Bigdye 3.1 kit

Purified DNA fragments were cloned into the pMD ® 19-T vector according to the manufacturer's instructions. Recombinant plasmids were transformed into E. coli DH5 competent cells and cultured on Luria-Bertani plates (containing 100 μg mL 1 ampicillin, 0.5 mmol L 1 Isopropyl -D-Thiogalactoside (IPTG), and 40 μg mL 1 X-gal). Bacterial colonies were picked using the blue-white screening method from LB plates, followed by cultured in liquid LB media at 37°C overnight. The recombinant plasmids were prepared using the TIANprep Mini plasmid kit (Tiangen Biotech, Beijing) followed by bidirected circular sequencing reactions using the Bigdye 3.1 kit (Applied Biosystems, USA). The plasmids were sequenced by TaKaRa Inc. using the sequencing primers M13-47 and RV-M, which flanked the insert position. The sequences obtained in this study were deposited in GenBank (accession No. KU570088-KU570133).

For those isolates that gave a positive carbapenemase test, β-lactamase genes were identified using the PCR. Plasmid DNA, and genomic DNA for Serratia marcescens, was purified by GenElute Bacterial Genomic DNA Kit (Sigma Life Science, St Louis, MO, USA). Genomic DNA from S. marcescens was screened for the presence of bla SME , 24, 25 and plasmids were screened for the presence of bla CTX-M , bla OXA , bla SHV , bla TEM , bla KPC , bla IMP , bla NDM and bla VIM by PCR amplification with published primer sets. [26] [27] [28] [29] [30] Sequencing of the PCR products that tested positive for bla genes was performed using the BigDye 3.1 kit (Applied Biosystems, Foster City, CA, USA) and analyzed by BLAST (http:// blast.ncbi.nlm.nih.gov/).

For highly sensitive amplification of the complete VP1 region of EV-D68 strains directly from clinical material a specific one-step reverse-transcription polymerase chain reaction (RT-PCR) assay was established at the German National Reference Centre for Poliomyelitis and Enteroviruses (NRZ PE). Amplification was performed using One-Step-RT-PCR Kit (Qiagen, Hilden, Germany) followed by a nested PCR using HotStarTaq-Mastermix (Qiagen, Hilden Germany) according to the manufacturer's protocol. RT-PCR and nested PCR were done with 600 nM of primers (Table 1). The RT-PCR was conducted with primers NRZ 267/268 and with the following temperature profile: 10 min 22 °C, 45 min 50 °C, 15 min 95 °C for RT followed by 40 cycles (30 s 94 °C; 30 s 55 °C; 90 s 72 °C) and final elongation for 10 min at 72 °C. The nested PCR was carried out with primers 269/270 by using a touchdown protocol with 10 cycles (30 s 94 °C; 30 s 60 °C; 90 s 72 °C) with a decrease of 1 °C per cycle of the initial 60 °C annealing temperature, followed by 30 cycles (30 s 94 °C; 30 s 50 °C; 90 s 72 °C) and final elongation for 10 min at 72 °C. The resulting product of 1,129 bp was treated with ExoSAP-IT (Affymetrix) before cycle sequencing with primers NRZ 269, NRZ 270 and NRZ 271 using the BigDye 3.1 kit (Applied Biosystems, Weiterstadt, Germany).