J Med Microbiol 56 (2007), 1346-1349; DOI: 10.1099/jmm.0.47235-0
© 2007 Society for General Microbiology
ISSN 1473-5644
Development of a real-time PCR assay for the detection and identification of Staphylococcus capitis, Staphylococcus haemolyticus and Staphylococcus warneri
Tadayuki Iwase,
Keiko Seki,
Hitomi Shinji,
Yoshimitsu Mizunoe and
Shogo Masuda
Department of Microbiology II, Jikei University School of Medicine, 3-25-8 Nishi-Shinbashi, Minato-Ku, Tokyo 105-8461, Japan
Correspondence
Tadayuki Iwase
iwase.tadayuki{at}jikei.ac.jp
Received 17 February 2007
Accepted 13 June 2007
Staphylococcus capitis, Staphylococcus haemolyticus and Staphylococcus warneri are coagulase-negative staphylococci. Each species has different characteristics, and a difference in pathology is also seen in compromised hosts. Therefore, the development of a species-specific simple detection method for the identification of these staphylococci is important. Here, a species-specific real-time PCR assay is reported that targets the superoxide dismutase A-encoding gene of these bacteria. Primers were designed with a base that was non-complementary with regard to the other bacteria. This base was at the 3' end of the primer (3' mismatch primer) and conferred high specificity. These primers were then evaluated using real-time PCR. They reacted only with the target bacterium. In addition, stable quantitative reactions were observed when experiments were performed using genomic DNA extracted from varying numbers of staphylococci cells (101–107 cells). These results indicate that this method is useful for the identification and quantitative analysis of S. capitis, S. haemolyticus and S. warneri.
Abbreviations: CNS, coagulase-negative staphylococci.
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INTRODUCTION
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Staphylococcus capitis, Staphylococcus haemolyticus and Staphylococcus warneri are coagulase-negative staphylococci (CNS) that inhabit human skin (Kloos & Schleifer, 1975). Although infection with these staphylococci is rare compared with Staphylococcus aureus, staphylococci infection increases gradually in compromised hosts (Buttery et al., 1997; Wang et al., 1999; Otto, 2004; Raponi et al., 2005; Stollberger et al., 2006). In addition, these bacteria have been reported to possess drug resistance (Froggatt et al., 1989; Monsen et al., 1999; Wang et al., 1999; Raponi et al., 2005; de Allori et al., 2006). Therefore, accurate detection is important (Pfaller & Herwaldt, 1988).
A conventional technique for the identification of staphylococci is the sugar fermentation test. However, this method often results in false positives (Skulnick et al., 1989; Bannerman et al., 1993; Perl et al., 1994; Lee & Park, 2001). In contrast, results obtained using PCR are more accurate; the 16S rRNA gene, a housekeeping gene, is generally targeted when species-specific detection by PCR is performed. However, the 16S rRNA gene of Staphylococcus epidermidis is highly similar to that in other CNS. In order to solve this problem, it is possible to use alternative single-copy target sequences that exhibit a higher sequence divergence than the 16S rRNA gene. Recently, the use of the highly conserved ubiquitous sodA gene of CNS, which encodes the manganese-dependent superoxide dismutase, was reported (Poyart et al., 2001). Therefore, to develop a species-specific detection method, we targeted the superoxide dismutase A-encoding (sodA) gene, another housekeeping gene useful for the identification of staphylococci (Poyart et al., 2001; Sivadon et al., 2004, 2005). We designed primers for S. capitis, S. haemolyticus and S. warneri each having a base that was non-complementary with regard to the two other bacteria. This base was at the 3' end of the primer (3' mismatch primer) for the sodA gene. Furthermore, to perform quantitative analysis, we adopted real-time PCR using SYBR Green chemistry. The result was a species-specific detection and quantification method for S. capitis, S. haemolyticus and S. warneri.
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METHODS
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Procedures for the design of species-specific primers.
We searched the sequences of the sodA genes of each strain registered in GenBank [GenBank accession nos: AJ343896 and AJ343939–AJ343941 (S. capitis); AJ343910, AJ343949 and AJ343950 (S. haemolyticus); and AJ343932, AJ343958, AY795889–AY795891 and AY485204 (S. warneri)] and an alignment analysis was conducted using the CLUSTAL W program. PRIMEREXPRESS (Applied Biosystems) was used to design appropriate specific primers.
Bacterial strains and preservation conditions.
The following type strains were used in the present study: S. epidermidis JCM 2414T, S. warneri JCM 2415T, S. haemolyticus JCM 2416T, Staphylococcus cohnii JCM 2417T, Staphylococcus xylosus JCM 2418T, S. capitis JCM 2420T, Staphylococcus intermedius JCM 2422T, Staphylococcus simulans JCM 2424T, Staphylococcus saprophyticus JCM 2427T, S. aureus JCM 2874T, Staphylococcus schleiferi JCM 7470T, Staphylococcus saccharolyticus GTC 181T, Staphylococcus auricularis GTC 326T, Staphylococcus caprae GTC 378T, Staphylococcus lugdunensis GTC 458T and Staphylococcus hominis GTC 485T. In addition, laboratory and clinically isolated strains of the following were prepared: 29 S. capitis strains, 5 S. haemolyticus strains, 4 S. warneri strains, 134 S. epidermidis strains, 14 S. hominis strains, 2 S. cohnii strains, 4 S. saprophyticus strains, 14 S. caprae strains, 2 S. lugdunensis strains, 34 S. aureus strains and 3 Micrococcus sp. strains. Bacteria were maintained by cultivation in tryptic soy agar medium (Difco).
Extraction of DNA.
Bacteria were cultivated with shaking in tryptic soy broth (Difco) for 16 h at 37 °C. After centrifugation of each bacterial culture medium, the pellets were washed twice with PBS. To extract DNA, a miniprep kit (Qiagen) was used and the DNA was dissolved in 50 µl TE buffer (10 mM Tris/HCl, 1 mM EDTA, pH 8.0). Human genomic DNA was obtained from Applied Biosystems.
Conventional (qualitative) PCR procedures.
For qualitative PCR, an ABI Prism 7700 (Applied Biosystems) was used without a fluorescence monitor. AmpliTaq Gold PCR master mix (Applied Biosystems) and a 100 nM concentration of each primer were used. The reaction mix volume was 25 µl. The reaction was performed for 35 cycles of 15 s at 94 °C, 30 s at the appropriate annealing temperature (Table 1
) and 30 s at 72 °C. A first step of 10 min at 94 °C was included for activation of the PCR enzyme. After the reaction was complete, PCR products were detected by agarose gel electrophoresis followed by visualization under a UV transilluminator. Experiments were performed at least three times.
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Table 1. PCR primers for species-specific detection and quantification using the sodA gene of S. capitis, S. haemolyticus and S. warneri
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Real-time PCR procedures.
An ABI Prism 7700 was used with SDS software v1.7 and the procedure was conducted according to the manufacturer's manual. Power SYBR PCR mix (Applied Biosystems) and a 100 nM concentration of each primer were used. The volume of each reaction mix was 50 µl. Genomic DNA prepared from staphylococci cells (0–107 cells) was used. The reaction was performed for 40 cycles of 10 s at 95 °C, 30 s at the appropriate annealing temperature (Table 1
) and 45 s at 72 °C. A first step of 10 min at 94 °C was included for activation of the PCR enzyme. Melting point analysis was then performed using dissociation curve analysis software. Experiments were performed at least three times.
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RESULTS AND DISCUSSION
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We first designed species-specific primers for the sodA gene (Table 1). The lengths of the amplicons were 208, 85 and 63 bp (Table 1). We determined experimentally that suitable annealing temperatures for the primers for S. capitis, S. haemolyticus and S. warneri were 59, 50 and 60 °C, respectively (Table 1). When PCR was performed using these primers and standard genomic DNA from S. capitis, S. haemolyticus or S. warneri, an amplification product of the expected length was observed in each case (data not shown). To evaluate the specificity of these primers, a test using other staphylococci and human genomic DNA was performed. In this test, no amplification products were observed (Table 2).
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Table 2. Results of testing of 18 species using a species-specific assay for S. capitis, S. haemolyticus and S. warneri
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Next, quantitative analysis of S. capitis, S. haemolyticus and S. warneri DNA was performed using these primers. Each set of primers targeted a specific bacterium and the results of real-time PCR showed the same specificity as conventional PCR (Table 2). The melting point of the amplicons was 75.8, 71.6 and 72.4 °C, respectively (Fig. 1). The specificity of these primers for other staphylococci and human genomic DNA was also evaluated. No amplification products (Table 2) or primer dimers were observed (Fig. 1). We calculated the regression line based on the results of quantitative PCR using genomic DNA extracted from S. capitis, S. haemolyticus and S. warneri cells (101–107 cells) and found linearity within the range of concentrations tested (Fig. 2).
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Fig. 1. Melting curve analysis of amplicons obtained using primers specific for S. capitis (a), S. haemolyticus (b) and S. warneri (c). The y axis indicates the negative derivative of fluorescence (F) with respect to temperature (T). Representative results are shown.
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Fig. 2. Mean threshold cycle (Ct) values from three replicates tested using primers specific for S. capitis (a), S. haemolyticus (b) and S. warneri (c) on DNA from the respective bacteria. The plot of Ct values and DNA inputs fits a linear function: (a) r2=0.998, (b) r2=0.998, (c) r2=0.996.
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This study reports a simple species-specific PCR method for studying S. capitis, S. haemolyticus and S. warneri. The primers designed in the present study were successful not only with conventional PCR but also with real-time PCR using SYBR Green chemistry.
PCR is usually considered to be a good method for bacterial detection as it is simple, sensitive and specific. However, it does have limitations. Although the 16S rRNA gene is generally targeted for the design of species-specific PCR primers for identification, designing primers is difficult when the sequences of the homologous genes have high similarity. In the case of staphylococci, therefore, ribotyping, internal transcribed spacer PCR and various other methods have been studied (Welsh & McClelland, 1992; Gribaldo et al., 1997; Gaszewska-Mastalarz et al., 1998; Edwards et al., 2001; Lee & Park, 2001; Sakai et al., 2004; Dobbins et al., 2002). However, the development of a species-specific quantitative PCR methodology has proved difficult.
In this study, 3' mismatch primers for the sodA gene made the selective identification of S. capitis, S. haemolyticus and S. warneri possible. The 3' mismatch primer is frequently used for highly specific detection of single-nucleotide polymorphisms (Ayyadevara et al., 2000; Papp et al., 2003). Each primer designed in the present study for S. capitis, S. haemolyticus and S. warneri was specific and reacted only with the target bacteria. No primer-dimer formation was observed. Moreover, these primers were also useful for species-specific quantitative analysis by real-time PCR using SYBR Green chemistry. The SYBR Green method is a new quantitative detection method in which the PCR product is detected directly, and quantified by measuring the increase in fluorescence caused by the binding of the SYBR Green dye to double-stranded DNA (Arya et al., 2005). When genomic DNA extracted from S. capitis, S. haemolyticus and S. warneri cells (0–107 cells) was used, the reactions were stable and quantitative in the range of 101–107 cells. Therefore, the present method is simple, sensitive and specific.
Our results indicate that the primers designed in the present study are useful for the identification of individual colonies and for the quantitative analysis of S. capitis, S. haemolyticus and S. warneri. In addition, this technique may be applied to other bacteria that are difficult to identify by standard methods.
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ACKNOWLEDGEMENTS
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We are grateful to Sadayori Hoshina and Hiroko Ikeshima-Kataoka.
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REFERENCES
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INTRODUCTION
METHODS
