In order to detect extra-chromosomally encoded ESBL factors, plasmid DNA was isolated by commercially available kit (Thermo-Scientific Gene Jet plasmid Miniprep Kit). ESBL factors including bla- TEM, bla-SHV and genes bla-OXA, bla-PSE were PCR amplified as described elsewhere [18 (link)]. Amplified products were then purified (Gel Band Purification Kit, Amersham, USA) and sequencing was done by automated DNA sequencer (ABI 3130, Perkin-Elmer Applied Biosystems, Foster City, California). Sequences were reported to the Gene Bank database (accession number; KX171170–171195). PCR amplifications and sequencing of bla-CTX-M allele was carried out, bla-CTX-M types were determined by comparing DNA sequences available in the database [19 ]. A total of 18 different virulence factors ( VF) corresponding to the main classes of extra-intestinal virulence associated genes (VAGs) including adhesins, toxins, siderophores, capsular proteins and uropathogenic-specific protein (usp) were scrutinized in all 155 isolates. VF genes were amplified by previously reported sets of primers and amplification conditions [20 (link)].
Gel band purification kit
Manufactured by Cytiva
Sourced in United States, Germany
The Gel Band Purification Kit is a laboratory tool used to extract and purify DNA fragments from agarose gels. It provides a simple and efficient method to recover DNA of interest after electrophoresis separation.
Automatically generated - may contain errors
8 protocols using gel band purification kit
1
Molecular Detection of ESBL Factors
2
Detecting Quinolone Resistance in E. coli
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PCR was carried out to amplify quinolone resistance determining regions (QRDRs) of the target genes (gyrA, and parC) to identify mutations in 43 FQ-resistant E. coli isolates using primers and conditions described previously [18 (link), 19 (link)]. The PCR products were purified using GFX PCR DNA and the gel band purification kit (Amersham Bioscience, Freiburg, Germany), and sequenced by an automatic sequencer (Cosmogenetech, Seoul, Korea). The sequences were aligned with those in the GenBank nucleotide database using the Basic Local Alignment Search Tool (BLAST) program available through the National Center for Biotechnology Information website (http://www.ncbi.nlm.nih.gov/BLAST ). PMQR genes (qnrA, qnrB, qnrC, qnrD, qnrS, aac(6’)-Ib-cr, and qepA) were detected by PCR amplification and sequencing analysis, as described in Table S1 .
3
Molecular Detection of β-Lactamase Genes
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Polymerase chain reaction amplification was performed using primers for the β-lactamase genes blaCTX-M (Pitout et al., 2004 (link), Ogutu et al., 2015 (link)), blaTEM (Briñas et al., 2002 (link)), blaSHV (Briñas et al., 2002 (link)), and blaOXA (Briñas et al., 2002 (link)). The PCR products were purified using GFX PCR DNA and the Gel Band Purification Kit (Amersham Bioscience, Freiburg, Germany) and sequenced using an automatic sequencer (Cosmogenetech, Seoul, Korea). The sequences were confirmed with those in the GenBank database using the Nucleotide Basic Local Alignment Search Tool (nBLAST) available at the National Center for Biotechnology Information website (http://www.ncbi.nlm.nih.gov/BLAST ). The presence of ISEcp1, IS26, orf477, and IS903 sequences surrounding the CTX-M–type genes was analyzed by PCR using primers and conditions as previously described (Eckert et al., 2006 (link); Sun et al., 2010 (link)).
4
Quinolone Resistance Profiling of E. coli
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PCR was carried out to amplify the target genes (gyrA, gyrB, parC, and parE) in quinolone resistance determining regions (QRDRs ) to identify mutations in 106 FQ-resistant E. coli isolates using primers and conditions described previously (Fendukly et al., 2003 (link); Dutta et al., 2005 (link); Bai et al., 2012 ). The PCR products were purified using GFX PCR DNA and the Gel band purification kit (Amersham Bioscience, Freiburg, Germany), and sequenced by automatic sequencer (Cosmogenetech, Seoul, Korea). The sequences were confirmed with those in the GenBank nucleotide database using the Basic Local Alignment Search Tool (BLAST) program available through the National Center for Biotechnology Information website (http://www.ncbi.nlm.nih.gov/BLAST ). PMQR genes (qnrA, qnrB, qnrC, qnrD, qnrS, aac(6’)-Ib-cr, and qepA) were also detected by PCR amplification and sequencing analysis, as described in previous studies (Yu et al., 2015 (link)).
5
Cloning and Sequencing of DNA Fragments
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The PCR products were purified using the Gel Band Purification kit (Amersham, USA), followed by Sepharose CL-6B affinity chromatography (Pharmacia Corp., NYC, NY, USA), and then cloned into a pCR Ò II-TOPO Ò vector (Invitrogen, USA). After transformation of E. coli cells, positive clones were screened by using agar plates containing ampicillin (50 ll/ml), X-Gal (40 mg/ml) and IPTG (40 ll of 100 mM IPTG -Isopropyl b-D-1-thiogalactopyranoside solution). Plasmids were extracted using the SDS plasmid isolation protocol (Sambrook et al. 1990) . In order to confirm the presence of the expected cloned fragment, plasmid DNA clones were digested before sequencing by the EcoRI restriction enzyme and visualized on agarose gel.
6
Identifying FQ-Resistance Mutations in APEC
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PCR was performed to amplify the gyrA and parC of the quinolone resistance-determining region to identify mutations in 33 FQ-resistant APEC isolates using primers and conditions described previously (Pons et al., 2014 (link)). The PCR products were purified using GFX PCR DNA and the Gel Band Purification Kit (Amersham Biosciences, Freiburg, Germany) and sequenced using an automatic sequencer (Cosmogenetech, Seoul, Korea). The sequences were compared with those in the GenBank nucleotide database using the Basic Local Alignment Search Tool program available through the National Center for Biotechnology Information website (http://www.ncbi.nlm.nih.gov/BLAST ).
7
Cloning and Sequencing of DNA Fragments
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The PCR products were purified using the Gel Band Purification kit (Amersham, USA), followed by Sepharose CL-6B affinity chromatography (Pharmacia Corp., NYC, NY, USA), and then cloned into a pCR Ò II-TOPO Ò vector (Invitrogen, USA). After transformation of E. coli cells, positive clones were screened by using agar plates containing ampicillin (50 ll/ml), X-Gal (40 mg/ml) and IPTG (40 ll of 100 mM IPTG -Isopropyl b-D-1-thiogalactopyranoside solution). Plasmids were extracted using the SDS plasmid isolation protocol (Sambrook et al. 1990) . In order to confirm the presence of the expected cloned fragment, plasmid DNA clones were digested before sequencing by the EcoRI restriction enzyme and visualized on agarose gel.
8
Amplification and Purification of Fungal ITS Regions
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The fragments of ITS1-5.8S-ITS2 were amplified by primers ITS1 and ITS4 (White et al., 1990) . The amplification of ITS regions of rDNA followed the methodology of Magnani et al. (2005) . The amplified fragments were analyzed by 1% agarose gel electrophoresis. The products of amplification of ITS1-5.8S-ITS2 region of rDNA of the endophyte fungi strains were purified with GFX PCR DNA and the Gel Band Purification Kit (Amersham Biosciences, USA).
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