DNA microarray-based identification and typing of Actinobacillus pleuropneumoniae

Abstract

A DNA microarray system was prepared and shown to facilitate identification and typing of Actinobacillus pleuropneumoniae. The DNA microarray, composed of 18 DNA polymerase chain reaction (PCR) amplicons printed on glass slides and arranged in 3 subarrays, was developed. These target DNA included 1 or multiple fragments of the outer membrane lipoprotein, apx toxin, capsular polysaccharide, and disulfide bound formation protein E (dsbE)-like genes of A. pleuropneumoniae. These arrayed target DNA retained their expected hybridization properties. The hybridization signal intensities ranged from the least-intense to the most-intense, 4626 to 9789 arbitrary fluorescence units, respectively. Cy3-probes of A. pleuropneumoniae strains labeled with multiplex PCR were hybridized to the DNA microarray. A total of 51 different A. pleuropneumoniae strains representing serotype 1 to 12 reference strains and clinical isolates were detected and typed by the DNA microarray. Twelve reference serotypes produced 11 distinct target DNA hybridization patterns, and hybridization patterns of serotypes 1 (n = 7), 3 (n = 5), and 7 (n = 6) field isolates were identical to hybridization patterns of reference serotypes 1, 3, and 7, respectively. Non-serotyped isolates 4, 6, and 11 (out of 21) from diseased pigs had identical hybridization patterns to reference serotypes 3, 7, and 1, respectively. The results show that the DNA microarray system described in the present study is a valuable tool for identifying and typing reference strains and isolates of A. pleuropneumoniae, and enables relatively rapid identification of non-serotyped isolates.

Figure 1

A selection of the resulting fluorescence images from 18 target genes individually hybridized to 3 subarrays. One target DNA was spotted 10 times in a 10 × 10-spot subarray. Cps1-cps1, omlA4-omlA4, and apxIIIA-apxIIIA were specific hybridization; apxID-apxIIID was cross-hybridization. Cps1-cps1 hybridization produced the most-intense signals and apxIIIA-apxIIIA hybridization produced the least-intense signals on these arrays at homologous targets. 3 × SSC was blank control.

Figure 2

Quantitation of specific hybridization signals of 18 target DNA fragments. A glass background value was automatically subtracted from the 260- to 300-μm region surrounding each spot. The mean intensity and standard deviation (n = 10 spots) were calculated for each target.

Figure 3

Agarose gel electrophoresis of products generated by multiplex polymerase chain reaction (PCR) with primer combination I for labeling serotypes 1, 2, 3, 4, and 6, reference strains. M: marker DL1500; Lane: 1 to 5, serotypes 1 to 4, 6, respectively.

Figure 4

Agarose gel electrophoresis of products generated by multiplex polymerase chain reaction (PCR) with primer combination I for labeling serotypes 7 to 10, reference strains. M: marker DL1500; Lane: 1 to 4, serotypes 7 to 10, respectively.

Figure 5

Agarose gel electrophoresis of products generated by multiplex polymerase chain reaction (PCR) with primer combination II for labeling serotypes 1 to 4, reference strains. M: marker DL1500; Lane: 1 to 4, serotypes 1 to 4, respectively.

Figure 6

Agarose gel electrophoresis of products generated by multiplex polymerase chain reaction (PCR) with primer combination II for labeling serotypes 6 to 10, reference strains. M: marker DL1500; Lane: 1 to 5, serotypes 6 to 10, respectively.