dental caries is a multifactorial infectious disease , related to biofilm accumulation on dental surface16 and frequent consumption of fermentable carbohydrates2 . by the fermentation of dietary carbohydrates , the bacteria in
the dental biofilm produce acids that decrease the ph and increase the biofilm potential in promoting dental demineralization3,4 .
additionally , the acid environment selects cariogenic bacteria , such as mutans streptococci16 . among them , streptococcus mutans is known to be one of the most important cariogenic microorganisms15,16 because , in addition to being acidogenic and acid - tolerant , it uses sucrose to produce insoluble glucans in biofilm matrix3 , which may play an important role in the development of caries3,5,17,22 .
different genotypes of s. mutans may present different expression levels of glucosyltransferases19 and higher production of insoluble polysaccharides has been reported by genotypes from caries - active individuals8,20 .
different genotypes of s. mutans have been found in saliva , and dental biofilm and ap - pcr technique has been widely used to discriminate this genotypic diversity1,6,7,9,11,12,18,23,26 . this technique has a discriminatory potential comparable to other techniques for genotypic identification of s. mutans 
 13,14,28 .
the application of more than one arbitrary primer was suggested to increase the discriminatory power of ap - pcr genotyping13,29 .
saarela , et al.28 ( 1996 ) have reported that primers opa 05 and opa 13 were efficient to identify s. mutans genotypes .
truong , et al.29 ( 2000 ) , investigating genotypic diversity among mutans streptococci , verified that primers opa 03 and opa 18 were capable to distinguish s. mutans from other oral microorganisms .
li and caufield13 ( 1998 ) , after evaluating various arbitrary primers in the genotyping of reference strains of s. mutans , suggested that the primer opa 02 showed the best power of genotype discrimination , giving the highest number of amplicons when compared to other primers .
nevertheless , there is always the question whether other primers should be used to ascertain this diversity and , in addition , the evaluation of different primers was performed with only reference strains13 .
thus , the aim of the present study was to evaluate the degree of agreement of genotypic identification between ap- pcr reactions performed with distinct arbitrary primers ( opa 02 , opa 03 , opa 05 , opa 13 and opa 18 ) of streptococcus mutans isolated from saliva of adult volunteers .
this research was approved by the research ethics committee of the dental school of piracicaba , state university of campinas ( protocol # 078/2007 ) , the volunteers were fully informed about the procedures , and written consent was obtained prior to the beginning of the study .
stimulated saliva from 11 volunteers , who participated in a previous study evaluating the effect of sucrose on genotypic diversity1 , was collected for isolation of mutans streptococci .
healthy volunteers ( 18 to 28 years old ) , who fulfilled inclusion criteria ( counts of mutans streptococci in saliva from 10 to 10 colony - forming units ( cfu ) per ml ) and exclusion criteria ( antibiotic use for the last 2 months prior to the beginning of the study , use of any form of medication that modifies salivary secretion , periodontal disease , general / systemic diseases ) took part in this study .
thereafter , pcr with specific primers ( gtfb and gbpb ) was conducted18,24 for identification of s. mutans , and then , these isolates were submitted to genotyping protocols by arbitrarily primed polymerase chain reaction ( ap - pcr ) with 5 different arbitrary primers : opa 02 , opa 03 , opa 05 , opa 13 and opa 18 . stimulated saliva samples were collected by parafilm chewing , in the morning , under fasting condition , and without previous toothbrushing .
saliva samples were diluted in sterile 0.9% nacl and plated on mitis salivarius - bacitracin agar ( msb ) ( difco , sparks , md , usa ) .
after incubation for 48 h at 37c in 10% co2 , 8 representative morphological types of s. mutans colonies were collected from each sample , subcultured on mitis salivarius agar ( msa ) ( difco , sparks , md , usa ) and pure cultures stored at -70c in 10% skim milk medium10 .
the purity and identity of the isolates were checked by gram 's stain and colonial morphology on msa .
then , aliquots were collected from skim milk and plated on brain heart infusion ( bhi ) ( difco , sparks , md , usa ) , which was incubated for 24 h at 37c in 10% co2 .
the colonies from bhi agar were inoculated into todd hewitt broth ( difco , sparks , md , usa ) and incubated for 18 h at 37c and 10% co2 .
cultures were then centrifuged at 4c for 15 min , genomic dna was extracted and purified from cell pellet18 , and stored at -20c .
integrities of the genomic dna samples were checked in samples electrophoretically resolved in 1% agarose gel and stained with ethidium bromide ( 5 g / ml ) .
isolates were confirmed for species identity in pcr reactions with primers specific for gtfb , enconding glucosyltransferase b ( 5 , actacactttcgggtggcttgg-3 and 5 , cagtataagcgccagtttcatc-3)24 , and specific to gbpb , enconding glucan - binding protein b ( 5 , caacagaagcacaaccatca-3 and 5- tgtccaccattaccccagt-3)18 .
although gtfb primers amplify s. mutans gtfb gene , a previous study revealed that gtfb primers yield some cross - amplification with several clinical isolates of s. sobrinus ( defined as s. sobrinus species by sequencing of 16srrna)10 . to overcome this problem ,
the strains were also tested with gbpb primers because these primers yield amplicons of predicted size in all s. mutans genotypes tested in a previous study18 , and do not amplify s. sobrinus sequences18 .
each reaction consisted of 1 l template dna , 10 m of each primer , 10 m of dntp , 2.5 l 1x pcr buffer , 50 mm mgcl2 and 5u/l taq dna polymerase in a total volume of 25 l .
the amplification reaction was performed in a thermocycler ( tc-412 , techne , duxford , cambridge , uk ) in 30 cycles as follows : denaturation 95c for 30 s , annealing at 59c for 30 s , and extension at 72c for 1 min , using s. mutans ua130 ( kindly provided by dr .
page w. caufield , new york university , ny , usa ) and s. sobrinus , and distilled / deionized water as positive and negative controls , respectively .
the resulting amplicons were submitted to electrophoresis in 2% agarose gels and the images were captured by a digital imaging system ( gel logic 100 imaging system , kodak , tokyo , japan ) .
ap - pcr assays were performed with the arbitrary primers : opa 02 ( 5-tgccgagctg-3)13 , opa 03 ( 5- agtcagccac-3)29 , opa 05 ( 5-aggggtcttg-3)28 , opa 13 ( 5-cagcacccac-3)28 and opa 18 ( 5- aggtgaccgt-3)29 .
the reactions were processed in 50 l mixtures containing 1x pcr buffer , 5 u / pl of taq dna polymerase , 10 mm dntp , 20 m primer , 50 mm mgcl2 and 2 l template dna .
reactions were performed with the following conditions : 
  opa 02 : one initial cycle of denaturation at 95c for 2 min , followed by 45 cycles of 94c for 30 s ( denaturation ) , 36c for 30 s ( annealing ) and 72c for 1 min ( extension ) and a final extension at 72c for 5 min; opa 03 and 18 : one initial cycle of denaturation at 95c for 2 min , followed by 30 cycles of 94c for 1 min ( denaturation ) , 32c for 1 min ( annealing ) and 72c for 2 min ( extension ) and a final extension at 72c for 5 min; opa 05 and 13 : one initial cycle of denaturation at 95c for 2 min , followed by 35 cycles of 95c for 1 min ( denaturation ) , 36c for 2 min ( annealing ) and 72c for 2 min ( extension ) and a final extension at 72c for 5 min . 
  
opa 02 : one initial cycle of denaturation at 95c for 2 min , followed by 45 cycles of 94c for 30 s ( denaturation ) , 36c for 30 s ( annealing ) and 72c for 1 min ( extension ) and a final extension at 72c for 5 min ;  opa 03 and 18 : one initial cycle of denaturation at 95c for 2 min , followed by 30 cycles of 94c for 1 min
( denaturation ) , 32c for 1 min ( annealing ) and 72c for 2 min ( extension ) and a final extension at 72c for 5 min ;  opa 05 and 13 : one initial cycle of denaturation at 95c for 2 min , followed by 35 cycles of 95c for 1 min ( denaturation ) , 36c for 2 min ( annealing ) and 72c for 2 min ( extension ) and a final extension at 72c for 5 min .
the ap - pcr products were electrophoretically resolved in 1.5% agarose gels , using s. mutans ua130 and distilled and deionized water as positive and negative control , respectively .
the gel was stained with a 5 g / ml of ethidium bromide solution for 10 min , and their images were captured by a digital imaging system ( gel logic 100 imaging system , kodak , tokyo , japan ) .
the amplicon profiles ( amplitypes ) of the same volunteer were always resolved side - by - side in the same gel for visual comparisons1,11,25 .
isolates were considered as having the same genotypic identity when presented identical ap - pcr product - size profiles .
the genotypes found were analyzed descriptively and their proportion , in relation to the number of colonies isolated in each sample and condition , was calculated . also , the number of bands from each genotype amplified by each of the arbitrary primers was counted and the mean value was calculated .
this research was approved by the research ethics committee of the dental school of piracicaba , state university of campinas ( protocol # 078/2007 ) , the volunteers were fully informed about the procedures , and written consent was obtained prior to the beginning of the study .
stimulated saliva from 11 volunteers , who participated in a previous study evaluating the effect of sucrose on genotypic diversity1 , was collected for isolation of mutans streptococci .
healthy volunteers ( 18 to 28 years old ) , who fulfilled inclusion criteria ( counts of mutans streptococci in saliva from 10 to 10 colony - forming units ( cfu ) per ml ) and exclusion criteria ( antibiotic use for the last 2 months prior to the beginning of the study , use of any form of medication that modifies salivary secretion , periodontal disease , general / systemic diseases ) took part in this study .
thereafter , pcr with specific primers ( gtfb and gbpb ) was conducted18,24 for identification of s. mutans , and then , these isolates were submitted to genotyping protocols by arbitrarily primed polymerase chain reaction ( ap - pcr ) with 5 different arbitrary primers : opa 02 , opa 03 , opa 05 , opa 13 and opa 18 .
stimulated saliva samples were collected by parafilm chewing , in the morning , under fasting condition , and without previous toothbrushing .
saliva samples were diluted in sterile 0.9% nacl and plated on mitis salivarius - bacitracin agar ( msb ) ( difco , sparks , md , usa ) .
after incubation for 48 h at 37c in 10% co2 , 8 representative morphological types of s. mutans colonies were collected from each sample , subcultured on mitis salivarius agar ( msa ) ( difco , sparks , md , usa ) and pure cultures stored at -70c in 10% skim milk medium10 .
the purity and identity of the isolates were checked by gram 's stain and colonial morphology on msa .
then , aliquots were collected from skim milk and plated on brain heart infusion ( bhi ) ( difco , sparks , md , usa ) , which was incubated for 24 h at 37c in 10% co2 .
the colonies from bhi agar were inoculated into todd hewitt broth ( difco , sparks , md , usa ) and incubated for 18 h at 37c and 10% co2 .
cultures were then centrifuged at 4c for 15 min , genomic dna was extracted and purified from cell pellet18 , and stored at -20c .
integrities of the genomic dna samples were checked in samples electrophoretically resolved in 1% agarose gel and stained with ethidium bromide ( 5 g / ml ) .
isolates were confirmed for species identity in pcr reactions with primers specific for gtfb , enconding glucosyltransferase b ( 5 , actacactttcgggtggcttgg-3 and 5 , cagtataagcgccagtttcatc-3)24 , and specific to gbpb , enconding glucan - binding protein b ( 5 , caacagaagcacaaccatca-3 and 5- tgtccaccattaccccagt-3)18 .
although gtfb primers amplify s. mutans gtfb gene , a previous study revealed that gtfb primers yield some cross - amplification with several clinical isolates of s. sobrinus ( defined as s. sobrinus species by sequencing of 16srrna)10 . to overcome this problem ,
the strains were also tested with gbpb primers because these primers yield amplicons of predicted size in all s. mutans genotypes tested in a previous study18 , and do not amplify s. sobrinus sequences18 .
each reaction consisted of 1 l template dna , 10 m of each primer , 10 m of dntp , 2.5 l 1x pcr buffer , 50 mm mgcl2 and 5u/l taq dna polymerase in a total volume of 25 l .
the amplification reaction was performed in a thermocycler ( tc-412 , techne , duxford , cambridge , uk ) in 30 cycles as follows : denaturation 95c for 30 s , annealing at 59c for 30 s , and extension at 72c for 1 min , using s. mutans ua130 ( kindly provided by dr .
page w. caufield , new york university , ny , usa ) and s. sobrinus , and distilled / deionized water as positive and negative controls , respectively .
the resulting amplicons were submitted to electrophoresis in 2% agarose gels and the images were captured by a digital imaging system ( gel logic 100 imaging system , kodak , tokyo , japan ) .
ap - pcr assays were performed with the arbitrary primers : opa 02 ( 5-tgccgagctg-3)13 , opa 03 ( 5- agtcagccac-3)29 , opa 05 ( 5-aggggtcttg-3)28 , opa 13 ( 5-cagcacccac-3)28 and opa 18 ( 5- aggtgaccgt-3)29 .
the reactions were processed in 50 l mixtures containing 1x pcr buffer , 5 u / pl of taq dna polymerase , 10 mm dntp , 20 m primer , 50 mm mgcl2 and 2 l template dna .
reactions were performed with the following conditions : 
  opa 02 : one initial cycle of denaturation at 95c for 2 min , followed by 45 cycles of 94c for 30 s ( denaturation ) , 36c for 30 s ( annealing ) and 72c for 1 min ( extension ) and a final extension at 72c for 5 min; opa 03 and 18 : one initial cycle of denaturation at 95c for 2 min , followed by 30 cycles of 94c for 1 min ( denaturation ) , 32c for 1 min ( annealing ) and 72c for 2 min ( extension ) and a final extension at 72c for 5 min; opa 05 and 13 : one initial cycle of denaturation at 95c for 2 min , followed by 35 cycles of 95c for 1 min ( denaturation ) , 36c for 2 min ( annealing ) and 72c for 2 min ( extension ) and a final extension at 72c for 5 min . 
  
opa 02 : one initial cycle of denaturation at 95c for 2 min , followed by 45 cycles of 94c for 30 s ( denaturation ) , 36c for 30 s ( annealing ) and 72c for 1 min ( extension ) and a final extension at 72c for 5 min ;  opa 03 and 18 : one initial cycle of denaturation at 95c for 2 min , followed by 30 cycles of 94c for 1 min ( denaturation ) , 32c for 1 min ( annealing ) and 72c for 2 min ( extension ) and a final extension at 72c for 5 min ;  opa 05 and 13 : one initial cycle of denaturation at 95c for 2 min , followed by 35 cycles of 95c for 1 min ( denaturation ) , 36c for 2 min ( annealing ) and 72c for 2 min ( extension ) and a final extension at 72c for 5 min .
the ap - pcr products were electrophoretically resolved in 1.5% agarose gels , using s. mutans ua130 and distilled and deionized water as positive and negative control , respectively .
the gel was stained with a 5 g / ml of ethidium bromide solution for 10 min , and their images were captured by a digital imaging system ( gel logic 100 imaging system , kodak , tokyo , japan ) .
the amplicon profiles ( amplitypes ) of the same volunteer were always resolved side - by - side in the same gel for visual comparisons1,11,25 .
isolates were considered as having the same genotypic identity when presented identical ap - pcr product - size profiles .
the genotypes found were analyzed descriptively and their proportion , in relation to the number of colonies isolated in each sample and condition , was calculated . also , the number of bands from each genotype amplified by each of the arbitrary primers was counted and the mean value was calculated .
a total of 88 representative colonies of s. mutans were isolated from saliva , being 8 from each volunteer .
all isolates were identified with all tested arbitrary primers were identified as s. mutans in the pcr reactions with specific primers .
most volunteers ( 9 out of 11 ) carried only one genotype ( table 1 ) .
the exceptions were volunteer 8 , who harbored 3 genotypes identified with all tested arbitrary primers ( figure 1 ) , and volunteer 11 , who presented 2 genotypes when evaluated using the following primers : opa 02 , 03 , 05 and 13 . using primer opa 18 ,
considering the proportions of genotypes in relation to the total number of isolates , in volunteer 8 , the predominant genotype was " h " ( 75% ) and the others , " i " and " j " , represented 12.5% each one of the 8 isolates of this volunteer . for volunteer 11 , the predominant genotype was " m " ( 87.5% ) and
the genotype " n " represented 12.5% . in addition , the genotypes were distinct among all volunteers .
table 1 also presents the number of bands produced after the ap - pcr reaction with each one of the arbitrary primers tested .
the amplification with opa 02 primer resulted in a higher number of bands ( mean of 11.9 ) , in relation to the other primers .
opa 03 primer presented around 9.7 bands and the other primers between 8.6 and 8 bands .
the proportion ( % ) of the genotypes in relation to the number of colonies isolated in each condition is represented within the parenthesis .
the genotypes m and n were identified by all primers , except for opa 18 .
it is well known that the oral cavity harbors distinct genotypes of s. mutans 
 9,12,18,20,27,28 . in the present study ,
only one genotype was found in the saliva of 9 out of 11 volunteers ( table 1 ) .
this agrees with the findings of previous studies in saliva or dental biofilm samples of children9,12,18,20,27,28 and adult subjects1,21 .
this low genotypic diversity could be related to the fact that other genotypes might be present in saliva in a proportion below to the detection limit of the microbiological method used26 .
also - certain genotypes present in the oral cavity could become permanently established , while other genotypes , due to their reduced ability of interacting with the host , form a transient population21 . despite this low genotypic diversity found in the present study ,
saliva samples harbor those genotypes present at higher proportions in dental biofilms1,12 . in the present study , although distinct genotypes were identified by all primers tested , the opa 02 showed the best results , considering the number of bands produced by reaction ( table 1 ) .
these data are in agreement with the study of li and caufield13 ( 1998 ) . despite the lower number of bands yielded in reactions with opa 03 , when compared to primer opa 02 , primer opa 03 allowed an efficient differentiation of genotypes .
this result is in contrast with those of li and caufield13 ( 1998)m who found that opa 03 presented a lower discriminatory capacity than opa 02 .
in addition , opa 05 , 13 and 18 showed a smaller number of bands than opa 02 and opa 03 ( table 1 ) . the reduced number of bands might decrease the differentiation among genotypes , since just one genotype was identified by opa 18 in volunteer 11 , compared to the other two genotypes identified with the other primers ( table 1 ) .
in addition , although opa 05 produced fewer bands than opa 02 and 03 , which might make difficult the differentiation of the genotypes , it seems that this characteristic did not impair the identification and differentiation of the genotypes in the samples analyzed . because of this finding , considering the results of the present study , an association between opa 02 and opa 03 or opa 02 and opa 05 may be useful in the identification of genotypic diversity .
nevertheless , further studies should evaluate a larger number of volunteers and different samples , such as biofilm , which present a higher diversity , using different primers .
in conclusion , the results of the present study suggest that primers opa 02 , 03 , 05 and 13 were suitable for genotypic identification of s. mutans isolates of saliva from adult volunteers .
 streptococcus mutans has been considered one of the main etiological agents of dental caries and the genotypic diversity rather than its salivary counts may be considered as a virulence factor of this bacterium . for genotyping with polymerase chain reaction ( pcr ) with arbitrary primers , 
 several primers have been used in order to improve complexity and specificity of amplicon patterns . 
 thus , the aim of this study was to evaluate the degree of agreement of genotypic identification among ap - pcr reactions performed with 5 distinct arbitrary primers of s. mutans isolated from saliva . stimulated saliva was collected from 11 adult volunteers for isolation of s. mutans , and a total of 88 isolates were genotyped with arbitrary primers opa 02 , 03 , 05 , 13 and 18 . 
 fourteen distinct genotypes were identified in the saliva samples . 
 most volunteers ( 9 out of 11 ) presented only one genotype . 
 the results of the present study suggest that primers opa 02 , 03 , 05 and 13 were suitable for genotypic identification of s. mutans isolates of saliva from adult volunteers . 