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 .
Gfx pcr dna
Manufactured by Cytiva
Sourced in Germany
The GFX PCR DNA is a laboratory equipment used for performing polymerase chain reaction (PCR) amplification of DNA samples. It provides a controlled environment for the thermal cycling necessary for the DNA amplification process.
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Lab products found in correlation
5 protocols using gfx pcr dna
1
Detecting Quinolone Resistance in E. coli
2
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)).
3
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)).
4
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 ).
5
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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