| Mutans streptococci, especially s. mutans and s. sobrinus, were believed as the chief cariogenic bacteria in oral cavity, and it might be useful to quantify them in saliva for screening children in high caries risk. But the traditional methods to detect them, including species detection, culturing, distinguishing their biochemical characters, were hard to fulfill the clinical needs. And there have been some new methods which can detect these microorganisms in the molecule level, such as polymerase chain reaction (PCR).The present study aimed to establish a real-time fluorescence quantitative polymerase chain reaction method (FQ PCR) to quantify mutans streptococci quickly in children's saliva.1. Two pairs of specific primers designed from s. mutans!6S rRNA gene and s. sobrinus dexA gene) were chosen based on our experiments in the first experiment, follows were their sequences:PI: 5'-GGT CAG GAA AGT CTG GAG TAA AAG GCT A-3\ P2: 5'-GCG TTA GCT CCG GCA CTA AGC C-3'(771-750), P3: 5'-CAA GAC TGG AAC ACT TGG A-3\ P4S 5'-GGT TGT AGT AGC GTT GGA-3\ PI, P2 were expected to be specific to s. mutans and P3, P4 to be specific to s. sobrinus accordingly. They were tested by PCR and compared with 12 species of standard mutans streptococci (serotype a~h) and other 23 strains oral bacteria for their specific abilities. The results showed that the 4 oligonucleotides were highly preserved in oral bacteria strains and could be used as specific primers. The PCR method could detect at least 10 bacteria copies and it was more sensitive compared with other methods. Also we found that the direct boiling method for purifying bacteria genome could fulfill the needs of templates making.2. Based on the study 1, we established real-tune fluorescence quantitativepolymerase chain reaction methods (FQ PCR) to quantify both s. mutans and s. sobrinus by adding a specific TaqMan oligonucleotide probe to each PCR reactions of the 1st study. The efficacy of the two FQ PCR methods were verified by amplifying standard bacteria template with different concentrations from 102~106 copies per milliliter, and the Ct value (cycle threshold) for different concentrations had a highly closed correlation in FQ PCR (r = -0.999609). Then 58 saliva samples from children of 3 to 5 years old were tested quantitatively by both FQ PCR methods and the culturing methods, and the latter method was chosen as a control. The evaluation indexes for FQ PCR were as follows compared with control: Se =100%, Sp =94.62%, PV+ =94.31%, PV. =100%, correct rate( JT ) = 97. 13%.3. A caries prediction model was achieved from our investigation of 203 preschool children aged from 3 to 5 years old. In the beginning of our investigation, the dental caries of objects were examined and recorded, and 1 ml unstimulated whole saliva were acquired from each individual, then they were tested for s. mutans and s. sobrinus quantification by the FQ PCR methods established in the previous studies. 6 months later, the children were reexamined for caries status. Then we could analysis these data using Logistic Regression Analysis. In the final Logistic prediction model, 3 factors were chosen for their high relationship with future caries, they were saliva s. mutans counts, saliva s. sobrinus counts and past caries experiences(dmfs value).The evaluation indexes for this caries prediction model were as follws: Se =81.52%, Sp =92.78% correct rate( it) = 89.24% .In brief, the FQ PCR methods builded in this study displayed a specific and accurate property, and it was more timesaving compared with the culturing method to detect both s. mutans and s. sobrinus.
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