Development of Streptococcus gordonii-specific quantitative real-time polymerase chain reaction primers based on the nucleotide sequence of rpoB

Authors

  • Soon-Nang Park,

    1. Korean Collection for Oral Microbiology, Department of Oral Biochemistry, and Oral Biology Institute, School of Dentistry, Chosun University, Dong-Gu, Gwangju, Korea
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  • Joong-Ki Kook

    Corresponding author
    • Korean Collection for Oral Microbiology, Department of Oral Biochemistry, and Oral Biology Institute, School of Dentistry, Chosun University, Dong-Gu, Gwangju, Korea
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Correspondence

Joong-Ki Kook, Department of Oral Biochemistry, School of Dentistry, Chosun University, 375 Seosuk-Dong, Dong-Gu, Gwangju 501-759, Korea. Tel: +82 62 230 6877; Fax: +82 62 224 3706; email: jkkook@chosun.ac.kr

ABSTRACT

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.

List of Abbreviations
ATCC

American Type Culture Collection

CCUG

Culture Collection, University of Göteborg

CT

cycle threshold

gtfG

glucosyltransferase gene

KCCM

Korean Culture Center of Microorganisms

KCOM

Korean Collection for Oral Microbiology

KCTC

Korean Collection for Type Cultures

OAT

optimal annealing temperature

OD

optical density

P. gingivalis

Porphyromonas gingivalis

qPCR

quantitative real-time polymerase chain reaction

rpoB

RNA polymerase β-subunit gene

S. gordonii

Streptococcus gordonii

TSB

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 [5]. 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 [12]. 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 [13].

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).

Table 1. The strains used in this study
SpeciesStrainsNo. in Fig. 1Sources
  • , all human except for Fusobacterium simiae from monkey dental plaque and Prevotella brevis from bovine rumen.
Streptococcus gordoniiCCUG 33482T32Blood, subacute endocarditis
 KCOM 134761Maxillary sinusitis
 KCOM 135762Maxillary sinusitis
 KCOM 136463Maxillary sinusitis
 KCOM 136964Maxillary sinusitis
 KCOM 138765Maxillary sinusitis
 KCOM 150666Acute pulpitis
 KCOM 185167Osteomyelitis
 KCOM 188268Gingival fluid, cementoblastoma
Streptococcus anginosusATCC 700231T26Mesial subgingival plaque in healthy adult twin
Streptococcus australisKCOM 137194Maxillary sinusitis
 KCOM 138695Maxillary sinusitis
 KCOM 143996Chronic osteomyelitis
Streptococcus constellatusATCC 27823T27Probably from case of purulent pleurisy
Streptococcus infantisKCOM 135891Maxillary sinusitis
 KCOM 137592Maxillary sinusitis
 KCOM 137793Maxillary sinusitis
Streptococcus intermediusKCTC 3268T28
Streptococcus mitisKCTC 3556T29Oral cavity
 KCOM 105069Maxillary sinusitis
 KCOM 129570Gingivitis
 KCOM 135571Maxillary sinusitis
 KCOM 137972Maxillary sinusitis
 KCOM 138873Maxillary sinusitis
Streptococcus mutansATCC 25175T35Carious dentine
Streptococcus oligofermentansKCOM 151297Acute pulpitis
 KCOM 151398Acute pulpitis
 KCOM 151699Endodontic infection
Streptococcus oralisCCUG 13229T31Mouth
 KCOM 140178Maxillary sinusitis
 KCOM 144779Chronic osteomyelitis
 KCOM 150580Acute pulpitis
 KCOM 151881Endodontic infection
 KCOM 157782Periapical abscess
Streptococcus parasanguinisCCUG 30417T34Throat
 KCOM 135988Maxillary sinusitis
 KCOM 129489Gingivitis
 KCOM 158590Periapical abscess
Streptococcus pneumoniaeCCUG 28588T30
 KCCM 4041074
 KCCM 4156775
 KCCM 4157076
 KCTC 393277
Streptococcus sanguinisCCUG 17826T33Subacute bacterial endocarditis
 KCOM 101483Maxillary sinusitis
 KCOM 117184Dental plaque
 KCOM 268885Chronic osteomyelitis
 KCOM 185586Osteomyelitis
 KCOM 157687Periapical abscess
Streptococcus sobrinusATCC 33478T36Oral cavity
Aggregatibacter actinomycetemcomitansATCC 33384T1Abscess
Actinomyces georgiaeCCUG 32935T6Healthy adult, gingival crevice
Actinomyces israeliiATCC 12102T5Brain abscess
Actinomyces meyeriCCUG 21024T7Purulent pleurisy
Actinomyces naeslundiiCCUG 35333T8Sinus after extraction
Actinomyces odontolyticusCCUG 20536T9Dental caries
Atopobium parvulumKCTC 3663T44Oral cavity
Atopobium rimaeKCTC 5749T45Gingival crevice
Capnocytophaga gingivalisATCC 33624T10Periodontal lesion
Capnocytophaga ochraceaKCTC 5787T11Oral cavity
Campylobacter rectusATCC 33238T12Periodontal pocket
Capnocytophaga sputigenaKCTC 5789T38Periodontal lesion
Eikenella corrodensKCOM 137851Maxillary sinusitis
Eubacterium limosumKCTC 2487T39Facet
Filifactor alocisATCC 35896T49Gingival sulcus
Fusobacterium canifelinumCCUG 49733T47Wound, purulent, dog-bite
Fusobacterium naviformeATCC 50052T50Head lesion, exudate
Fusobacterium necrophorumATCC 25286T46
Fusobacterium nucleatumATCC 25586T18Cervico-facial lesion
Fusobacterium periodonticumATCC 33693T19Periodontitis
Fusobacterium simiaeCCUG 16798T48Monkey dental plaque
Gemella haemolysansKCOM 138152Maxillary sinusitis
Haemophilus aphrophilusATCC 33389T2Cheekbone abscess
Haemophilus parainfluenzaeATCC 33392T4Wound, septic finger
Haemophilus paraphrophilusATCC 29242T3Paronychia
Leptotrichia buccalisCCUG 34316T17Supragingival calculus
Neisseria meningitidisATCC 13077T15Cerebrospinal fluid, fatal meningitis
Neisseria mucosATCC 19696T13Sputum, chronic bronchitis
Neisseria siccaATCC 29256T16Pharyngeal mucosa, healthy individual
Neisseria subflavaATCC 49275T14Throat
Propionibacterium acnesKCTC 3314T25Facial acne
Propionibacterium propionicumKCTC 5342T24Lacrimal duct, small concretions
Porphyromonas endodontalisATCC 35406T22Infected root canal
Porphyromonas gingivalisATCC 33277T23Gingival sulcus
Prevotella biviaATCC 29303T55Endometrium
Prevotella brevisATCC 19188T56Bovine rumen
Prevotella buccaeATCC 33574T57Gingival crevice, moderate periodontitis
Prevotella buccalisCCUG 15557T58Dental plaque
Prevotella corporisATCC 33547T59Cervical swab
Prevotella dentalisATCC 49559T60Dental root canal
Prevotella intermediaATCC 25611T21Empyema
Prevotella nigrescensATCC 33563T20Gingivitis
Rothia dentocariosaKCTC 3204T42Caries lesion
Selenomonas artemidisKCTC 5742T43Gingival crevice, juvenile localized periodontitis
Selenomonas noxiaKCTC 5746T40Gingival crevice, severe periodontitis
Tannerella forsythiaATCC 43037T37Periodontal pocket
Veillonella disparKCOM 130153Subgingival dental plaque, gingivitis
Veillonella parvulaKCTC 5019T41Intestinal 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 [18], 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.

Figure 1.

(a) Specificity test of the S. gordonii-specific PCR primers, RTSgo-F2/RTSgo-R2, with 4 ng of each bacterial genomic DNA. The PCR reaction products were electrophoresed on 1.5% agarose gels. Lanes: S, size marker (100 bp ladder); 1 through 99 corresponds to number of strain in Table 1; 100, negative control (deionized distilled water). (b) Sensitivity test of the S. gordonii-specific qPCR primers, RTSgo-F2/RTSgo-R2. (i) Standard curve, (ii0 amplification plot and (iii) melting curve were obtained by qPCR using the RTSgo-F2/RTSgo-R2 primers from 10-fold serial dilutions of S. gordonii CCUG 33482T genomic DNA ranging from 4 ng to 4 fg. In the standard curve, the regression equation for the standard curve was: Y = −0.2669X + 9.9569. The R2 value was 0.9976.

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 [19]. 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 [20]. In that study, there was no significant difference between the TaqMan and SYBR Green methods in specificity, precision and sensitivity [20]. 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.

Table 2. Standard curve of the qPCR primers with serially 10-fold diluted genomic DNA of S. gordonii CCUG 33482T
Genomic DNA amountCell number corresponding to DNA amountCT
  1. ND, not detected.
4 ng1.7 × 10614.7
400 pg1.7 × 10518.8
40 pg1.7 × 10422.3
4 pg1.7 × 10326.3
400 fg1.7 × 10230.1
40 fg1.7 × 10133.0
4 fg1.7 × 100ND
00ND

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 [23]. In addition, S. gordonii has been established as a cause of subacute bacterial endocarditis [6] and has also been isolated from septic arthritis lesions [7]. 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.

ACKNOWLEDGMENTS

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).

DISCLOSURE

All authors have no conflicts of interest.

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