Identification of Staphylococcus spp. by PCR-restriction fragment length polymorphism analysis of dnaJ gene

Abstract

A PCR-restriction fragment length polymorphism (RFLP) analysis method that analyzes a part of the dnaJ gene was designed for the rapid and accurate identification of Staphylococcus spp. XapI or Bsp143I digestion of the PCR-generated products rendered distinctive RFLP patterns that allowed 41 reference species and subspecies to be identified with a high degree of specificity. The novel method was validated by the identification of 23 clinical staphylococcal strains, and the results were compared with those obtained by other genotypic identification methods. A 100% concordance of the results was shown. Therefore, PCR-RFLP analysis of the dnaJ gene is proposed as a reliable and reproducible method for the identification of Staphylococcus spp.

FIG. 1.

(A and B) Agarose gel electrophoresis of the fragments produced by XapI digestion of the PCR amplification products from Staphylococcus reference strains (Table 1). Lanes: 1, S. arlettae; 2, S. aureus subsp. anaerobius; 3, S. aureus subsp. aureus; 4, S. auricularis; 5, S. capitis subsp. capitis; 6, S. capitis subsp. urealyticus; 7, S. caprae; 8, S. carnosus subsp. carnosus; 9, S. carnosus subsp. utilis; 10, S. chromogenes; 11, S. cohnii subsp. cohnii; 12, S. cohnii subsp. urealyticus; 13, S. condimenti; 14, S. epidermidis; 15, S. equorum subsp. equorum; 16, S. equorum subsp. linens; 17, S. felis; 18, S. fleurettii; 19, S. gallinarum; 20, S. haemolyticus; 21, S. hominis subsp. hominis; 22, S. hominis subsp. novobiosepticus; 23, S. hyicus; 24, S. intermedius; 25, S. kloosii; 26, S. lentus; 27, S. lugdunensis; 28, S. muscae; 29, S. nepalensis; 30, S. pasteuri; 31, S. pettenkoferi; 32, S. piscifermentans; 33, S. pseudintermedius; 34, S. saccharolyticus; 35, S. saprophyticus subsp. bovis; 36, S. saprophyticus subsp. saprophyticus; 37, S. schleiferi subsp. coagulans; 38, S. sciuri subsp. carnaticus; 39, S. sciuri subsp. rodentium; 40, S. sciuri subsp. sciuri; 41, S. simulans; 42, S. succinus subsp. casei; 43, S. succinus subsp. succinus; 44, S. vitulinus; 45, S. warneri; 46, S. xylosus; 47, S. simiae; M, DNA molecular mass markers, 50-bp ladder (bottom band, 50 bp).

FIG. 2.

Agarose gel electrophoresis of the fragments produced by Bsp143I digestion of the PCR amplification products from Staphylococcus reference strains (Table 1). Lanes: 1, S. auricularis; 2, S. felis; 3, S. caprae; 4, S. pasteuri; 5, S. carnosus subsp. carnosus; 6, S. warneri; 7, S. cohnii subsp. cohnii,; 8, S. epidermidis; 9, S. kloosii; 10, S. saprophyticus subsp. bovis; 11, S. nepalensis; 12, S. pettenkoferi; M, DNA molecular mass markers, 50-bp ladder (bottom band, 50 bp).

FIG. 3.

Agarose gel electrophoresis of the fragments produced by XapI digestion of the PCR amplification products from clinical Staphylococcus isolates. Lanes: 1, S. aureus; 2, S. haemolyticus; 3, S. cohnii subsp. urealyticus; 4, S. epidermidis; 5, S. sciuri; 6, S. vitulinus; M, DNA molecular mass markers, 50-bp ladder (bottom band, 50 bp).