Detection and identification of Actinobacillus pleuropneumoniae serotype 5 by multiplex PCR.
Serotyping of Actinobacillus pleuropneumoniae is based on detection of the serotype-specific capsular antigen. However, not all isolates can be serotyped, and some may cross-react with multiple serotyping reagents. To improve sensitivity and specificity of serotyping and for early detection, a multiplex PCR assay was developed for detection of A. pleuropneumoniae and identification of serotype 5 isolates. DNA sequences specific to the conserved export and serotype-specific biosynthesis regions of the capsular polysaccharide of A. pleuropneumoniae serotype 5 were used as primers to amplify 0.7- and 1.1-kb DNA fragments, respectively. The 0.7-kb fragment was amplified from all strains of A. pleuropneumoniae tested with the exception of serotype 4. The 0.7-kb fragment was not amplified from any heterologous species that are also common pathogens or commensals of swine. In contrast, the 1.1-kb fragment was amplified from all serotype 5 strains only. The assay was capable of amplifying DNA from less than 10(2) CFU. The A. pleuropneumoniae serotype 5 capsular DNA products were readily amplified from lung tissues obtained from infected swine, although the 1.1-kb product was not amplified from some tissues stored frozen for 6 years. The multiplex PCR assay enabled us to detect A. pleuropneumoniae rapidly and to distinguish serotype 5 strains from other serotypes. The use of primers specific to the biosynthesis regions of other A. pleuropneumoniae serotypes would expand the diagnostic and epidemiologic capabilities of this assay.
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Center for Molecular Medicine and Infectious Disease, Virginia-Maryland Regional College of Veterinary Medicine, Virginia Polytechnic Institute and State University, Blacksburg 24061-0342, USA.
Ward
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Inzana
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TJ
D000189:Actinobacillus Infections / Q000175:diagnosis / Q000382:microbiology / Q000662:veterinary*; D016977:Actinobacillus pleuropneumoniae / Q000145:classification* / Q000235:genetics / Q000302:isolation & purification*; D000818:Animals; D016667:Bacterial Capsules / Q000235:genetics; D015373:Bacterial Typing Techniques; D015139:Blotting, Southern; D017931:DNA Primers; D004269:DNA, Bacterial / Q000032:analysis / Q000302:isolation & purification; D004587:Electrophoresis, Agar Gel; D008168:Lung / Q000382:microbiology; D009666:Nose / Q000382:microbiology; D011001:Pleuropneumonia / Q000382:microbiology / Q000662:veterinary*; D016133:Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D012703:Serotyping; D013045:Species Specificity; D013552:Swine; D013553:Swine Diseases / Q000382:microbiology*
D017931:DNA Primers; D004269:DNA, Bacterial