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Development of species-specific primers for detection of Streptococcus mutans in mixed bacterial samples

Zhou Chen, Deepak Saxena, Page W. Caufield, Yao Ge, Minqi Wang, Yihong Li
DOI: http://dx.doi.org/10.1111/j.1574-6968.2007.00756.x 154-162 First published online: 1 July 2007

Abstract

Streptococcus mutans is the major microbial pathogen associated with dental caries in children. The objectives of this study were to design and evaluate species-specific primers for the identification of S. mutans. Validation of the best primer set, Sm479F/R, was performed using seven S. mutans reference strains, 48 ATCC non-S. mutans strains, 92 S. mutans clinical isolates, DNA samples of S. mutansStreptococcus sobrinus or S. mutansStreptococcus sanguinis, and mixed bacterial DNA of saliva samples from 33 18-month-old children. All of the S. mutans samples tested positive, and no PCR products were amplified from members of the other streptococci or nonstreptococci strains examined. The lowest detection level for PCR was 10−2 ng of S. mutans DNA (c. 4.6 × 103 copies) in the test samples. The results of this study suggest that the Sm479F/R primer pair is highly specific and sensitive for identification of S. mutans in either purified or mixed DNA samples.

Keywords
  • Streptococcus mutans
  • PCR
  • species-specific primer
  • dental caries

Introduction

Dental caries has a polymicrobial etiology, with Streptococcus mutans being the major pathogen. Conventionally, studies of S. mutans have relied heavily upon cultivation to identify and characterize S. mutans in the oral cavity. The major limitations of culture methods include a finite threshold of detection of S. mutans in clinical samples; an inconsistent morphology depending on the culture medium used; and its high cost and labor intensiveness. Moreover, cultivation requires viable samples, making its application in field epidemiological studies and high-throughput research impractical.

Because conventional culture methods can limit population-based field studies of S. mutans colonization and its interaction with other bacteria in the oral cavity, a number of DNA-based probes and primers have been developed. Many of the specific probes or primers were targeted to specific genes that are associated with virulence in S. mutans, such as glucosyltransferases (Colby et al., 1995; Yano et al., 2002), fructosyltransferases (Smorawinska & Kuramitsu, 1992), dextranase (Igarashi et al., 1996), glucan-binding protein B (Smith et al., 2003), cell surface protein (Lee & Boran, 2003), the phosphoenolpyruvate-dependent sucrose phosphotransferase system (Macrina et al., 1991; Cvitkovitch et al., 1995), and protein antigen (Okahashi et al., 1989, 1993). Several other sets of primers for PCR were designed to amplify specific regions of the 16S rRNA genes of S. mutans (Bentley et al., 1991; Shiroza et al., 1998; Oho et al., 2000; Rupf et al., 2001; Aguilera Galaviz et al., 2002; Becker et al., 2002; Wang et al., 2002; Yano et al., 2002; Yoshida et al., 2003; Arakawa et al., 2004; Hoshino et al., 2004). After an extensive literature review, it was found that many primers for PCR assays work well for pure S. mutans cultures. However, there was very little information as to whether the PCR-targeted regions might also be present in other bacterial species found in the same habitat as S. mutans or whether these primers can detect S. mutans in mixed clinical specimens. Indeed, some of these genetic loci may not be unique to S. mutans (Hamada & Slade, 1980; Russell, 1991).

Previously, the maternal influence on mother-to-child transmission of S. mutans was investigated using a chromosomal DNA fingerprinting technique in various populations (Li & Caufield, 1995; Emanuelsson et al., 1998; Li et al., 2000, 2001; Caufield et al., 2007). In hundreds of S. mutans chromosomal DNA fingerprints, the consistent presence of a 14-kb HaeIII restriction fragment was observed as illustrated in Fig. 1a. The main objective of the present study was to identify unique sequence information in this 14-kb fragment for development of S. mutans-specific PCR primers. One such primer pair, Sm479F/R, was evaluated for its sensitivity and specificity for S. mutans in various mixed bacterial samples. The PCR results were further compared against the findings from conventional culture methods archived in a natural history database (Li et al., 2005a).

Figure 1

Development of species-specific primers for Streptococcus mutans. (a) Chromosomal DNA fingerprint profiles of different Streptococcus species after HaeIII restriction enzyme digestion and electrophoresis in a 0.55% agarose gel. Lanes 1–5, S. mutans reference strains 10449, KPSK2, Ingbritt, UA159, and OMZ175. Lane 6, Streptococcus sobrinus reference strain OMZ65. Lane 7, Streptococcus sanguinis reference strain ATCC10556. The unique 14-kb fragment was observed among all S. mutans strains, but not other streptococcus strains. (b) Locations of the Sm479F/R primers. The primers were designed to anneal to sequences within the unique 13 693-bp fragment, which encompasses 2 021 910–4 682 nt of the UA159 genome (AE014133). The targeted segment comprises a portion of the htrA locus and a part of an intergenic locus of the S. mutans genome. The final size of the PCR amplicon is 479 bp.

Materials and methods

This study protocol was approved by the IRB of the University of Alabama at Birmingham on Activities Involving Human Subjects and the IRB of New York University. Written parental consent was obtained for each child in this study.

Bacterial samples

Four sets of bacterial samples were included in this study.

  1. Based on their association with dental diseases, a variety of bacterial reference strains (mol% of G+C content ranged from 27% to 71%) were selected (Table 1): seven S. mutans reference strains, 16 other streptococci reference strains, and 32 nonstreptococci gram-positive and gram-negative oral bacteria reference strains. All but 11 of the DNA samples were isolated using a commercial DNA extraction kit (Genomic-tip 100/G, Qiagen, Valencia, CA), followed by an additional phenol–chloroform–isoamyl alcohol extraction. Genomic DNA samples of 11 of the 55 reference strains, indicated by ‘D’ in Table 1, were directly purchased from American Type Culture Collection (ATCC, Manassas, VA).

  2. For testing the specificity and sensitivity of the primer sets, 92 clinical isolates of S. mutans were randomly selected from the archived S. mutans collection. All were confirmed previously as being S. mutans based on both phenotypic and genotypic profiles (Li & Caufield, 1995; Li et al., 2001, 2005a). The genomic DNAs of these isolates were purified using the same DNA extraction kit above.

  3. Mixtures of DNA samples of S. mutans plus Streptococcus sobrinus and S. mutans plus Streptococcus sanguinis were prepared. A serial dilution (10 ng µL−1–10−3 ng µL−1) of purified DNA samples of S. mutans (UA159) was added to known concentrations (10−3 ng µL−1–10 ng µL−1) of genomic DNA samples of either S. sobrinus (OMZ65) or S. sanguinis (ATCC10556). The mixed samples were used to determine, by PCR, the lowest detectable concentration of S. mutans DNA in the presence of other oral streptococcus species.

  4. Thirty-three bacterial samples obtained from an MM10-sucrose blood medium (Syed & Loesche, 1973) were also selected for testing the species specificity and the limit of detection of the new primer set for identifying S. mutans in mixed bacterial samples. These saliva samples have been previously collected from 33 18-month-old children; five of the 33 children (15%) were positive for S. mutans by using a conventional culture assay. The procedures for sample collection, bacterial cultivation, and DNA isolation were published elsewhere (Li et al., 2005a, b).

View this table:
Table 1

List of bacterial samples used in this study

Bacterial speciesSources and codeSources of isolation and clinical significance
Streptococcus mutans strains
    S. mutans UA159ATCC 700610Caries-active child
    S. mutans NCTC10449ATCC 25175Human carious dentine
    S. mutans AF199This studyCaries-active child
    S. mutans IngbrittB. KrasseDental plaque of highly caries-active person
    S. mutans GS5R.J. GibbonsHuman carious lesions
    S. mutans LM7R.J. GibbonsCaries-active child
    S. mutans OMZ175B. GuggenheimHuman carious lesions
Non-Streptococcus mutans strains
    S. agalactiaeATCC BAA-611DHuman clinical specimen
    S. criceti AHTB. KrasseHuman dental plaque
    S. cristatusATCC 49999Human oral cavity and throat
    S. gordoniiATCC 10558Patient with bacterial endocarditis
    S. oralisATCC 10557Patient with bacterial endocarditis
    S. oralisATCC 9811Human mouth
    S. parasanguinisATCC 15911Human throat
    S. pyogenesATCC 12344DHuman pharyngitis
    S. rattiATCC 19645Caries lesion in rat
    S. ratti BHTT. ShiotaCaries lesion in rat
    S. salivariusATCC 7073Patient with acute articular rheumatism
    S. sanguinisATCC 10556Patient with bacterial endocarditis
    S. sobrinus OMZ176B. GuggenheimHuman carious lesions
    S. sobrinus OMZ65B. GuggenheimHuman carious lesions
    S. sobrinusATCC 33478Human dental plaque
    S. vestibularisATCC 49124Human oral cavity
Gram-positive rods
    Actinomyces naeslundiiATCC 12104Human sinus
    A. odontolyticusATCC 17929Deep carious lesions around teeth
    A. viscosusATCC 15987Naturally occurring periodontal disease in hamsters
    A. israeliiATCC 12102Human brain abscess
    A. meyeriATCC 35568Human with purulent pleurisy
    A. gerencseriaeATCC 29322Cervicofacial actinomycosis
    A. odontolyticusATCC 29323Dental plaque
    A. georgiaeATCC 49285Healthy subgingival plaque
    A. radingaeATCC 51856Human perianal abscess
    A. bovisATCC 13683Typical case of lumpy jaw in a cow
    A. bernardiaeATCC 51728Human eye infection
    Bifidobacterium infantisATCC 15697DIntestine of infant
    Lactobacillus caseiATCC 393Dairy products (cheese)
    L. rhamnosusATCC 7469Human infective endocarditis and bacteremia
    L. salivarius ssp. salivariusATCC 11741Oral cavity
    L. caseiATCC 11578Oral cavity
    L. fermentumATCC 14931Fermented beets
    L. paracasei ssp. paracaseiATCC 25598Milking machine
    L. acidophilusATCC 4356Human mouth
Gram-negative cocci
    Veillonella parvulaATCC 10790DIntestinal tract
Gram-negative rods
    Actinobacillus actinomycetemcomitansATCC 43718Subgingival dental plaque
    A. actinomycetemcomitansATCC 29522Mandibular abscess
    Campylobacter jejuni ssp. jejuniATCC 33560DFeces, animal (bovine feces)
    Escherichia coliATCC 10798DFeces from diphtheria convalescent
    Fusobacterium nucleatum ssp. vincentiATCC 49256Human periodontal pocket
    F. nucleatum ssp. polymorphumATCC 10953Inflamed gingiva, adult male
    Aggregatibacter actinomycetemcomitansATCC 700685DSubgingival plaque with juvenile periodontitis
    Helicobacter pyloriATCC 43504DHuman gastric antrum
    Prevotella intermediaATCC 25611DEmpyema
    Porphyromonas gingivalisATCC 33277Human gingival sulcus
    Shigella flexneriATCC 29903DPathogen of acute gastroenteritis
    Tannerella forsythensisATCC 43037DHuman periodontal pocket
Additional bacterial samples
    S. mutans+S. sobrinusThis studyPure culture mixed
    S. mutans+S. sanguinisThis studyPure culture mixed
    S. mutans clinical isolatesThis studyN=92; randomly selected from archived bacterial database
    DNA of total cultivable bacteria in salivaThis studyN=33; salivary samples of children aged 18 months
Human DNA samples
    Genomic DNAThis studyWhole blood cells
    Genomic DNAThis studyBuccal mucosa epithelial cells from oral cavity
  • American Type Culture Collection, Manassas, VA, USA. The code with ‘D’ at the end indicates that DNA samples were directly purchased.

  • Department of Cariology, Faculty of Odontology, University of Goteborg, Goteborg, Sweden.

  • Forsyth Dental Center, Boston, MA.

  • Institute for Oral Biology, Section for Oral Microbiology and General Immunology, University of Zurich, Zurich, Switzerland.

  • Department of Microbiology, University of Alabama, Birmingham, AL.

PCR and real-time quantitative PCR assays (real-time qPCR)

The species specificity of each newly developed primer set was evaluated initially against the seven S. mutans and 48 non-S. mutans reference strains (Table 1), and further validated using the randomly selected purified S. mutans DNA of clinical isolates and the mixed bacterial samples described above. The limit of detection of the primers was evaluated using PCR against a set of 10-fold serially diluted concentrations of UA159 genomic DNA samples and further validated using the mixed S. mutans DNA samples containing known concentrations of S. sobrinus or S. sanguinis DNA.

PCR assays were performed using a standardized protocol in a thermal cycler (GeneAmp PCR system 9700, Applied Biosystems, Foster City, CA). Each reaction mixture (25 µL total volume) contained 1 × PCR buffer (10 mM Tris-HCl, 50 mM KCl, pH 8.3), 1.5 µL of 2.5 mM dNTP mixture, 1 mM MgCl2, 10 pmoles each of forward and reverse primers, 1.5 U of Taq DNA polymerase, and 10 ng of template DNA. The reaction was conducted as follows: 95°C for 2 min, followed by 40 cycles of 95°C for 30 s, 60±5°C for 30 s, and 72°C for 1 min, and then finally 5 min at 72°C for extension. The PCR amplicons were evaluated in a 1.5% agarose gel in TBE (Tris-borate-EDTA) buffer and stained with ethidium bromide solution (1 µg mL−1). The final images of the gels were captured by a digital camera (AlphaImager 3300 System, Alpha Innotech Corp., San Leandro, CA).

The specificity and limit of detection of the primers in identifying S. mutans colonization in 33 18-month-old children were determined using real-time qPCR. Briefly, real-time qPCR was performed using an Opticon real-time machine (Monitor-2, MJ Research Inc., Alameda, CA) with low-profile 96-well polypropylene microplates. Ten-fold serially diluted, known DNA concentrations of S. mutans UA159 were used as an external standard for absolute quantification. Each tube contained 25 µL of reaction mixture, including 1 × PCR Master Mix (QuantiTect SYBR Green, Qiagen Inc.), 10–100 ng of the mixed bacterial DNA samples obtained from MM10 culture plates, and 0.4 µM of each primer. The cycling conditions were 15 min at 95°C for activation of HotStar Taq DNA polymerase, 45 cycles of 15 s at 94°C for denaturation, 30 s at 56°C for annealing, and 30 s at 72°C for extension, followed by a melting curve analysis of the PCR product. All reactions were carried out in duplicate and the final analysis was based on the mean of the two reactions. Furthermore, the PCR products were reconfirmed for correct size by electrophoresis in a 1.5% agarose gel alongside molecular size standards. The real-time qPCR results were compared with the results obtained previously using conventional culture methods.

DNA sequencing analysis

To further confirm the species specificity of the primers, 50% of the PCR products of the S. mutans reference strains, the clinical isolates, and the mixed bacterial DNA samples were randomly selected, purified, and sequenced from both directions (ABI Prism cycle sequencing kit, BigDye Terminator chemistries with AmpliTaq DNA polymerase FS; Perkin-Elmer, Wellesley, MA). A sequence similarity search of the nonredundant GenBank database was performed using the standard nucleotide–nucleotide blast (blastn) search, and sequences were aligned using clustalW (Chenna et al., 2003).

Statistical analyses

Analyses were performed using a computer statistics program (spss 13.0, SPSS Inc. Chicago, IL). The differences in the species specificity and the limit of detection between the different bacterial samples were evaluated using Pearson chi-square and Fisher's Exact Tests. All P values of <0.05 were two tailed.

Results

As illustrated in Fig. 1a, a unique 14-kb fragment is present in chromosomal DNA fingerprints of S. mutans isolates after HaeIII restriction enzyme digestion. This fragment was further characterized according to the HaeIII restriction site map of the whole genome sequence of the S. mutans reference strain UA159 (AE014033) using sequencher Software version 4.1 (Gene Codes Corporation, Ann Arbor, MI). The precise length of this unique fragment is 13 693 bp, which encompasses the end of the circular genome (2 021 910–2 030 921 nt) and the contiguous starting region (1–4 682 nt) (Fig. 1b). The protein map of UA159 revealed 10 ORF within this region, including core housekeeping genes involved with the origin and execution of DNA replication. Because each of the ORF shared some degree of similarity to homologues in other streptococci, it was decided to construct primers that spanned from known ORFs to within intergenic spacer regions (ISR). Based on the sequence information, six sets of primers were designed (Supplementary Table S1) using primer3 software (Rozen & Skaletsky, 2000) and evaluated against a panel of prototype strains for species specificity.

After systematically testing each of the six primer sets, it was found that Sm479F: 5′-TCGCGAAAAAGATAAACAAACA-3′ and Sm479R: 5′-GCCCCTTCACAGTTGGTTAG-3′ were highly specific for identification of S. mutans in either purified or mixed DNA samples. The results showed that all of the S. mutans reference strains and S. mutans clinical isolates were PCR positive. No PCR products were detected in other Streptococcus species, including S. sobrinus, Streptococcus criceti, and Streptococcus ratti, Streptococcus salivarius, Streptococcus vestibularis, S. sanguinis, Streptococcus parasanguinis, Streptococcus gordonii, Streptococcus oralis, and Streptococcus cristatus, or in the other oral bacterial strains (Fig. 2a). In contrast to the Sm479F/R primer pair, the other five sets of primers showed positives among the other streptococci ornonstreptococci strains tested. In the serially diluted S. mutans DNA samples, the lowest detectable concentration of the Sm479F/R primers was 0.01 ng µL−1 (c. 4.6 × 103 cell copies) (Fig. 2b). A similar limit level of detection was also obtained in the S. mutans–S. sanguinis or S. mutansS. sobrinus mixed DNA samples (Fig. 2c).

Figure 2

Evaluation of the species specificity and the limit of detection of the primers by PCR. (a) Gel electrophoresis of PCR products of the reference strains (16 out of a total of 55 are illustrated) of mutans streptococci and other nonmutans streptococci species using the primers Sm479F/R. The agarose gel shows the PCR-amplified target DNA to be present in the Streptococcus mutans type-strains and absent in the non-S. mutans strains tested with a high degree of specificity. The molecular size standard consisting of a 100-bp DNA ladder is shown in the first lane. (b) Detection of S. mutans DNA by PCR using the Sm479F/R primers against fivefold serially diluted concentrations of pure UA159 DNA samples. The minimum detectable level was ≥1.6 × 10−2 ng. (c) Detection of S. mutans DNA by PCR using the Sm479F/R primers against serially diluted UA159 genomic DNA samples mixed with Streptococcus sanguinis (ATCC10556) or Streptococcus sobrinus (OMZ65) DNA. The lowest detection level for S. mutans was 0.01 ng.

To exclude the possibility that Sm479F/R might encounter unexpected cross-reactivity in reactions applied to mixed clinical samples containing bacterial and human DNA, PCR assays were performed against 10 ng of human genomic DNA sample (isolated from 1 mL blood sample) and 10 ng of hEt cell line DNA sample (derived from human buccal mucosa epithelial cells). Both human DNA samples showed negative PCR results (Fig. 3).

Figure 3

Evaluation of the specificity Sm479F/R primers by PCR. DNA amplification was observed from the Streptococcus mutans (UA159) strain (lane 2), but not from human buccal mucosa epithelial cells (hEt) (lane 3), nor from a human whole-blood sample (lane 4) or the negative control (lane 5). The results further support the conclusion that the Sm479F/R primers are not only specific for S. mutans but also do not display cross-reactivity with human DNA samples.

Sequencing analysis revealed that the Sm479F/R primers target an amplicon of 479 bp (2 029 599–2 030 077 nt of AE014133), with a 5′ within the htrA gene and a 3′ within the ISR but outside the putative spoJ gene (SMU.2165; Fig. 1b). Homologues of both genes are widely distributed among Gram-positive bacteria. A search of the nonredundant GenBank database for sequences similar to the 479-bp PCR amplicon yielded only a single hit, which was within the S. mutans UA159 complete genome and showed 100% identity. The results of a multiple sequence alignment revealed that the products amplified by the Sm479F/R primers were 98–100% identical among the S. mutans serotype c strains UA159, ATCC25175, Ingbritt, and GS5; the serotype e strain LM7; the serotype f strain OMZ175; and randomly selected S. mutans clinical isolates (Supplementary Table S2). The nucleotide sequences targeted by the Sm479F/R primers in each serotype of S. mutans have been deposited in GenBank under accession numbers EF533872, EF533873, EF533874, EF533875, EF533876, EF533877, EF533878 and EF533879.

Furthermore, S. mutans DNA was detected by real-time qPCR in 14 of the 33 mixed bacterial samples (42.4%), whereas in the results from culture, five of the 33 children (15.2%) had detectable S. mutans in their saliva. All of the samples that tested positive for S. mutans by culturing also tested positive by real-time qPCR (100% agreement). A homogeneous melting peak at 78°C indicated that the amplified target DNA products were specific for S. mutans. Real-time qPCR with the Sm479F/R primers significantly improved the sensitivity of detecting S. mutans in the clinical samples (42.4% vs. 15.2%; P=0.008; Fisher's Exact Test).

Discussion

The results of this study demonstrate that the Sm479F/R primer set is highly sensitive and species-specific for PCR-based detection and evaluation of S. mutans colonization in the oral cavity. The species specificity of the primers was first tested in different types of pure S. mutans DNA samples, and then systematically evaluated and validated in mixed bacterial samples, including S. mutansS. sobrinus, S. mutansS. sanguinis, and mixtures of total bacterial colonies from MM10-medium. The reasons for using bacterial samples from MM10-medium were threefold: (1) Culture methods have served as the ‘gold standard’ for bacterial detection for decades. (2) Previous studies of the colonization of S. mutans, S. sobrinus, and S. sanguinis were all based on conventional culture methods, including the use of MM10-medium. There had been full access to a well-archived bacteriological database to conduct various validation experiments, including testing the newly designed S. mutans-specific primers. (3) The same bacterial samples obtained from the same individuals were utilized for the validation tests to minimize, by design, potential experimental bias. The authors acknowledge that testing of whole saliva samples from the same individuals would yield additional information but longitudinal samples were not available for this study. Overall, the present data demonstrate consistent results among the different sets of bacterial samples; interestingly, the primers can be used to identify not only S. mutans serotype c strains but also serotype e and f strains. Furthermore, the species specificity was confirmed by DNA sequence analysis. These findings suggest that this S. mutans species-specific primer set is reliable and can be applied to evaluate S. mutans colonization for clinical studies.

The S. mutans-specific primer set was based on the discovery of a unique 14-kb HaeIII restriction fragment of UA159, although the significance of this fragment present in S. mutans is not well understood. As the fragment consists of the end of the circular genome with a number of unknown genes and intergenic space regions and a conserved 4-kb segment after the origin, the 14-kb fragment became the starting point for finding a unique signature DNA sequence from S. mutans. In this study, it was found that the Sm479F/R primer set targeted region is directly associated with HtrA and genetic competence in S. mutans of UA159 as demonstrated by Ahn (2005). HtrA homologues have been identified in many gram-positive bacteria including streptococci. Most evidence suggests that HtrA acts as a housekeeping protease to degrade unfolded proteins during heat shock (Pallen & Wren, 1997). Biswas and coworkers (Biswas & Biswas, 2005) found that the HtrA protease is associated with the ability to survive under different stress conditions and is essential for stress tolerance, such as a high or low temperature and under acidic conditions, in S. mutans. Other studies suggest that htrA may also be involved in the biogenesis of extracellular proteins, biofilm formation, and genetic transformation (Diaz-Torres & Russell, 2001; Ahn et al., 2005; Biswas & Biswas, 2005).

In addition to the htrA gene, the Sm479F/R primers target an intergenic locus of unknown function that is unique to S. mutans species. PCR amplification of the 16S–23S rRNA gene intergenic spacer region (IGSR) was found to be a useful tool for bacterial species-specific typing because of the considerable variability in size and sequence among organisms (Bourque et al., 1995; Leys et al., 1999; Kwon et al., 2005; Grattard et al., 2006; Valcheva et al., 2007). A similar assumption was implied, the Sm479F/R was constructed to target one of the major IGSRs of the 14-kb fragment for species selectivity, and a high specificity of the PCR amplification in this study was observed. The particular combination of the 479-bp amplicon, which includes a potential virulence locus (htrA) and an S. mutans species-specific locus (IGSR), may offer a new unique biomarker for PCR-based S. mutans identification and S. mutans DNA quantification.

As the conventional culture method is considered to be the ‘gold standard’ for detecting S. mutans colonization, The present real-time qPCR results were compared with data obtained previously by culture methods. One of the significant findings was that the real-time qPCR with the Sm479F/R primers significantly increased the sensitivity of detecting S. mutans in clinical samples by nearly threefold. Previously, both Loesche's group and this study reported that the average detection levels of S. mutans in saliva range from 104 to 106 CFUs per milliliter using conventional culture methods (Syed & Loesche, 1973; Li et al., 2005a). Oho et al. showed that a PCR method could detect S. mutans in saliva with a detection threshold of >104 CFUs (Oho et al., 2000). In this study, it was observed that 64.3% of the children who were S. mutans negative by the culture method were S. mutans positive by real-time qPCR. As little as 10−2 ng of S. mutans DNA, c. 4.6 × 103 of copies of S. mutans, in the saliva that might not be able to produce cultures under standard laboratory conditions, could be detected by PCR. As population-based caries studies begin to move away from culture methods towards recently developed DNA-based molecular methods, it is critical to develop S. mutans-specific primers that can accurately identify and quantify S. mutans in clinical samples. The present findings suggest that the Sm479F/R primer set has these abilities and may be used for conducting high-throughput epidemiological studies of S. mutans infection and for a better understanding of the microbial role of S. mutans associated with dental caries.

Supplementary material

The following supplementary material is available for this article online:

Table S1. Sequences of the candidate primers, designed from the 13 693-bp HaeIII restriction fragment of S. mutans UA159.

Table S2. Alignment of the nucleotide sequences of the 479-bp amplicons from UA159, ATCC25175 (10449), Ingbritt, GS5, LM7, OMZ175, and two randomly selected mixed bacterial samples (25&#x2013;2 and 25&#x2013;18).

Acknowledgements

This study was supported by the NIDCR Grant DE015706, National Institutes of Health, Bethesda, MD, 20892, USA. The authors thank Dr Joseph Guttenplan, Professor of Basic Science Department at the New York University College of Dentistry, for providing us with the human epithelia cell DNA.

Footnotes

  • Editor: William Wade

References

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