Multiplex PCR assay that identifies the major lipooligosaccharide serotype expressed by Moraxella catarrhalis clinical isolates

Abstract

A heterologous cluster of glycosyltransferase genes was identified in the three Moraxella catarrhalis LOS serotype strains. Multiple PCR primers designed to this region amplified products that differentiate between the serotypes more rapidly and efficiently than previously described serological analyses. This assay will be valuable for clinical and research-based studies.

FIG. 1.

Organization of lgt clusters in strains of M. catarrhalis expressing different LOS serotypes and binding sites of the primers used in the multiplex PCRs. (A) Representative (putative) lgt cluster in a LOS serotype A genome from M. catarrhalis strain ATCC 25238 (NCBI accession number DQ137417). (B) Representative lgt cluster in a LOS serotype B genome from M. catarrhalis strain 7169 (Edwards, 2005). (C) Representative (putative) lgt cluster in a LOS serotype C genome from M. catarrhalis strain RS10 (NCBI accession number DQ137418). The 5′ end of the cluster in serotype A and C strains contained DNA sequence that is homologous to the 5′ region of the lgt cluster in 7169, as depicted by gray shading in all three clusters (A to C). Black sequence portion in panel A represents a region of sequence divergence at the 3′ end of lgt2_A.

FIG. 2.

Representative multiplex PCR test using primers 406, 408, and 649 (Table 1) with chromosomal DNA isolated from various M. catarrhalis isolates. Lanes 1 to 3 show amplicons from the three LOS serotype control strains: LOS serotype A, ATCC 25238 (lane 1); LOS serotype B, 7169 (lane 2); and LOS serotype C, RS10 (lane 3). Lanes 4 to 8 contain amplicons from five COPD patients' isolates: 3P3B1 (lane 4), 51P9B1 (lane 5), 5P26B1 (lane 6), 18P6B1 (lane 7), and 12P80B1 (lane 8). Lanes 9 to 13 contain amplicons from five children's OME isolates: HF-039 (lane 9), HF-2246 (lane 10), HF-165 (lane 11), HF-218 (lane 12), and HF-006 (lane 13). Lanes 14 to 18 contain amplicons from five geographically diverse (global) isolates: 2951 (lane 14), 035E (lane 15), CCUG 3292 (lane 16), ATCC 43617 (lane 17), and CCUG 26391 (lane 18).