Identification of oral streptococci using PCR-based,reverse-capture,checkerboard hybridization

A PCR based, reverse capture checkerboard hybridization methodology was designed to rapidly detect and enumerate oral species of Streptococcus and other genera in plaque samples to assess the bacterial etiology of root surface caries or other oral diseases. The procedure circumvents the need for in vitro bacterial cultivation. Up to 30 reverse capture probes that target regions of 16S rRNA genes are deposited on a nylon membrane in separate horizontal lanes using a MiniSlot apparatus. 16S rRNA genes of DNA from plaque or pure cultures are PCR amplified using a digoxigenin-labeled primer. Hybridizations are performed in vertical channels in a Miniblotter apparatus with digoxigenin-labeled amplicons from up to 45 samples. Consequently, 1350 hybridizations are performed simultaneously using a single membrane. Hybridiza-tion signals are detected using chemifluorescence procedures. Bacterial numbers are determined by analyzing hybridization signals using a Storm system. A genus-specific probe is used to assess the total number of streptococci and universal probes are used to assess total bacterial counts. Use of this procedure enables laboratories to analyze thousands of clinical or environmental samples with many probes in a relatively short period. This methodology has broad range applications in microbiology to monitor population distributions in a given environment.