 50 pigs of an A. pleuropneumoniae-negative herd. The sensitivity assessed by agarose gel analysis of the PCR product was 102 CFU/PCR test tube. The specificity and sensitivity of this PCR compared to those of culture suggest the use of this PCR for routine identification of A. pleuropneumoniae.

* Corresponding author. Mailing address: Danish Veterinary Laboratory, Bülowsvej 27, DK-1790 Copenhagen V, Denmark. Phone: 45 35300100. Fax: 45 35300120. E-mail: tg{at}svs.dk.

Journal of Clinical Microbiology, February 1998, p. 443-448, Vol. 36, No. 2
0095-1137/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.


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