Download Quantitative detection of Streptococcus mutans and bacteria of

Quantitative detection of Streptococcus mutans and bacteria of dental caries and no caries groups in
permanent teeth from a north China population
Key words: Real-time PCR; Streptococcus mutans; Dental caries
Background Streptococcus mutans(S. mutans) is the most major pathogen of dental caries. There are
less reports about the relationship between the Streptococcus mutans(S. mutans) , bacteria and dental
caries in permanent teeth than those in primary teeth. The aim of the present paper was to detect
Streptococcus mutans(S. mutans) and bacteria of dental caries and non-caries groups in permanent teeth
from a north China population by real-time polymerase chain reaction (PCR)and compare the
relationship between the number of these bacteria and the prevalence of dental caries in permanent teeth
from a north China population.
Methods Human saliva samples were collected from 142 subjects with permanent teeth. According to the
dental tooth (DT), 142 subjects were divided into dental caries group (DT ≥ 1) and non-caries group (DT
= 0). With specific primers for S. mutans and 16S rRNA , the total number of S. mutans and total bacteria
of 142 saliva samples were detected by real-time PCR and statistically analyzed.
Results There were no significant difference between the detection rates of S. mutans (P=0.118) and
medians of S. mutans (P=0.115). The ratio of S. mutans to total bacteria from the people with dental
caries was significantly higher than those without caries(P<0.001), but the total number of bacteria from
the people with dental caries was significantly lower than those without caries(P<0.001).
Conclusions S. mutans had different effects on different individual caries in permanent teeth from a
North China population. The ratios of S. mutans to total bacterial in saliva detected by real-time PCR with
Sm479F/R and 16S RNA primers were closely associated with the prevalence of dental caries in the same
population. These assays may be useful for the assessment of an individual’s risk of dental caries.
Dental caries is one of the most common infectious diseases afflicting humans and has a polymicrobial
etiology. The mutans streptococci are generally accepted as one of the principal aetiological agents of
dental caries 1~3. The mutans group of oral streptococci consists of 7 species, which are Streptococcus
cricetus, S. rattus, S. mutans, S. sobrinus, S. downei, S. macacae and S. ferus, and 8 serotypes (a~h).
Among the group, S. mutans including of serotype c, e, f is generally considered to be the principal
aetiological agent for dental caries 4, 5, which possesses a variety of mechanisms to colonize tooth surfaces.
The total amount of S. mutans in saliva more than 108 CFU L-1 is considered as a high-risk critical region
for the formation of dental caries 6, and the quantification of S. mutans in the plaque has been proposed as
a method to recognize the high-risk population of dental caries. Moreover, the total amount of bacteria
and the proportion of S. mutans in bacteria can contribute to the assessment of the individual caries risk
and dental caries incidence.
Various methods have been used to identify S. mutans, including conventional culture methods,
biochemical tests and immunological methods. These techniques are time-consuming and low sensitive
and therefore are inappropriate in situations where rapid diagnostic decisions are required. Molecular
approaches based on polymerase chain reaction (PCR) have therefore been developed for the detection
and identification of S. mutans in oral samples, which are more rapid, highly sensitive and specific than
methods mentioned above7, 8. In the study of mother-to-child transmission of S. mutans from the different
population, the consistent presence of a 14-kb HaeIII restriction fragment was observed in hundreds of S.
1
mutans chromosomal DNA fingerprints 9~11. The aim of this study was to detect S. mutans using specific
primers (Sm479F/R) for a unique sequence of a 14-kb HaeIII restriction fragment 12 and bacteria of dental
caries and no caries in permanent teeth from a north China population by real-time PCR and compare the
relationship between the number of these bacteria and the prevalence of dental caries in different people.
MATERIALS AND METHODS
Baterial strains and DNA Extraction
Serotype Inbrieeing (c), LM 7 (e), DMZ-175 (f), Ss, BHR (d), KI (g), Porphyromonas gingivalis strain
(ATCC 33277), Porphyromonas pulp bacteria (ATCC 35406), Actinomyces viscosus (ATCC19246),
Fusobacterium nucleatum (ATCC 10953), Fusobacterium nucleatum (Fn1011) , saliva streptococci ,
Stenotrophomonas maltophilia, Escherichia coli, Klebsiella pneumoniae, moraxella catarrhalis(MC),
Acinetobacter baumannii(Ab), Streptococcus agalactiae, Citrobacter braakii, Enterobacter cloacae,
Staphylococcus epidermidis and methicillin-resistant Staphylococcus aureus were used in this study.
Genomic DNA was isolated and purified using a Puregene DNA isolation kit (BioDevTech, Co. Ltd,
China) in accordance with the manufacturer’s instructions for gram-positive bacteria. Serotype C was
used as a standard strain. The nucleic acid concentration was determined spectrophotometrically
(Shimadzu UV-1206).
Subject population and saliva sampling
According to simple stochastic sampling method, 142 subjects for conventional oral examination from
October 2008 to March 2010 have participated in this study (49 male and 93 female). The subjects
received complete information regarding the objectives and procedures of the study and provided written
informed consent. According to the dental tooth (DT), they were divided into dental caries group (DT ≥ 1)
and non-caries group (DT = 0) (69 and 73, respectively), ranging in age from 14 to 77 years (39.9 ±
13.8years). The population presented good general health, had not received treatment with antibiotics
within the past 3 months and with gargle liquid within the past 4 weeks prior to the study and had any
food within 2 hours before sampling. Subjects presenting systemic diseases, immuno-deficiency and/or
use of orthodontic or prosthetic devices were excluded from this study. Each participator collected 1.5 ml
of saliva in oral cavity for PCR detection and took an examination for dental caries. Sample saliva in the
microtube centrifuged for 10 min at 12,000 rpm (centrifugal radius of 8 cm, Sigma 3K15, Germany), and
the genomic DNA from the resulting pellet were extracted using the same kit mentioned above.
Conventional and real-time PCR analysis
The universal primers for a broad range of bacteria were designed from 16S rRNA 13 and the S.
mutans-specific primers were designed from Sm479F/R 12. Oligonucleotide primers used in this study
were synthesized by Shanghai Sangon Biological Engineering Technology & Services Co. Ltd. and listed
in Table 1. Conventional PCR assays were performed using a standardized protocol in a thermal cycler
(iCycler Thermal Cycler, Bio-Rad, U.S.A). Reaction reagents were purchased in a kit (Takara Ex Taq
polymerase, Japan). Each reaction mixture (20 µL total volume) contained 2µL 10×PCR buffer (Ex Taq),
0.25 mM dNTP mixture, 10µM each of forward and reverse primers, 1U Ex Taq DNA polymerase, and
2µL template DNA. The reaction was conducted as follows: 95℃ for 2 min, followed by 40 cycles of
denaturation for 30 s at 95℃, primer annealing for 30 s at 63℃ for S. mutans, 68℃ for bacterial and
extension for 50 s at 72℃, and then finally 5 min at 72℃ for extension. The PCR amplicons were
evaluated in a 2% agarose gel in TBE (Tris-borate-EDTA) buffer and stained with ethidium bromide
solution (0. 5 g L-1). The final images of the gels were captured by a digital camera ( Biosepctrum ® AC
System with Gel Camera, Upland CA, USA).
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The real-time PCR was performed using a thermalcycler (ABI PRISM® 7000, USA). Ten-fold serially
diluted, known DNA concentrations of S. mutans Inbrieeing (c) were used as an external standard for
absolute quantification. Reaction reagents were purchased in a kit (SYBR ® Green Realtime PCR Master
Mix -Plus，TOYOBO, Japan):Each tube contained 50 µL of reaction mixture, including 25 µL SYBR
®
Green Realtime PCR Master Mix -Plus-, 5µLplus solution, 10µM each of forward and reverse primers,
5µL template DNA. The cycling conditions were 5 min at 95℃, 40 cycles of 15 s at 95℃ for
denaturation, 1 min at 56 ℃ for S. mutans, and 68℃ for bacterial, followed by a melting curve analysis of
the PCR product.
Statistical analysis
Categorical data were described with number (percentile), and were compared with chi-square test.
Normally distributed numeric data were described with mean (standard deviation), and were compared
with t test. Skewed distributed numeric data were described with median, 25th percentile (Q1) and 75th
percentile (Q3) and comparisons of those data were conducted with Wilcoxon rank sum test. Two-tailed
P≤0.05 was considered statistically significant. All statistical analyses were performed by SAS 9.13
software.
RESULTS
Specificity and sensitivity
(1) Specific detection results:
The conventional PCR electrophoresis results showed that no amplicons of any bacteria except of
serotype c, e, f were observed on gel electrophoresis gel at 479 bp when a pair of S. mutans primers was
used (data not shown). The results of the real-time PCR melting curve analysis showed that only S.
mutans showed a specific peak at 83±0.5℃. These indicated that primers Sm479F/R were highly specific
to S. mutans (data not shown).
(2) Sensitivity detection results
Tenfold serial dilutions starting from 10 µg L-1 of DNA were investigated to determine the sensitivity and
detection range of the real-time PCR. The assay was able to detect bacterial DNA over a linear range of
more than 0.01µg L-1 (data not shown).Ten-fold serially diluted serotype c was amplified with the primers
Sm479F/R by the routine PCR (detection range from100 µg L-1 to 0.01 µg L-1) (data not shown) and the
real-time PCR (detection range from 10 µg L-1 to 1 ng L-1), and the results of standard curve were showed
in figure 1. The lowest detectable concentration of the routine and real-time PCR were 1 µg L-1(the
number of bacteria: 4.6×102 cell copies) and 0.1 µg L-1(the number of bacteria: 4.6×10 cell copies),
respectively.
Statistical analysis of saliva samples the real-time PCR results
The total contents of S. mutans and bacteria were detected respectively with the real-time PCR for 142
saliva samples. The sex ratios and age distribution were not significantly different. The sex ratios were
20:49 in dental caries group and 29:44 in non-caries group, Χ2=1.811，P=0.178. The average ages were
39.5±14.0 and 40.4±13.6 respectively, t=0.414，P=0.680. No significant difference were found about the
detection rates of S. mutans (81.2% and 69.9%, P=0.118) and medians of S. mutans (5.6×107 L-1 and
10.8×107 L-1, P=0.115), as shown in table 2. In table 3, we found that the number of total bacteria
(51.4×108 L-1 and 523.1×108 L-1) and the ratios of S. mutans to bacteria (0.0164and 0.0021) were
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significant different between two groups, P<0.001.
DISCUSSION
S. mutans is generally considered to be the principal pathogen for human dental caries. Quick, sensitive
and specific assays are essential for the early diagnosis and effective assessment of an individual’s risk of
dental caries. It is of great importance to detect the presence of S. mutans early for dental caries prediction
and subsequent treatment. Except classical phenotypic identification the methods used to detect and
identify S. mutans include DNA probe technique, PCR for different target genes and monoclonal antibody
detection of saliva sample3,14-18. We have ever built a semi-quantitative PCR method to detect S. mutans
(serotype c, e and f) in which dexA gene was selected as the target gene and the reference gene was
amplified and assayed by gel electrophoresis at the same time 19-20. Previous studies have indicated that
when the transmission of S. mutans was investigated using a chromosomal DNA fingerprinting technique
in various populations, a 14-kb HaeIII restriction fragment was constantly observed in S. mutans positive
population 9-12. This study further confirmed that primers Sm479F/R which 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, were highly sensitive and species-specific for PCR-based detection and evaluation
of S. mutans.
In the mutans streptococci, S. mutans is the most major pathogen of dental caries and play an important
role in caries production. In view of the high prevalence of dental caries among children, previous
studies8,17,19,21-22 about S. mutans have paid more attention to preschool children, but there were few
reports about the relationship between S. mutans and permanent teeth caries. In this study, all samples
came from the population with permanent teeth. There were no significant difference between caries
group and non-caries group about sex ration and age distribution, which showed good consistency
between two groups. We have detected the S. mutans and total bacterial of 142 saliva samples using the
real-time quantitative PCR and explored the function of S. mutans on permanent teeth caries. Three
interesting characteristic of the profiles in this study should be paid more attention. First, although the
detection rate in dental caries group and non-caries group were 81.2% and 69.9% respectively, there was
not significantly different between the rate of two groups, which were inconsistent with studies of Okada
22
and Zhi HQ et al 17. The results of Zhi HQ et al showed that the detection rates of S. mutans in two
groups were 100% and 75%, respectively and had a significantly difference. Moreover the proportions of
CFU>108 L-1 were approximately 40% at both groups, which was inconsistent with our previous results
19-20
. In previous studies, saliva samples of many children were detected with semi- quantitative PCR and
the detection rate of S. mutans >108 L-1, which were significantly higher. All the subjects of Okada, Zhi
HQ and our previous study were preschool children with severe caries, which was different from our
present study. S. mutans has a variety of virulence factors as a potent initiator of caries. S. mutans can
bind to tooth surfaces in the presence of sucrose by the formation of water-insoluble glucans, a
polysaccharide that aids in binding the bacterium to the tooth, produce lactic acid to enamel
demineralization, and thrive under acidic condition 23-25. In comparison with permanent teeth the
calcification and acid-tolerating ability were poor for primary teeth. In the light of children oral health
habit, we got a conclusion that it was easier in primary teeth than in permanent teeth for S. mutans to play
a role on the formation of caries, which was in line with the results of Wang LJ et al 26. Secondly, the
median (Q1, Q3) of S. mutans was 5.6（2.5，21.2）×107 L-1 in caries group, which was consistent with
Yoshida’s study 27. The median (Q1, Q3) of S. mutans was 10.8(4.5，27.0)×107 L-1 in non-caries group,
which was higher than caries group but there was no significance. In this study, the non-caries group
4
includes the subjects who have never had caries and who had once been sufferring from caries but have
treated and filled and have no fresh dental caries. The interaction between different bacteria may be one
cause. Strahinic et al 28 determined the species and subspecies levels by using sequencing of 16S rDNA
genes and PCR in dental plaques and found the antagonism between fermentative lactobacillium and S.
mutans. And the results of Zhao YZ 29 et al showed that Candida albicans was negatively associated with
S. mutans. For fermentative lactobacillium and Candida albicans were acid-forming bacteria, the
antagonism between fermentative lactobacillium, Candida albicans and S. mutans had no effect on the
formation of dental caries. So the antagonism of different cariogenic bacteria could cause the lower
content of S. mutans in caries group. In addition, the results further validated the study of Emilson30 et al
and Pieralisi 5 et al, which showed the genetic diversity of S. mutans was positively associated with dental
caries, for another possible explanation for S. mutans median (Q1, Q3) of two groups was that S. mutans
virulence factors can differ between populations with contrasting caries prevalence. The early reports5,
30-32
showed that the preschool children with dental caries have more genotypes than the caries-free
children. The existence of several genotypes could merely be a consequence of favorable circumstances
for S. mutans. Moreover, it is possible that the simultaneous action of different genotypes, with distinct
virulence potential, further increases the risk of caries. This may be attributed to heavy colonization and
growth of multiple genotypes in the same oral cavity is likely to be consequences of frequent
consumption of fermentable carbohydrates. Different clonal types of S. mutans detected within the oral
cavity of one subject may exhibit different phenotypic and genetic properties. In addition, the high clonal
diversity of S. mutans can result in colonization by clones with different virulence attributes. Finally, the
ratio of S. mutans to total bacteria should be paid more attention. In accordance with the study of Hata et
al, our study also demonstrated that the ratio of S. mutans to total bacteria was positively associated with
dental caries. In present study, the median ratio of S. mutans to total bacterial was 0.0164, which was low.
But previous results showed that the proportions of S. mutans to mutans streptococcis in dental plaques
ranged from 0.01 to 0.5 17-18 and the ratio of S. mutans to total bacterial was less. This may be because S.
mutans was Gram-positive bacteria and the outer envelope was too hard to be broken 19. The result of this
study showed that both ratios of S. mutans to total bacterial in two groups were low, but it was
significantly higher in caries group. In the formation and development of dental caries, the actual number
of S. mutans was importantly significant for the evaluation of caries-susceptible population and the
formation of dental caries, but the ratio of S. mutans to total bacterial in this study showed the tightly
association with the prevalence of dental caries, and we should attach due importance to the total number
of bacteria in cavity and the combined action of other bacteria and factors.
In conclusion, quantitative amplification of S. mutans with specific sequence for 14kb HaeIII fragment
held better specificity and sensitivity. The detection results of S. mutans to total bacterial in 142 saliva
samples showed that S. mutans had different effects on different individual caries in permanent teeth from
a north China population. The ratios of S. mutans to total bacterial indicated that the formation of caries
teeth was influenced by many factors, so the indicators of predicting the caries risk should be diversified.
ACKNOWLEDGEMENTS
This study was supported by the 863 program Grant 2006AA027.434, China. The authors thank School of
Stomatology, Beijing Stomatological Hospital, Capital Medical University, for providing us with bacteria
strains( including Serotype Inbrieeing (c), LM 7 (e), DMZ-175 (f), Ss, BHR (d), KI (g), ATCC 33277,
ATCC 35406, ATCC19246, ATCC 10953, Fn1011, saliva streptococci) and Microbiology Laboratory,
Beijing Chaoyang Hospital, Capital University of Medical, for providing us with bacteria
strains(including Stenotrophomonas maltophilia, Escherichia coli, Klebsiella pneumoniae, moraxella
5
catarrhalis(MC), Acinetobacter baumannii(Ab), Streptococcus agalactiae, Citrobacter braakii,
Enterobacter cloacae, Staphylococcus epidermidis and methicillin-resistant Staphylococcus aureus).
REFERENCE
1. Loesche WJ. Role of Streptococcus mutans in human dental decay. Microbiol Rev1986; 50:353–380.
2. Becker MR, Paster BJ, Leys EJ, Moeschberger ML, Kenyon SG, Galvin JL, et al. Molecular analysis of bacterial species associated with
childhood caries. J Clin Microbiol 2002; 40:1001–1009.
3. Kamiya RU, Napimoga MH, Höfling JF, Gonçalves RB. Frequency of four different mutacin genes in Streptococcus mutans genotypes
isolated from caries-free and caries-active individuals. J Med Microbiol 2005; 54: 599–604
4. Whiley RA, Beighton D. Current classification of the oral streptococci. Oral Microbiol Immunol 1998;13:195-216.
5. Pieralisi FJS, Rodrigues MR, Segura VG, Maciel SM, Ferreira FBA, Garcia JE, et al. Genotypic Diversity of Streptococcusmutans in
Caries-Free and Caries-Active Preschool Children. International Journal of Dentistry 2010; 1-5
6. Bian YJ. Oral Preventive Medicine. The 3rd Edition. Beijing: People's Medical Publishing House. 2000; 91-93.
7. Ono T, Hirota K, Nemoto K, Fernandes EJ, Ota F, Fukui K. Detection of Streptococcus mutans by PCR amplification of spaP gene. J Med
Microbiol 1994;41:231–235
8. Igarashi T, Yamamoto A, Goto N. Direct detection of Streptococcus mutans in human dental plaque by polymerase chain reaction. Oral
Microbiol Immunol 1996;5: 294–298.
9. Emanuelsson IR, Li Y, Bratthall D. Genotyping shows different strains of mutans streptococci between father and child and within
parental pairs in Swedish families. Oral Microbiol Immunol 1998;13: 271–277.
10. Li Y, Wang W, Caufield PW. The fidelity of mutans streptococci transmission and caries status correlate with breast-feeding experience
among Chinese families. Caries Res 2000;34: 123–132
11. Caufield PW, Saxena D, Fitch D, Li Y. Population structure of plasmid-containing strains of Streptococcusmutans, a member of the
human indigenous biota. J Bacteriol 2007;189: 1238–1243.
12. Chen Z, Saxena D, Caufield PW, Ge Y, Wang M, Li Y. Development of species-specific primers for detection of Streptococcus mutans in
mixed bacterial samples. FEMS Microbiol Lett 2007;272:154-162.
13. Rupf S, Merte K, Eschrich, K. Quantification of bacteria in oral samples by competitive polymerase chain reaction. J Dent Res 1999; 78:
850–856.
14.
Li Y, Caufield P, Emanuelsson I, Thornqvist E. Differentiation of Streptococcus mutans and Streptococcus sobrinus via genotypic and
phenotypic profiles from three different populations. Oral Microbiol Immunol 2001; 16: 16–23.
15. Matsumoto YN, Sugihara M, Koseki Y,Maki Y. Rapid and Quantitative Detection System for Streptococcus mutans in Saliva Using
Monoclonal Antibodies. Caries Res 2006; 40:15–19
16. Smorawinska M, Kuramitsu HK. DNA probes for detection of cariogenic Streptococcus mutans[J]. Oral Microbiol Immunol 1992; 7:
177-181
17.
Zhi QH, Lin HC, Zhang R, Liao YD, Tu JZ. Arbitrarily primed-PCR detection of Streptococcus Mutans and Streptococcus Sobrinus in
dental plaque of children with high dmft and no caries. Chin J Stomatol 2007; 42:219-222
18.
Tan HP, Bian Z, Fan MW, Du MQ, Fan B. Simultanous Detection of Streptococcus Mutans and Streptococcus Sobrinus by Polymerase
Chain Reaction. J Oral Science Research 2006; 22:598-600
19. Xiao B, Wang JQ, Liu JZ, Li CY. Technique and application of cariogenic Streptococcus mutans detection by semi-quantitative PCR.
Chin J Infect Control 2002; 1: 8-11.
20. Wang JQ, Li CY, Xiao B, Liu JZ. Detection of cariogenic Streptococcus mutans by quantitative polymerase chain reaction. Chin J
Stomatol 2002; 37: 281-283.
21. Hata S, Hata H, Miyasawa-Hori H, Kudo A, Mayanagi H. Quantitative detection of Streptococcus mutans in the dental plaque of
Japanese preschool children by real-time PCR. Lett Appl Microbiol 2006; 42:127-131.
22. Okada M, Soda Y, Hayashi F, Doi T, Suzuki J, Miura K, et al. PCR detection of Streptococcus mutans and S. sobrinus in dental plaque
samples from Japanese pre-school children. J Med Microbiol 2002; 51:443-447.
23. Tanzer JM, Chassy BM, Krichevsky MI. Sucrose metabolism by Streptococcus mutans, SL-I. Biochim Biophys Acta. 1971;261:379–387
24. Hamada S, Koga T, Ooshima T. Virulence factors of Streptococcus mutans and dental caries prevention.J Dent Res 1984;63:407-11.
6
25. Simon L. The Role of Streptococcus mutans And Oral Ecology in the Formation of Dental Caries. Lethbridge Undergraduate Research
Journal 2007; 2: 2
26. Wang LJ, Tang R, Bonstein T, Bush P, Nancollas GH. Enamel demineralization in primary and permanent teeth. J Dent Res
2006;85(4):359-63.
27. Yoshida A, Suzuki N, Nakano Y, Kawada M, Oho T, Koga T. Development of a 5' nuclease-based real-time PCR assay for quantitative
detection of cariogenic dental pathogens Streptococcus mutans and Streptococcus sobrinus. J Clin Microbiol 2003; 41:4438-4441.
28. Strahinic I, Busarcevic M, Pavlica D, Milasin J, Golic N, Topisirovic L. Molecular and biochemical haracterizations of human oral
lactobacilli as putative probiotic candidates[J]. Oral Microbiol Immunol 2007; 22:111 - 117.
29. Zhao YZ, Gong YB. The correlation between oral Candida albicans and putative cariogenic bacteria. Chinese Journal of Practical
Stomatology 2010; 3: 3170-3172
30. Emilson CG, Carlsson P, Bratthall D. Strains of mutans streptococci isolated in a population with extremely low caries prevalence are
cariogenic in the hamster model. Oral Microbiol Immunol 1987; 2:183–186
31. Guo LH, Shi JN, Zhang Y, Liu XD, Duan J, Wei S. Identification of genetic differences between two clinical isolates of Streptococcus
mutans by suppression subtractive hybridization. Oral Microbiol Immunol 2006; 21: 372–380,.
32. Caufield PW. Dental caries—a transmissible and infectious disease revisited: a position paper. Pediatr Dent 1997; 19: 491–498,.
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Table 1
The primer sequence
Amplicon
Primer sequence
Target gene
size（bp）
Primers for S. mutans
Sm479F
2 029 599 –
5’-TCGCGAAAAAGATAAACAAACA-3’
2 030 077 nt of
479
Sm479R
AE014133
5’-GCCCCTTCACAGTT GGTTAG-3’
Universal primers for bacteria
Ub f 5￠-ACT ACG TGC CAG CAG CC
296-300
16S rRNA
Ub r 5￠-GGA CTA CCA GGG TAT CTA ATC C
Table 2 The detection results S. mutans in saliva samples
Total
Positive number
Median（Q1,Q3）of S. mutans
（Detection rate）
（×107 /L）*
Dental caries
69
56（81.2%）
5.6（2.5，21.2）
Non-caries
73
51（69.9%）
10.8（4.5，27.0）
Total
142
107（75.4%）
7.7（2.7，23.4）
Statistics
Χ2=2.437
Z=1.575
P
0.118
0.115
* Only including subjects with positive S. mutans.
8
Table 3 Bacteria detection results of siliva samples（Only including subjects with positive S. mutans）
Median（Q1,Q3）of bacteria Median（Q1,Q3）of S. mutans Percentile of
（×108/L）
/ bacteria
CFU>108/L
Dental caries
51.4（13.8，141.6）
0.0164(0.0032,0.0734)
41.1%
Non-caries
523.1（185.4，1505.4）
0.0021(0.0010,0.0109)
56.9%
Total
Z=5.264
Z=4.460
Χ2=2.664
Statistics
<0.001
<0.001
0.103
2~7：The concentrations of serotype c were ranging from 104µg/L to10-1 µg/L
Fig 1 The standard curve of ten-fold serially diluted S. mutans standard strain with the primers Sm479F/R
by the real-time qPCR
9