Mutans Streptococci Genotyping by High Throughput REP-PCR

Mutans streptococci (MS) play an important role in initiating dental caries. Previous use of genetic techniques to identify MS has revealed that many young children appear to be infected with MS from their mothers. Objectives: This study is the initial report of a longitudinal study to determine MS genotype diversity, stability and commonality for primary and newly erupting permanent molars. We report the numbers and diversity of genotypes within the oral cavity for plaque samples collected from children and their household family members. Methods: This study consisted of ten families, which included 5-6 year old children (index children) prior to eruption of at least one permanent molar and household family member, i.e., mother, siblings, and other relatives living with the index child. Plaque samples from molars plated on Gold's media, grown anaerobically, and bacterial colonies were transferred to Todd Hewitt (TH) plates. Single isolated colonies from a TH plate were subsequently inoculated into Todd Hewitt broth. DNA was extracted for repetitive extragenic palindromic-polymerase chain reaction (rep-PCR) (Bacterial barcodes, Inc, Atlanta, GA) followed by microfluidics-based DNA amplicon fractionation and characterization using an Agilent 2100 Electrophoresis Bioanalyzer. Analysis was performed with the DiversiLab software (v3.3) using the Pearson correlation coefficient method to create DNA fingerprints of virtual gel images to aid in data interpretation. Results: Of 20 isolates from each subject, less than 4 distinct genotypes were demonstrated. Conclusion: Highly integrated rep-PCR and microfluidics LabChip® assay is efficient for high throughput screening of MS genotypes between family members and continual database construction. These findings will be used for future longitudinal comparisons within and between index children and family members as new permanent molar teeth erupt.

Supported by grant #DE016684 from the NIDCR