Endodontic P. acnes isolates identified by partial 16S rRNA gene sequencing were typed by partial recA gene sequencing [18 (link)]. All selected P. acnes isolates were subcultured on FAA and grown for 24 h.
The P. acnes recA gene was amplified using primer PAR-1 (positions −96 to −75; 5′-AGCTCGGTGGGGTTCTCTCATC-3′) and primer PAR-2 (positions +1105 to +1083; 5′-GCTTCCTCATACCACTGGTCATC-3′), which generated a 1201-bp amplicon [18 (link)]. The reaction mix (final volume, 25 μl) comprised of 0.5 μl of PAR-1 (concentration 10 pmol/μl; Sigma), 0.5 μl of PAR-2 (concentration 10 pmol/μl; Sigma), 23 μl of Reddymix (Thermo Scientific) and 1 μl DNA extract.
The thermal cycling conditions included initial denaturation at 95 °C for 3 min, denaturation at 95 °C for 1 min, annealing at 55 °C for 30 s, and extension at 72 °C for 90 s, repeated for 35 cycles and a final extension at 72 °C for 10 min. Amplified products were run on a 0.5 % agarose gel and visualized under UV transillumination.
Sequencing was performed as described above. The P. acnes recA sequences were compared with GenBank sequences AY642055 (type IA), EU687255 (type IB), AY642061 (type II) and DQ672252 (type III). NJ trees were constructed using the Jukes-Cantor method with MEGA (version 4.1) software (www.megasoftware.net /).
The P. acnes recA gene was amplified using primer PAR-1 (positions −96 to −75; 5′-AGCTCGGTGGGGTTCTCTCATC-3′) and primer PAR-2 (positions +1105 to +1083; 5′-GCTTCCTCATACCACTGGTCATC-3′), which generated a 1201-bp amplicon [18 (link)]. The reaction mix (final volume, 25 μl) comprised of 0.5 μl of PAR-1 (concentration 10 pmol/μl; Sigma), 0.5 μl of PAR-2 (concentration 10 pmol/μl; Sigma), 23 μl of Reddymix (Thermo Scientific) and 1 μl DNA extract.
The thermal cycling conditions included initial denaturation at 95 °C for 3 min, denaturation at 95 °C for 1 min, annealing at 55 °C for 30 s, and extension at 72 °C for 90 s, repeated for 35 cycles and a final extension at 72 °C for 10 min. Amplified products were run on a 0.5 % agarose gel and visualized under UV transillumination.
Sequencing was performed as described above. The P. acnes recA sequences were compared with GenBank sequences AY642055 (type IA), EU687255 (type IB), AY642061 (type II) and DQ672252 (type III). NJ trees were constructed using the Jukes-Cantor method with MEGA (version 4.1) software (