Design and evaluation of specific PCR primers for rapid and reliable identification of Staphylococcus xylosus strains isolated from dry fermented sausages.
Rapid and reliable identification of Staphylococcus xylosus was achieved by species-specific PCR assays. Two sets of primers, targeting on xylulokinase (xylB) and 60 kDa heat-shock protein (hsp60) genes of S. xylosus, respectively, were designed. Species-specificity of both sets of primers was evaluated by using 27 reference strains of the DSM collection, representing 23 different species of the Staphylococcus genus and 3 species of the Kocuria genus. Moreover, 90 wild strains isolated from different fermented dry sausages were included in the analysis. By using primers xylB-F and xylB-R the expected PCR fragment was obtained only when DNA from S. xylosus was used. By contrast, amplification performed by using primers xylHs-F and xylHs-R produced a single PCR fragment, of the expected length, when DNA from S. xylosus, S. haemolyticus, S. intermedius and S. kloosii were used as template. Nevertheless, AluI digestion of the xylHs-F/xylHs-R PCR fragment allowed a clear differentiation of these 4 species. The rapidity (about 4 h from DNA isolation to results) and reliability of the PCR procedures established suggests that the method may be profitably applied for specific detection and identification of S. xylosus strains.
Blaiotta
Giuseppe
G
Dipartimento di Scienza degli Alimenti, Sezione di Microbiologia Agraria, Alimentare, Ambientale e di Igiene, Stazione di Microbiologia Industriale, Università degli Studi di Napoli "Federico II", Portici, Italy.
Pennacchia
Carmelina
C
Parente
Eugenio
E
Villani
Francesco
F
D001426:Bacterial Proteins / Q000235:genetics; D018834:Chaperonin 60 / Q000235:genetics; D017931:DNA Primers; D004269:DNA, Bacterial / Q000032:analysis* / Q000302:isolation & purification; D004275:DNA, Ribosomal / Q000737:chemistry / Q000302:isolation & purification; D005516:Food Microbiology; D008461:Meat Products / Q000382:microbiology*; D008969:Molecular Sequence Data; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000235:genetics; D016133:Polymerase Chain Reaction; D012150:Polymorphism, Restriction Fragment Length; D012680:Sensitivity and Specificity; D017422:Sequence Analysis, DNA; D013210:Staphylococcus / Q000145:classification* / Q000235:genetics / Q000302:isolation & purification*
D001426:Bacterial Proteins; D018834:Chaperonin 60; D017931:DNA Primers; D004269:DNA, Bacterial; D004275:DNA, Ribosomal; D017853:Phosphotransferases (Alcohol Group Acceptor); C032700:xylulokinase