Discrimination of major capsular types of Campylobacter jejuni by multiplex PCR

Abstract

The polysaccharide capsule (CPS) of Campylobacter jejuni is the major serodeterminant of the Penner serotyping scheme. There are 47 Penner serotypes of C. jejuni, 22 of which fall into complexes of related serotypes. A multiplex PCR method for determination of capsule types of Campylobacter jejuni which is simpler and more affordable than classical Penner typing was developed. Primers specific for each capsule type were designed on the basis of a database of gene sequences from the variable capsule loci of 8 strains of major serotypes sequenced in this study and 10 published sequences of other serotypes. DNA sequence analysis revealed a mosaic nature of the capsule loci, suggesting reassortment of genes by horizontal transfer, and demonstrated a high degree of conservation of genes within Penner complexes. The multiplex PCR can distinguish 17 individual serotypes in two PCRs with sensitivities and specificities ranging from 90 to 100% using 244 strains of known Penner type.

Fig. 1.

Schematic of variable CPS loci from representative Penner serotypes. CPS loci were sequenced from the strains listed in Table 1. Gene names were attributed if the predicted protein showed >80% sequence identity with other known C. jejuni proteins. Genes are color coded as shown in the key to the figure on the basis of best homology to any predicted protein in databases by BLAST analyses. Genes containing homopolymeric tracts of >8 G or C residues are marked with an asterisk.

Fig. 2.

Comparison of sequences of CPS loci of C. jejuni strains. (A) Comparison of the sequences of the CPS loci of C. jejuni HS4/13/64 (strain GC8486) and the HS4 type strain; (B) comparison of the CPS loci of C. jejuni HS4/13/64 (strain GC8486), the HS10 type strain, and a strain of HS41 (strain 176.83) (17); (C) comparison of the CPS loci of C. jejuni HS4/13/64 (strain GC8486), the HS15 type strain, and an HS41 strain (strain 176.83); (D) comparison of the CPS loci of strains C. jejuni HS41 (strain 176.83) and the type strains of HS8, HS17, and HS42. Vertical bars represent identical regions between loci. The red bars correspond to regions that are oriented similarly, and blue bars indicate regions oriented in opposite directions. The outermost genes in red represent kpsC (left) and kpsF (right), two conserved genes in CPS synthesis that bracket the hypervariable CPS loci. The comparison was made using the Artemis comparison tool (ACT).

Fig. 3.

Representative multiplex PCR with alpha and beta mixes separated on 2% agarose. (A) Lane 1, mixture of PCR products amplified with the alpha primer sets on HS4A, HS15, HS41, HS53, HS10, HS6, HS3, and HS2 DNAs; lane 2, 100-bp NEB DNA standard; lane 3, mixture of PCR products amplified with the beta primer sets on HS4B, HS1, HS42, HS17, HS23, and HS44 DNAs. Amplicons were separated on 2% agarose as described in Materials and Methods. The position of each CPS-specific amplicon is shown. (B) Typical PCR products obtained with primer mixes alpha and beta. Lane 1, alpha primer sets on HS3 DNA; lane 2, alpha primer sets on HS4 DNA; lane 3, alpha primer sets on HS6 DNA; lane 4, 100-bp NEB DNA standard; lane 5, alpha primer sets on HS10 DNA; lane 6, alpha primer sets on HS15 DNA; lane 7, alpha primer sets on HS41 DNA; lane 8, alpha primer sets on HS53 DNA; lane 9, 100-bp NEB DNA standard; lane 10, PCR beta primer sets on HS1 DNA; lane 11, beta primer sets on HS4/13/64 DNA; lane 12, beta primer sets on HS8 DNA; lane 13, beta primer sets on HS23 DNA.