title: Development of a novel multiplex PCR assay for rapid detection of virulence associated genes of Pasteurella multocida from pigs.
abstract: As the pathogenicity of Pasteurella multocida is associated with various virulence factors (VFs), the aim of the study was to develop a novel multiplex PCR (m-PCR) assay for the rapid detection of important virulence associated genes (VAGs) of P. multocida isolates from pigs. The target recognized VFs used in the study were diverse adhesins (ptfA and pfhA), toxins (toxA), siderophores (tonB and hgbA), sialidases (nanB, nanH) and outer membrane proteins (ompA, ompH, oma87 and plpB). The primers for the genes encoding these VFs were designed by primer3 software (http://bioinfo.ut.ee/primer3-0.4.0/) using gene sequences available in Genbank. The detection limit of the developed assay was 10(2)  CFU ml(-1) . The m-PCR did not produce any nonspecific amplification products when tested against Bordetella bronchiseptica which also commonly infects pigs. We applied m-PCR to the field samples, and the results obtained were the same as the single PCR results. The developed assay would be very useful for veterinary diagnostic laboratories and for others interested in the rapid virulence profiling of porcine P. multocida isolates circulating in the piggeries.
  
  
  CONCLUSIONS
  The study reports the development and evaluation of a novel multiplex PCR assay for the rapid detection of 11 important VAGs of Pasteurella multocida isolates from pigs. Rapid and simultaneous detection of recognized VFs of the organism are essential to know the virulo-types of P. multocida isolates circulating in the piggeries. The developed novel assay will be very useful for the rapid detection of VAGs of P. multocida isolates from pigs.
authors.affiliation: National Research Centre on Pig, Indian Council of Agricultural Research, Guwahati, Assam, India.
authors.forename: S
authors.initials: S
authors.lastname: Rajkhowa
mesh_terms: D000818:Animals; D016950:Bordetella bronchiseptica / Q000235:genetics; D017931:DNA Primers / Q000235:genetics; D005798:Genes, Bacterial; D008969:Molecular Sequence Data; D060885:Multiplex Polymerase Chain Reaction / Q000379:methods*; D010326:Pasteurella Infections / Q000175:diagnosis / Q000662:veterinary*; D016979:Pasteurella multocida / Q000235:genetics / Q000302:isolation & purification* / Q000472:pathogenicity; D013552:Swine / Q000382:microbiology*; D013553:Swine Diseases / Q000175:diagnosis* / Q000382:microbiology; D037521:Virulence Factors / Q000235:genetics*
chemical_list: D017931:DNA Primers; D037521:Virulence Factors
keywords: Pasteurella multocida; multiplex PCR; porcine; rapid detection; virulence associated genes