The final molecular identification of the strain was performed by PCR for 16S rRNA gene with specific primers: UNI 16SF (5-GAG AGT TTG ATC CTG GC-3) and UNI 16SR (5-AGG AGG TGA TCC AGC CG-3). PCR products were purified with GeneJET PCR Purification Kit (Thermo Scientific, Lithuania) and sequenced by the Macrogen DNA sequencing service (Macrogen Inc., Netherlands). Obtained sequences were aligned in the NCBI database using BLAST. PFGE was performed as previously described [15 ]. Genomic DNA was digested with XbaI enzyme (Thermo Scientific, Lithuania), and obtained macrorestriction profiles were subject to statistical analysis. MLST was performed as was described in Kaiser et al [16 (link)] and the primers and protocols were downloaded from the website of the S. maltophilia MLST database (http://pubmlst.ors/smaltophilia/ ). Briefly, MLST was performed by PCR and sequencing of seven housekeeping genes: atpD (H (+)-transporting two-sector ATPase), gapA (NAD-dependent glyceraldehyde-3-phosphate dehydrogenase), guaA (GMP synthase), mutM (DNA-formamidopyrimidine glycosylase), nuoD (NADH dehydrogenase), ppsA (pyruvate, water dikinase), recA (RecA protein). Allele profiles obtained after sequencing were used to determine specific sequence type (ST) for analyzed isolates using MLST Database hosted by the University of Freiburg, Germany [16 (link)].
Xbai enzyme
Manufactured by Thermo Fisher Scientific
Sourced in United States, Lithuania
XbaI is a type II restriction enzyme that recognizes and cleaves the DNA sequence 5'-TCTAGA-3'. It is commonly used in molecular biology for DNA manipulation, such as DNA cloning and restriction fragment analysis.
Automatically generated - may contain errors
Lab products found in correlation
Chef dr 3, by Bio-Rad (4 mentions)
Wizard genomic dna purification kit, by Promega (2 mentions)
Seakem gold, by Bio-Rad (2 mentions)
Genejet pcr purification kit, by Thermo Fisher Scientific (1 mentions)
Chef dr 3 pfge apparatus, by Bio-Rad (1 mentions)
Gel doc camera system, by Bio-Rad (1 mentions)
Sybr safe dna gel stain, by Thermo Fisher Scientific (1 mentions)
Ethidium bromide, by Merck Group (1 mentions)
Chef bacterial genomic dna plug kit, by Bio-Rad (1 mentions)
Chef mapper xa pfge system, by Bio-Rad (1 mentions)
11 protocols using xbai enzyme
1
Molecular Identification and Typing of Stenotrophomonas maltophilia
2
Fingerprinting E. coli Strains by RAE-PFGE
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The above-mentioned eleven E. coli strains were fingerprinted by using the RAE-PFGE method, as per the PulseNet protocol developed by the US Centers for Diseases Control and Prevention [64 ] (PulseNet). Total DNA was prepared for restriction analysis with the application of the XbaI enzyme (Thermo Fisher Scientific, Vilnius, Lithuania). DNA separation was performed with CHEF DR III (Bio-Rad) under the following conditions: 1% agarose gel (Prona Agarose) in 0.5-M Tris–Borate–EDTA buffer at 14 °C for 19 h at 6.0 V/cm (200 V). Pulse time ranged between 2.2 and 63.8 s. Molecular weight marker ProMega-Markers® Lambda Ladders was used for analysis (Promega, Madison, USA). The gels were stained and visualized as per the PCR reactions above. RAE-PFGE patterns were analyzed by visual assessment, and the dendrograms were generated with the UPGMA method using online software available at http://insilico.ehu.es/ .
3
Bacterial Fingerprinting by PFGE Analysis
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All bacterial isolates were fingerprinted by the PFGE method, using the PulseNet protocol developed by Centers for Diseases Control and Prevention [61 ]. Chromosomal DNA was subjected to restriction analysis with application of XbaI enzyme (Thermo Fisher Scientific, USA). PFGE analysis was conducted with CHEF DR III PFGE apparatus (Bio-Rad, USA). DNA separation was performed with the following parameters: 1% agarose gel (Prona Agarose) on 0.5 M Tris–Borate–EDTA buffer at 14°C for 19 h at 6.0 V/cm (200 V). Pulse time was ranging of 2.2–63.8 s. The gels were stained with SYBR® Safe - DNA Gel Stain (Thermo Fisher Scientific, Germany) and band patterns were visualized under UV light and photographed using a Gel Doc camera system (Bio-Rad, USA). Molecular Weight Marker ProMega-Markers® Lambda Ladders was used for analysis (Promega, USA). PFGE patterns were analyzed via visual assessment and the dendrograms were generated with UPGMA method using on-line software http://insilico.ehu.es/ .
4
Molecular Characterization of K. pneumoniae
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The DNA of each isolate was extracted using the Wizard® Genomic DNA Purification kit (Promega) following manufacturer's instructions. K.pneumoniae isolates were confirmed by the amplification of the khe gene, and possible contamination with other species was ruled out by amplifying the genes pehX (K.oxytoca), uidA (E.coli), ehe (Enterobacter spp.), wosA (P.mirabilis) by multiplex PCR (table S1 ). Presence of relevant β-lactamases genes in all isolates was assessed by multiplex PCR (blaTEM,blaCTX-M,blaGES, blaIMP, blaVIM, blaKPC and blaNDM) using previously described protocols21 (link),22 (link). The genetic relatedness between isolates was determined by genome macro-restriction using the XbaI enzyme (Thermo Scientific) and PFGE separation according to the protocol reported by Herschleb et al.23 (link). PFGE pulsotypes were grouped in the same clonal complex (CC) when they presented no more than three different bands. Sequence type (ST) to some representative isolates of the most frequent clonal complexes was determined using the protocol reported by Diancourt et al.24 (link).
5
Bacterial Genomic DNA Isolation and Analysis
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According to the manufacturer’s instructions, DNA isolation was performed using the CHEF Bacterial Genomic DNA Plug Kit (Bio-Rad, Hercules, CA, USA). Restriction digestion of isolated DNA was carried out using Tango buffer and XbaI enzyme (Thermo Scientific, Waltham, MA, USA). A solution was prepared with 1.2 g of agarose (DNA Gdansk, Poland) dissolved in 100 mL of TBE buffer to prepare the agarose gel. Electrophoresis was conducted in 0.5× concentrated TBE buffer at 14 °C for 24 h. After electrophoretic separation, the gel was stained with 0.5 μg/mL ethidium bromide (Sigma-Aldrich, Darmstadt, Germany) for 30 min. A GelDoc-It2 Imager system (Upland, CA, USA) was used to read the results.
6
Genotyping Brucella Strains by PFGE
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Genomic DNA of Brucella strains was prepared in agarose plugs and digested by XbaI enzyme (Thermo Fisher Scientific, Waltham, MA) as described previously with minor modifications (Ridler et al., 2005 (link)). The plugs were placed in lysis buffer (50 mMTris–HCl [pH 8.0], 50 mM EDTA [pH 8.0], 1% Sodium lauroyl sarcosine, 1% SDS and 0.1 mg/ml of proteinase K) and incubated overnight at 55 °C. The plugs were digested with 30 U of XbaI overnight at 37 °C and after washing, completely digested genomic DNA were embedded into pre-formed wells of 1% agarose gel electrophoretic. Separation of the DNA genome was done with Rottaphor Biometra electric fields (version 6, Germany) using TBE buffer (45 mM Tris–HCl, 45 mM boric acid, 1.0 mM EDTA, pH 8.0) at 14 °C for 22 h at 120 v and 5 s and 35 s. Lambda Ladder PFGE Marker (NEB: N0340) was used as the molecular reference marker. Dendrograms for PFGE and PCR-RFLP were constructed according to obtained DNA fragment genotypes using BioNumerics total lab software version7.5 (Applied Maths, StMartens-Latem, and Belgium).
The analysis of similarity was calculated using the unweighted pair group average (UPGMA) and the Dice coefficient for cluster analyses. The criteria for related clones or classifications as similar types were considered when 80% or more of the patterns were similar (Tenover et al., 1995 (link)).
The analysis of similarity was calculated using the unweighted pair group average (UPGMA) and the Dice coefficient for cluster analyses. The criteria for related clones or classifications as similar types were considered when 80% or more of the patterns were similar (Tenover et al., 1995 (link)).
7
Pulsed-Field Gel Electrophoresis for EHEC Genotyping
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A total of 41 EHEC isolates were used in the PFGE using restriction enzyme digestion with XbaI enzyme (Thermo Fisher, USA) according to previously published protocols [19 ]. Gel electrophoresis was performed using the Chef Mapper XA PFGE system (Bio-Rad) under the following conditions: Initial switch time: 2.2 s; final switch time: 54.2 s; run time: 18 h; angle: 120°; gradient: 6.0 V/cm; temperature: 14°C; and ramping factor: linear. The electrophoresis gels were stained using ethidium bromide and visualized under ultraviolet light. The genotypic relatedness was determined using PFGE DNA fingerprint subtypes. The analysis of the bands was carried out using the Dice coefficient and the unweighted pair group method with arithmetic averages clustering methods with an optimization and position tolerance of 1.0%. Analysis of PFGE gel patterns was performed using BioNumerics software version 3.5 (Applied Maths, Belgium).
8
Molecular Characterization of Klebsiella Isolates
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The DNA of each isolate was extracted using the Wizard® Genomic DNA Purification kit (Promega) following manufacturer's instructions. K. pneumoniae isolates were confirmed by the amplification of the khe gene, and possible contamination with other species was ruled out by amplifying the genes pehX (K. oxytoca), uidA (E. coli), ehe (Enterobacter sp.), wosA (P. mirabilis) by multiplex PCR (table S1). Presence of relevant β-lactamases genes in all isolates was assessed by multiplex PCR (blaTEM, blaCTX-M, blaGES, blaIMP, blaVIM, blaKPC and blaNDM) using previously described protocols [21, 22] . The genetic relatedness between isolates was determined by genome macro-restriction using the XbaI enzyme (Thermo Scientific) and PFGE separation according to the protocol reported by Herschleb et al [23] . PFGE pulsotypes were grouped in the same clonal complex (CC) when they presented no more than three different bands. Sequence type (ST) to some representative isolates of the most frequent clonal complexes was determined using the protocol reported by Diancourt et al [24] .
9
Pulsed-Field Gel Electrophoresis for Bacterial Typing
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The PulseNet protocol, according to Ribot et al. [49 (link)] was used to conduce pulsed-field gel electrophoresis (PFGE). Bacteria grown at 37 °C overnight on TSA (OXOID®) were suspended in tubes containing 2mL of phosphate-buffered saline (PBS: 0.01 M phosphate buffer; pH 7.2; 0.85% NaCl). After agarose blocking, genomic DNA digestion was performed with 30 U of XbaI enzyme (Invitrogen®) for 2 h at 25 °C.The DNA fragments were separated on 1% agarose gel (SeaKem Gold®) in 0.5X TBE buffer in CHEF DRIII (Bio-Rad®, Hercules, CA, USA) for 18h with the following parameters: 200 V, 120° angle, 6 V/cm gradient, and 14 °C buffer temperature. The comparison of the band patterns was performed by the UPGMA analysis method, using the Dice similarity coefficient with a tolerance of 1.5% in the comparison of the position of the bands.
10
Pulsed-field Gel Electrophoresis for Bacterial Typing
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The isolates were characterised by the PulseNet standard PFGE protocol, as described by Ribot et al. (2006) with minor modifications in relation to the number of washes in the phase preceding the enzymatic lysis and the electrophoretic running time: there were three washes with buffer and one with water, and the run was carried out for 21 h. The system used was the CHEF DR-III (Bio-Rad, USA), with the chromosomal DNA being digested with the XbaI enzyme (Invitrogen, USA) and the electrophoresis performed on a Pulsifield Certified 1% agarose gel (Bio-Rad, USA), with a voltage of 6 V cm À1 , at an angle of 1208, with an initial polarity reversal time of 2.2 s and a final polarity reversal time of 54.2 s. The Salmonella Braenderup (H9812) strain was used as a standard, and the gels were submitted to electrophoresis for 21 h at a temperature of 14 8C. Fragment similarities were compared using the Dice coefficient at 1% tolerance and 0.5% optimisation. The dendrogram was calculated using the UPGMA grouping method, using Bio-Numerics Software, version 7.1 (Applied Mathematics, Sint-Martens-Latem, Belgium).
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