Rapid and accurate identification of human-associated staphylococci by use of multiplex PCR

Abstract

Although staphylococci are identified by phenotypic analysis in many clinical laboratories, these results are often incorrect because of phenotypic variation. Genetic analysis is necessary for definitive species identification. In the present study, we developed a simple multiplex-PCR (M-PCR) for species identification of human-associated staphylococci, which were as follows: Staphylococcus aureus, S. capitis, S. caprae, S. epidermidis, S. haemolyticus, S. hominis, S. lugdunensis, S. saprophyticus, and S. warneri. This method was designed on the basis of nucleotide sequences of the thermonuclease (nuc) genes that were universally conserved in staphylococci except the S. sciuri group and showed moderate sequence diversity. In order to validate this assay, 361 staphylococcal strains were studied, which had been identified at the species levels by sequence analysis of the hsp60 genes. In consequence, M-PCR demonstrated a sensitivity of 100% and a specificity of 100%. By virtue of simplicity and accuracy, this method will be useful in clinical research.

Fig. 1.

Electrophoresis after multiplex PCR for species identification of human-associated staphylococci on a 1.0% agarose gel. Lanes: 1, S. hominis; 2, S. epidermidis; 3, S. aureus; 4, S. haemolyticus; 5, S. capitis; 6, S. lugdunensis; 7, S. saprophyticus; 8, S. warneri; 9, S. caprae; 10, water (negative control).

Fig. 2.

The phylogenetic tree based on amino acid sequences of the nuc genes of staphylococci (staphylococcal thermonuclease group), staphylococcal nuclease family proteins of Gram-positive bacteria (staphylococcal nuclease group), and thermonuclease family proteins of thermophilic bacteria (thermophilic bacterial thermonuclease group). The tree was constructed by the neighbor-joining method using CLUSTAL W. The genes on plasmids and a prophage are indicated by open circles and a filled circle, respectively.