ABSTRACT
The aim of this study was to develop and evaluate a padlock probe based on the Rolling Circle Amplification (RCA), which targeted to 16S-23S rDNA region of S. mutans. The specificity of developed padlock probe was tested for DNA within a panel strains, including S. mutans isolated from the saliva and reference strains of the genus Streptococcus, as well as total DNA samples of biofilm and saliva. The results were positive either for DNA samples of S. mutans or DNA samples recovered from the biofilm and saliva revealing the specificity of designed padlock probe. The padlock probe based on the RCA was proved to be an effective, reproducible method for S. mutans detection and demonstrated the possibility of a rapid detection and accurate identification of S. mutans infection.
ABSTRACT
Objective To develop a novel padlock probe and rolling circle amplification(RCA) to detect 5 common cell culture contaminant Mollicute( Mycoplasma arginini, Mycoplasma fermentans, Mycoplasma hyorhinis, Mycomplasma orale, and Acholeplasma laidlawii ). Methods "Universal" primers ( SPS1, SPA2 and SPS2, SPA1) were used to amplify the Mollicute 16S-23S rRNA intergenic spacer region.Amplicon was ligate Mollicute specific padlock probe. Probe amplified and monitored using a Corbett RotorGeneTM 6000 machine. Results Five reference Mollicute strains were correctly detected by RCA method.There was no cross-reaction. RCA method can sensitive detect 10 copies templates and show strong positive signal. Sixty-two cell culture specimens were detected. Thirty-seven samples were contained single specie Mollicute and 14 samples contained two Mollicutes. Eleven samples did not contained Mollicute. RCA detection results were concordant with previously species-specific PCR. Conclusion RCA can rapid, sensitive and specific detect contamination Mollicute.