Abi 2700 thermal cycler

MLST was performed based on analyses and comparison of internal fragment sequences from seven housekeeping genes (gltA, gyrB, gdhB, recA, cpn60, gpi and rpoD). 21 Amplification reactions were carried out in an ABI 2700 thermal cycler (Applied Biosystems) with 30 cycles of denaturation (30 s at 94 °C), annealing (30 s at 54 °C) and extension (1 min at 72 °C) after 5-min denaturation at 94 °C and followed by a final extension at 72 °C for 10 min. All PCR products were purified and sequenced with the assistance of JIE LI Bio-Technologies (Shanghai, China). The sequences of these seven housekeeping genes were analyzed by means of the PubMed database (http://pubmlst.org/ abaumannii/). The sequence type (ST) was designated according to the allelic profiles in the database. The eBURST algorithm (version 3; http://eburst.mlst. net/) was used to assign STs to clonal complexes (CCs) and to assess the genetic relationship with definition of groups sharing alleles at ⩾ 6 of the 7 loci. 22

A. baumannii genomic DNA was extracted as described previously. 17 The primer pair ERIC1 (5′-ATGTAAGCTCCTGGGGATTCAC-3′) and ERIC2 (5′-AAGTAAGTGACTGGGGTGAGCG-3′) were used to amplify intervening fragments of ERIC in the genomic DNA. Amplification reactions were performed in a final volume of 25 μl. Each reaction system contained 2.5 μl of 10 × PCR buffer, 2 μl dNTP mixture, 0.125 μl Taq enzyme, 0.5 μl each primer (10 μmol l -1 ) and 1 μl DNA template, added to 25 μl of ddH 2 O. Amplification reactions were carried out in an ABI 2700 thermal cycler (Applied Biosystems, Carlsbad, CA, USA) with initial denaturation (5 min at 94 °C), followed by four cycles of denaturation (1 min at 94 °C), annealing (1 min at 26 °C) and extension (1 min at 72 °C) and 40 cycles of denaturation (30 s at 94 °C), annealing (30 s at 40 °C) and extension (1 min at 72 °C), with a final extension at 72 °C for 10 min. Amplified products of each sample were subjected to electrophoresis in 1.2% agarose gel containing ethidium bromide.