Multiplex PCR Assay for Direct Identification of Group B Streptococcal Alpha-Protein-Like Protein Genes

DOI: 10.1128/JCM.42.3.1326-1329.2004

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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.
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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.).

TABLE 1.

Nucleotide primer sequences and amplicon size expected for each S. agalactiae surface protein gene considered in this study

Primer	Sequence (5′-3′)	GenBank accession no.	Position from start codon (nt)^a	Amplicon size (bp)
Universal forward	TGATACTTCACAGACGAAACAACG		30
Alpha-C reverse	TACATGTGGTAGTCCATCTTCACC	M97256	428	398
Rib reverse	CATACTGAGCTTTTAAATCAGGTGA	U583333	325	295
Epsilon reverse	CCAGATACATTTTTTACTAAAGCGG	U33554	230	200
Alp2/3 reverse	CACTCGGATTACTATAATATTTAGCAC	AF208158	364	334
Alp4 reverse	TTAATTTGCACCGGATTAACACCAC	AJ488912	140	110

TABLE 2.

Characteristics of GBS strains used in this study

↵a The designation of the alpha-protein-like protein encoded by the gene is based both on PCR results and sequencing of the PCR product.
↵b The differentiation between the proteins encoded by the alp2 and alp3 genes was performed by sequencing entire genes as described in reference 10.