Nbt bcip colour detection kit

To confirm transfer had occurred, plasmids were visualized following S1 nuclease treatment via PFGE, and the locations of resistance genes were confirmed via Southern blot as described previously [28 (link)]. Briefly, genomic DNA was digested with S1 nuclease (TaKaRa, Kusatsu, Japan) at 37 °C for 20 min. Treated DNA was loaded on a 1 % agarose Gold gel and PFGE was performed at 14 °C for 18 h, with 6 V/cm and pulse times from 2.16 to 63.8 s using the Bio-Rad CHEF-Mapper XA machine (Bio-Rad, CA, USA). DNA was transferred to a positively charged nylon membrane (Millipore, Billerica, MA, USA) by the capillary method and hybridized with digoxigenin-labelled bla_OXA-58 and bla_NDM-1-specific probes with an NBT/BCIP colour detection kit (Roche, Mannheim, Germany) according to the manufacturer’s instructions. XbaI-treated genomic DNA from Salmonella enterica H9812 was used as a size marker.

S1-PFGE and Southern blotting were performed to analyse the size of the NDM-1-carrying plasmids in the K. pneumoniae strains as previously described and the results were analyzed according to the criteria proposed by Tenover et al. [18 (link), 19 (link)] Plasmid DNA of the isolates embedded in agarose gel plugs were digested with S1 nuclease and separated by PFGE. Plasmids obtained by PFGE were then transferred to a positively charged nylon membrane. The membrane was hybridized with digoxigenin-labelled bla_NDM-1-specific probes and the signals were detected using an NBT/BCIP colour detection kit (Roche Applied Sciences, Penzberg, Germany).

Transconjugants were further confirmed by Southern blotting. SmaI- and S1-PFGE analyses were performed as described previously (Tomita et al., 2002 (link); Yan et al., 2011 (link)). Southern blotting was performed using a DIG High Prime DNA labelling and Detection Starter Kit (Roche, Basel, Switzerland) according to the manufacturer’s instructions. The digoxigenin-labelled lsa(E)-specific probe was prepared using primers (forward 5′-ACAGCGAGTTGTTTCCTGCT-3′; and reverse 5′-GCACGTTTCATCGCTTTTGC-3′) that amplified a 410-bp region of the lsa(E) gene. After S1-PFGE, the DNA was transferred to a nylon membrane (Hybond N, Amersham, United Kingdom) that was hybridised with the prepared lsa(E)-specific probe. Detection was performed using an NBT/BCIP colour detection kit (Roche, Switzerland).