PI35:16S rRNA genes pyrosequensing of peri-implantitis
Sub-mucosal and sub-gingival plaque samples were obtained from the deepest pockets at the peri-implanttitis and periodontitis. After three paper points were inserted into a pocket for 30 seconds, all paper points were removed and placed in a sterile tube with 1 ml of sterile distilled water. Samples were mixed for 1 min using a vortex mixer. After removing the paper point, each sample was collected by centrifugation at 12,000g for 5 min.The Bacterial DNA was extracted and purified from the pellet, using Mora extract kit (Kyokuto Pharmaceuticals, Tokyo, Japan)  in accordance with the manufacturer?s instructions. Purification of the crude DNA was carried out according to the manufacturer?s instructions. PCR amplification of the V4 region of the 16S rRNA gene was carried out with bacterial universal primers 515F and 806R, which contained a 454 Life Sciences FLX sequencing primer and a 10-bp barcode sequence for multiplex. Forward: 5?-CCATCTCATCCCTGCGTGTCTCCGACTCAG-10-bp barcode-GTGCCAGCMGCCGCGGT-3? Reverse:5?-CCTATCCCCTGTGTGCCTTGGCAGTC-GGACTACCAGGGTATCTAAT-3? The PCR was carried out in a final volume of 50 ?L including 1?2 ?L template DNA using Takara Ex Taq HS (Takara Bio Inc., Otsu, Japan). Each amplification began with a denaturation step of 94 ? for 1 min. This was followed by 35 cycles of 98 ? for 10 s, 55 ? for 30 s and 72 ? for 2 min. Amplification cycles were followed by a final extension at 72 ? for 1 min. PCR products were purified by NucleoSpin Gel NucleoSpin Extract ? (Macherey? Nagel, D?ren, Germany) followed by AMPure paramagnetic beads (Agencourt Bioscience Corporation, Beverly, MA, USA), according to manufacturer?s protocol. Amplicons were quantified using the using KAPA Library Quantification Kit for Roche 454 GS Titanium (KAPA Biosystems, Woburn, MA). Quantified and equally mixed amplicons were sequenced by using a 454 GS Junior (Roche Diagnostics, Basel, Switzerland) using protocols recommended by the manufacturer.