Multiplex PCR-based reverse line blot hybridization assay to identify 23 Streptococcus pneumoniae polysaccharide vaccine serotypes

Abstract

We developed a multiplex PCR-based reverse line blot assay to identify 23 pneumococcal serotypes represented in the polysaccharide vaccine, using 334 well-characterized isolates, representing all 90 serotypes, and 268 "unknowns." The assay identified all target serotypes, but 11, which cross-react with 1 to 4 nonvaccine serotypes, could be distinguished using serotype-specific antisera.

FIG. 1.

mPCR/RLB results for a representative sample of 43 clinical isolates. See Table 1 for descriptions and specificities of probes listed at left. The choice of probes for this membrane, in addition to two pairs of S. pneumoniae-specific probes, was based on two for each of the serotypes represented in the 11-valent conjugate vaccine (serotypes 1, 3, 4, 5, 6B, 7F, 9V, 14, 18C, 19F, and 23F [shown in the top half of the membrane]) and others that are identified relatively commonly (serotypes 8, 9N, 19A, 20, and 33F) and single probes for each of the less common serotypes among those represented in the 23-valent vaccine (serotypes 2, 10A, 11A, 12, 15B, 17F, and 22F). The conventional serotypes of the 43 isolates shown on this membrane are (from left to right): 14, NT, 19F, NT, 19F, 22F, 9V, 6A, 19F, 18C, NSP, 19F, 16F, 11A, 4, 19F, 19F, 19F, 19F, 6B, 10A, 19F, 23F, 19F, 23F, 19A, 23F, 6A, 6B, 9V, 3, 14, 19F, 6B, 11A, 6A, 14, 23F, 19F, 46, 47F, 47A, and 48. NT, nontypeable. NSP, not S. pneumoniae.