Genetic organization of Pasteurella multocida cap loci and development of a multiplex capsular PCR typing system

Current serotyping methods classify Pasteurella multocida into five capsula r serogroups (serogroups A, B, D, E, and F) and 16 somatic serotypes (serot ypes 1 to 16), In the present study, we have developed a multiplex PCR assa y as a rapid alternative to the conventional capsular serotyping system. Th e serogroup-specific primers used in this assay were designed following ide ntification, sequence determination, and analysis of the capsular biosynthe tic loci of each capsular serogroup, The entire capsular biosynthetic loci of P. multocicia A:1 (X-73) and B:2 (M1401) have been cloned and sequenced previously (J, Y. Chung, Y, M. Zhang, and B. Adler, FEMS Microbiol. Lett, 1 66:289-296, 1998; J. D. Boyce, J, Y. Chung, and B, Adler, Vet. Microbiol, 7 2:121-134, 2000), Nucleotide sequence analysis of the biosynthetic region ( region 2) from each of the remaining three serogroups, serogroups D, E, and F, identified serogroup-specific regions and gave an indication of the cap sular polysaccharide composition. The multiplex capsular PCR assay was high ly specific, and its results, with the exception of those for some serogrou p P strains, correlated well with conventional serotyping results, Sequence analysis of the strains that gave conflicting results confirmed the validi ty of the multiplex PCR and indicated that these strains were in fact capsu lar serogroup A. The multiplex PCR will clarify the distinction between clo sely related serogroups A and F and constitutes a rapid assay for the defin itive classification of P. multocida capsular types.Current serotyping meth ods classify Pasteurella multocida into five capsular serogroups (serogroup s A, B, D, E, and Fl and 16 somatic serotypes (serotypes I to 16), In the p resent study, we have developed a multiplex PCR assay as a rapid alternativ e to the conventional capsular serotyping system. The serogroup-specific pr imers used in this assay were designed following identification, sequence d etermination, and analysis of the capsular biosynthetic loci of each capsul ar serogroup, The entire capsular biosynthetic loci of P. multocida A:1 (X- 73) and B:2 (M1404) have been cloned and sequenced previously (J, Y. Chung, Y, M. Zhang, and B. Adler, FEMS Microbiol. Lett, 166:289-296, 1998; J. D. Boyce, J, Y. Chung, and B, Adler, Vet. Microbiol, 72:121-134, 2000), Nucleo tide sequence analysis of the biosynthetic region (region 2) from each of t he remaining three serogroups, serogroups D, E, and F, identified serogroup -specific regions and gave an indication of the capsular polysaccharide com position. The multiplex capsular PCR assay was highly specific, and its res ults, with the exception of those for some serogroup P strains, correlated well with conventional serotyping results, Sequence analysis of the strains that gave conflicting results confirmed the validity of the multiplex PCR and indicated that these strains were in fact capsular serogroup A. The mul tiplex PCR will clarify the distinction between closely related serogroups A and F and constitutes a rapid assay for the definitive classification of P. multocida capsular types.