Nbt bcip color detection kit

Genomic DNA was digested with the S1 enzyme following the protocol of Barton et al. (1995) (link).
The DNA fragments were transferred to a positively charged nylon membrane (Millipore, United States), hybridized with a DIG-labeled specific probe and detected with an NBT/BCIP color detection kit (Roche, Germany). The hybridization probe was designed to bind within the middle portion of the tetA gene and was synthesized using the primers listed in Supplementary Table S1.

Plasmids were isolated from the 19 transconjugants using a QIAGEN Plasmid Midi Kit (Qiagen, Germany). The manufacturer’s instructions were followed with the inclusion of the lysozyme treatment (10 mg/mL, 60 min at 37°C) prior to the lysis step with buffer P2 [200 mM NaOH, 1% SDS (w/v)].
The restriction enzyme BsaI (New England Biolabs, United States), for which a target site is present within the fosB gene, was used to digest plasmid DNA. The hybridization probe was designed to bind within the latter half of the fosB gene after BsaI digestion (Supplementary Figure S1A) and was synthesized using the primers fosB-latter-F (5′-GTG GTA TAT GGT TAG CTT TGA ACG AAG-3′) and fosB-latter-R (5′-TGA GGT TTA GCC TCT TTA TAA TAA CTC-3′).
Plasmid DNA was first digested by BsaI at 37°C in a water bath overnight. After the digested DNA was agarose gel electrophoresed for 2 h, the DNA fragments were transferred to a positively charged nylon membrane (Millipore, United States), hybridized with a DIG-labeled fosB-latter specific probe, and then was detected with a NBT/BCIP color detection kit (Roche, Germany).

The plasmid location of the bla_KPC–2 gene was determined by Southern blot experiments according to the previous study (Wang et al., 2020 (link)). Briefly, whole chromosomal DNA was digested with S1-nuclease (TaKaRa, Japan). The digested fragments were electrophoresed on a CHEF-mapper XA pulsed-field gel electrophoresis (PFGE) system (Bio-Rad, United States) for 18 h at 14°C. The DNA fragments were transferred to a positively charged nylon membrane (Millipore, United States) and then hybridized with a digoxigenin-labeled bla_KPC–2-specific probe. The fragments was detected by an NBT/BCIP color detection kit (Roche, Germany). The Salmonella enterica serotype Braenderup H9812 was used as the size marker.

To estimate the sizes of mcr-positive plasmids, S1-PFGE and Southern hybridization were performed. Briefly, bacterial whole-cell DNA of mcr-positive isolates and their transconjugants was prepared in agarose plugs and digested with S1 nuclease (TaKaRa, Dalian, China). The DNA was separated using the CHEF-Mapper PFGE system (Bio-Rad) under the following conditions: 14°C, 6 V/cm, and a 120° pulse angle for 16 h, with the initial and final pulses conducted for 2.16 and 63.8 s, respectively. The separated DNA fragments were transferred to nylon membranes, hybridized with digoxigenin-labeled mcr-10- or mcr-8-specific probes, and detected using the nitroblue tetrazolium–5-bromo-4-chloro-3-indolylphosphate (NBT-BCIP) color detection kit (Roche, catalog no. 11745832910).

Southern blotting was employed to confirm the location of fosA3 gene. Total genomic DNA was digested with S1 nuclease and electrophoresed with a CHEF-Mapper XA PFGE system (Bio-Rad, USA) for 16 h at 14 °C and 6 V/cm, with alternating pulses in 2.16–63.8 s pulse time. The DNA fragments were transferred to nylon membranes (Millipore, USA) and hybridized with digoxigenin-labelled fosA3-specific probe. An NBT/BCIP color detection kit (Roche Applied Sciences, Germany) was employed to detect the fragments [31 (link)].

To further investigate whether the virulence and drug-resistance genes were situated on the plasmid and to determine their fragment sizes, we treated the entire chromosomal DNA of the K54-Kp strain with S1 nuclease (Takara, Otsu, Japan) and subsequently transferred the DNA fragments onto a nylon membrane. To ascertain if the two genes were present on the same plasmid, indicating a fusion plasmid, we hybridized with digoxin-labeled rmpA2 and KPC-specific probes. The fragments were then detected using an NBT/BCIP color detection kit (Roche, Mannheim, Germany) as described by Xu et al. (Xu et al., 2019 (link)).

According to literature [30] (link), bacterial DNA was prepared in agarose blocks and digested with S1 nuclease, and then separated by PFGE with conditions of 14 h at 6 V/cm and 14°, with a pulse angle of 120° and a switch time from 1 to 10 s. The gel was stained with ethidium bromide. The DNA fragments were transferred to nylon membranes (Hybond N, Amersham, UK), hybridized with digoxigenin-labeled bla_NDM-1-specific probes and detected using an NBT/BCIP color detection kit (Roche, Basel, Switzerland).