Multiplex PCR assay for direct identification of group B streptococcal alpha-protein-like protein genes

Abstract

We developed a group B streptococcus multiplex PCR assay which allows, by direct analysis of the amplicon size, determination of the surface protein antigen genes of alpha-C protein, epsilon protein, Rib, Alp2, Alp3, and Alp4. The multiplex PCR assay offers a rapid and simple method of subtyping Streptococcus agalactiae based on surface protein genes.

FIG. 1.

Amino acid multisequence alignment of the N-terminal portions of S. agalactiae alpha-C protein, Alp2, Alp3, Rib, and the epsilon protein. The nucleotide sequence corresponding to the string from positions 11 to 17, indicated in grey, was used as the forward universal primer. The reverse primers sequences, specific for each protein gene (except for Alp2/3), are also indicated in grey.

FIG. 2.

Gel electrophoresis of multiplex PCR amplification products. Direct analysis of amplicon size allowed the determination of the genes encoding the surface protein of each GBS strain as follows: Rib (lanes 1, 2, and 8), alpha-C protein (lanes 3 and 10), Alp2/3 (lanes 4, 6, 9, and 12), Alp4 (lane 7), and the epsilon protein (lanes 5 and 1l). Lane 13 is the negative control. M, 2-log DNA ladder (0.1 to 10.0 kb; New England Biolabs, Inc.).