In this study, Streptococcus gordonii-specific quantitative real-time polymerase chain reaction (qPCR) primers, RTSgo-F2/RTSgo-R2, were developed based on the nucleotide sequences of RNA polymerase β-subunit gene (rpoB). The specificity of the RTSgo-F2/RTSgo-R2 primers was assessed by conventional PCR on 99 strains comprising 63 oral bacterial species, including the type strain and eight clinical isolates of S. gordonii. PCR products were amplified from the genomic DNAs of only S. gordonii strains. The qPCR primers were able to detect as little as 40 fg of S. gordonii genomic DNA at a cycle threshold value of 33. These findings suggest that these qPCR primers detect S. gordonii with high specificity and sensitivity.
American Type Culture Collection
Culture Collection, University of Göteborg
Korean Culture Center of Microorganisms
Korean Collection for Oral Microbiology
Korean Collection for Type Cultures
optimal annealing temperature
- P. gingivalis
quantitative real-time polymerase chain reaction
RNA polymerase β-subunit gene
- S. gordonii
tryptic soy broth
Bacteria in dental plaque (biofilm) are the major causative agents of oral infectious diseases such as dental caries, periodontal diseases and endodontic infectious diseases [1-4]. S. gordonii, one of the pioneer species for formation of dental biofilm on tooth surfaces, has been widely used in studies of the mechanism initiating dental biofilm formation . In addition, S. gordonii has also been implicated in systemic diseases [6, 7].
Of the available microbial detection methods, conventional PCR or qPCR are widely used to detect bacteria qualitatively and/or quantitatively [8-10]. The 16S rDNA gene has been generally used as a target gene for designing a conventional PCR or qPCR for detecting bacterial species [9, 11]. The nucleotide sequences of 16S rDNA are well conserved among bacterial species . However, some mitis group streptococci bacterial species, such as Streptococcus mitis and Streptococcus oralis, are too homogeneous to allow their discrimination at the species-level by PCR .
Recently, the DNA-dependent RNA polymerase gene (rpoB) has been used instead of 16S rDNA to identify bacteria from genus to subspecies levels [14-17]. From a phylogenetic point of view, the nucleotide sequence of rpoB, one of the housekeeping genes, is well conserved within the strains of species, similarly to 16S rDNA. The rpoB is longer than that of 16S rDNA. In addition, it is more heterogeneous between bacterial species than is 16S rDNA.
In this study, S. gordonii-specific qPCR primers based on the nucleotide sequence of rpoB were developed for the quantitative detection of S. gordonii in epidemiological studies of infectious diseases related to S. gordonii.
The bacterial strains used in this study are listed in Table 1. They were obtained from the ATCC (Manassas, VA, USA), the CCUG (Göteborg, Sweden), KCCM (Seoul, Korea), the KCTC (Biological Resource Center, Daejeon, Korea), and the KCOM (Gwangju, Korea).
|Species||Strains||No. in Fig. 1||Sources†|
|Streptococcus gordonii||CCUG 33482T||32||Blood, subacute endocarditis|
|KCOM 1347||61||Maxillary sinusitis|
|KCOM 1357||62||Maxillary sinusitis|
|KCOM 1364||63||Maxillary sinusitis|
|KCOM 1369||64||Maxillary sinusitis|
|KCOM 1387||65||Maxillary sinusitis|
|KCOM 1506||66||Acute pulpitis|
|KCOM 1882||68||Gingival fluid, cementoblastoma|
|Streptococcus anginosus||ATCC 700231T||26||Mesial subgingival plaque in healthy adult twin|
|Streptococcus australis||KCOM 1371||94||Maxillary sinusitis|
|KCOM 1386||95||Maxillary sinusitis|
|KCOM 1439||96||Chronic osteomyelitis|
|Streptococcus constellatus||ATCC 27823T||27||Probably from case of purulent pleurisy|
|Streptococcus infantis||KCOM 1358||91||Maxillary sinusitis|
|KCOM 1375||92||Maxillary sinusitis|
|KCOM 1377||93||Maxillary sinusitis|
|Streptococcus intermedius||KCTC 3268T||28||—|
|Streptococcus mitis||KCTC 3556T||29||Oral cavity|
|KCOM 1050||69||Maxillary sinusitis|
|KCOM 1355||71||Maxillary sinusitis|
|KCOM 1379||72||Maxillary sinusitis|
|KCOM 1388||73||Maxillary sinusitis|
|Streptococcus mutans||ATCC 25175T||35||Carious dentine|
|Streptococcus oligofermentans||KCOM 1512||97||Acute pulpitis|
|KCOM 1513||98||Acute pulpitis|
|KCOM 1516||99||Endodontic infection|
|Streptococcus oralis||CCUG 13229T||31||Mouth|
|KCOM 1401||78||Maxillary sinusitis|
|KCOM 1447||79||Chronic osteomyelitis|
|KCOM 1505||80||Acute pulpitis|
|KCOM 1518||81||Endodontic infection|
|KCOM 1577||82||Periapical abscess|
|Streptococcus parasanguinis||CCUG 30417T||34||Throat|
|KCOM 1359||88||Maxillary sinusitis|
|KCOM 1585||90||Periapical abscess|
|Streptococcus pneumoniae||CCUG 28588T||30||—|
|Streptococcus sanguinis||CCUG 17826T||33||Subacute bacterial endocarditis|
|KCOM 1014||83||Maxillary sinusitis|
|KCOM 1171||84||Dental plaque|
|KCOM 2688||85||Chronic osteomyelitis|
|KCOM 1576||87||Periapical abscess|
|Streptococcus sobrinus||ATCC 33478T||36||Oral cavity|
|Aggregatibacter actinomycetemcomitans||ATCC 33384T||1||Abscess|
|Actinomyces georgiae||CCUG 32935T||6||Healthy adult, gingival crevice|
|Actinomyces israelii||ATCC 12102T||5||Brain abscess|
|Actinomyces meyeri||CCUG 21024T||7||Purulent pleurisy|
|Actinomyces naeslundii||CCUG 35333T||8||Sinus after extraction|
|Actinomyces odontolyticus||CCUG 20536T||9||Dental caries|
|Atopobium parvulum||KCTC 3663T||44||Oral cavity|
|Atopobium rimae||KCTC 5749T||45||Gingival crevice|
|Capnocytophaga gingivalis||ATCC 33624T||10||Periodontal lesion|
|Capnocytophaga ochracea||KCTC 5787T||11||Oral cavity|
|Campylobacter rectus||ATCC 33238T||12||Periodontal pocket|
|Capnocytophaga sputigena||KCTC 5789T||38||Periodontal lesion|
|Eikenella corrodens||KCOM 1378||51||Maxillary sinusitis|
|Eubacterium limosum||KCTC 2487T||39||Facet|
|Filifactor alocis||ATCC 35896T||49||Gingival sulcus|
|Fusobacterium canifelinum||CCUG 49733T||47||Wound, purulent, dog-bite|
|Fusobacterium naviforme||ATCC 50052T||50||Head lesion, exudate|
|Fusobacterium necrophorum||ATCC 25286T||46||—|
|Fusobacterium nucleatum||ATCC 25586T||18||Cervico-facial lesion|
|Fusobacterium periodonticum||ATCC 33693T||19||Periodontitis|
|Fusobacterium simiae||CCUG 16798T||48||Monkey dental plaque|
|Gemella haemolysans||KCOM 1381||52||Maxillary sinusitis|
|Haemophilus aphrophilus||ATCC 33389T||2||Cheekbone abscess|
|Haemophilus parainfluenzae||ATCC 33392T||4||Wound, septic finger|
|Haemophilus paraphrophilus||ATCC 29242T||3||Paronychia|
|Leptotrichia buccalis||CCUG 34316T||17||Supragingival calculus|
|Neisseria meningitidis||ATCC 13077T||15||Cerebrospinal fluid, fatal meningitis|
|Neisseria mucos||ATCC 19696T||13||Sputum, chronic bronchitis|
|Neisseria sicca||ATCC 29256T||16||Pharyngeal mucosa, healthy individual|
|Neisseria subflava||ATCC 49275T||14||Throat|
|Propionibacterium acnes||KCTC 3314T||25||Facial acne|
|Propionibacterium propionicum||KCTC 5342T||24||Lacrimal duct, small concretions|
|Porphyromonas endodontalis||ATCC 35406T||22||Infected root canal|
|Porphyromonas gingivalis||ATCC 33277T||23||Gingival sulcus|
|Prevotella bivia||ATCC 29303T||55||Endometrium|
|Prevotella brevis||ATCC 19188T||56||Bovine rumen|
|Prevotella buccae||ATCC 33574T||57||Gingival crevice, moderate periodontitis|
|Prevotella buccalis||CCUG 15557T||58||Dental plaque|
|Prevotella corporis||ATCC 33547T||59||Cervical swab|
|Prevotella dentalis||ATCC 49559T||60||Dental root canal|
|Prevotella intermedia||ATCC 25611T||21||Empyema|
|Prevotella nigrescens||ATCC 33563T||20||Gingivitis|
|Rothia dentocariosa||KCTC 3204T||42||Caries lesion|
|Selenomonas artemidis||KCTC 5742T||43||Gingival crevice, juvenile localized periodontitis|
|Selenomonas noxia||KCTC 5746T||40||Gingival crevice, severe periodontitis|
|Tannerella forsythia||ATCC 43037T||37||Periodontal pocket|
|Veillonella dispar||KCOM 1301||53||Subgingival dental plaque, gingivitis|
|Veillonella parvula||KCTC 5019T||41||Intestinal tract|
Streptococcus spp. and Haemophilus spp. were grown in TSB medium (Difco Laboratories, Detroit, MI, USA) supplemented with 0.5% yeast extract, 0.5 mg/mL hemin, and 2 μg/mL vitamin K1 at 37°C in an air atmosphere containing 5% CO2. Aggregatibacter actinomycetemcomitans was grown in TSB medium (Difco Laboratories) supplemented with 0.6% yeast extract, 5% horse serum, 75 μg/mL bacitracin, and 5 μg/mL vancomycin (Sigma − Aldrich, St. Louis, MO, USA) at 37°C in an anaerobic atmosphere (5% H2, 10% CO2, and 85% N2). Fusobacterium spp. were cultivated on Schaedler broth (Difco Laboratories) at 37°C for 48 hrs in an anaerobic chamber (Model Bactron I, Sheldon Manufacturing, Cornelius, OR, USA). The other bacterial species were cultured in TSB supplemented with 0.5% yeast extract, 0.05% cysteine HCl − H2O, 0.5 mg/mL hemin and 2 μg/mL vitamin K1 at 37°C in an anaerobic atmosphere.
Bacterial genomic DNAs were prepared using a G-spin Genomic DNA Extraction kit (iNtRON, Seoul, Korea) according to the manufacturer's instructions. The DNA concentrations were determined by measuring the OD at 260 and 280 nm using an Ultraspec 2000 ultraviolet spectrophotometer (Pharmacia Biotech., Cambridge, UK).
A pair of qPCR primers was designed based on the nucleotide sequence of rpoB of S. gordonii str. Challis substr. CH1 (GenBank accession number NC_009785, region: 1997060–2000626 nt) using the program PRIMERSECT (DNASTAR, Madison, WI, USA). The primer sequences were as follows: forward primer (RTSgo-F2), 5′-TGT ACC CCG TAT CGT TCC TGT G-3′; and reverse primer (RTSgo-R2), 5′-AAA GAC TGG AGT TGC AAT GTG AAT A-3′. The expected product length for S. gordonii was 175 bp.
The specificity of the qPCR primers (RTSgo-F2/RTSgo-R2) for S. gordonii was investigated by conventional PCR with the genomic DNAs of 45 strains (nine species) of mitis group streptococci, including nine strains of S. gordonii and 54 strains (54 species) of other oral bacterial species. PCR was performed with 4 ng of bacterial genomic DNA and AccuPower PCR PreMix (Bioneer, Daejeon, Korea) using a MyGenie 96 Gradient Thermal Block cycler (Bioneer) under the following conditions: initial denaturation at 95°C for 10 mins; 30 cycles of denaturation at 95°C for 30 s, primer annealing and extension at 65°C for 30 s; followed by final extension at 72°C for 5 mins. Six μL of PCR product was analyzed by 1.5% agarose gel electrophoresis in Tris-acetate buffer (0.04 M Tris-acetate, 0.001 M EDTA, pH 8.0) at 100 V for 30 mins. The amplification products were stained with ethidium bromide and visualized using an ultraviolet transilluminator.
To determine the detection limit (sensitivity) of the RTSgo-F2/RTSgo-R2 primers, qPCR was performed with serially 10-fold diluted genomic DNA of S. gordonii CCUG 33482T, ranging from 4 ng to 4 fg, using TOPreal qPCR 2 × PreMIX (SYBR Green) kit (Enzynommics, Daejeon, Korea) and an Exicycler 96 Real-Time Quantitative Thermal Block (Bioneer). Each qPCR was performed in a total volume of 20 µL containing 2 µL each of the forward and reverse primers (final concentration, 500 nM each), 5 µL of genomic DNA, 1 µL of sterilized DNase-RNase-free water, and 10 µL of TOPreal qPCR 2 × PreMIX. The qPCR conditions were initial denaturation at 95°C for 10 mins, 40 cycles of denaturation at 95°C for 10 s, primer annealing and extension at 65°C for 30 s, and final cooling at 25°C for 1 min. The reaction specificities were confirmed by melting curve analysis with a progressive increase in temperature from 65 to 94°C at a 1°C/s transition rate and continuous fluorescence acquisition.
According to the definition of the OAT, the highest temperature at which the PCR products could be amplified only from the genomic DNA of the target bacterial strain and at which the amount of PCR product was the greatest , the OAT of the RTSgo-F2/RTSgo-R2 primers was determined by gradient PCR performed with the genomic DNAs of S. gordonii CCUG 33482T. The tested annealing temperatures ranged from 61–72°C in 12 intervals. The density of the PCR products was the same between 61 and 65°C, but their density decreased at 66°C and above 67°C the PCR product was not amplified (data not shown). Therefore, the OAT of the RTSgo-F2/RTSgo-R2 primers was determined as 65°C.
The specificity of the RTSgo-F2/RTSgo-R2 primers was assessed by conventional PCR with 99 strains comprising 63 oral bacterial species, including the type strain and eight clinical isolates of S. gordonii. The PCR products were amplified from the genomic DNAs of only S. gordonii strains (Fig. 1a). These results indicate that the qPCR primers could be used to detect S. gordonii with high specificity.
The sensitivity of the RTSgo-F2/RTSgo-R2 primers was assessed with serially 10-fold diluted genomic DNA of S. gordonii CCUG 33482T. The RTSgo-F2/RTSgo-R2 primers were able to detect 40 fg of the genomic DNA of S. gordonii CCUG 33482T at a CT value of 33 (Fig. 1b and Table 2). We considered a CT value below 35 as positive data that ruled out the possibility of non-specific amplification. Considering that the genome size of the S. gordonii str. Challis substr. CH1 is 2.2 Mb (http://www.ncbi.nlm.nih.gov/nuccore/157149651?report=genbank), the RTSgo-F2/RTSgo-R2 qPCR primers could detect 17 bacterial cells of S. gordonii (Table 2). S. gordonii-specific qPCR primers designed based on the nucleotide sequences of gtfG were introduced using the TaqMan probe method . It is generally accepted that the SYBR Green method is slightly less sensitive than the TaqMan method. Recently, the efficiency of these two methods was evaluated by detecting P. gingivalis . In that study, there was no significant difference between the TaqMan and SYBR Green methods in specificity, precision and sensitivity . In addition, the SYBR Green method has some advantages over the TaqMan method; these include lower cost and ease of design of the PCR primers. The present results show that qPCR using RTSgo-F2/RTSgo-R2 primers detects S. gordonii with high sensitivity.
|Genomic DNA amount||Cell number corresponding to DNA amount||CT|
|4 ng||1.7 × 106||14.7|
|400 pg||1.7 × 105||18.8|
|40 pg||1.7 × 104||22.3|
|4 pg||1.7 × 103||26.3|
|400 fg||1.7 × 102||30.1|
|40 fg||1.7 × 101||33.0|
|4 fg||1.7 × 100||ND†|
Mitis group streptococci inhabit dental plaque and play an important role in initiating dental plaque formation. Of this group, S. gordonii is one of pioneer bacteria in dental plaque formation; this organism binds the saliva protein of acquired pellicle [21, 22]. S. gordonii can bind P. gingivalis, one of the major periodontopathogens . In addition, S. gordonii has been established as a cause of subacute bacterial endocarditis  and has also been isolated from septic arthritis lesions . Thus, accurate detection and identification of S. gordonii would be valuable for diagnostic purposes with these diseases.
In summary, RTSgo-F2/RTSgo-R2 primers designed based on the nucleotide sequence of rpoB are suitable for detecting S. gordonii with high specificity and sensitivity and could be useful in epidemiological studies of infectious diseases caused by S. gordonii.
This research was supported by the Basic Science Research Program through the National Research Foundation of Korea funded by the Ministry of Education, Science and Technology (2010-0025411).
All authors have no conflicts of interest.