All of the isolates that showed a key hole effect or had cefoxitin resistance with non-susceptibility to cefepime were subjected to real-time PCR analysis for the detection of SHV, TEM and CTX-M encoding genes (Roschanski et al., 2014 (link)). Simplex PCRs for the genes encoding AmpC β-lactamases FOX, MOX, ACC, EBC, DHA, and CMY were conducted for all strains showing non-susceptibility to cefoxitin (Dallenne et al., 2010 (link)). Simplex PCR was also used to test the ADC ampC β-lactamase gene in A. baumannii (Liu and Liu, 2015 (link)). DNA extraction was performed according to the manufacturer’s instructions using EZ1 DNA extraction kits (Qiagen, Courtaboeuf, France) with the EZ1 Advanced XL biorobot.
Ez1 dna extraction kit
Manufactured by Qiagen
Sourced in France, Germany
The EZ1 DNA extraction kits are a series of automated sample preparation systems designed for the rapid and efficient extraction of DNA from a variety of sample types. The kits use magnetic particle technology to isolate and purify DNA, and can be operated using the EZ1 Advanced XL or EZ1 workstations.
Automatically generated - may contain errors
Lab products found in correlation
Ez1 advanced xl biorobot, by Qiagen (2 mentions)
Abi 3130xl automated sequencer, by Thermo Fisher Scientific (1 mentions)
Ez1 advanced xl, by Qiagen (1 mentions)
Multiplexing sample preparation oligonucleotide kit, by Illumina (1 mentions)
E220 focused ultrasonicator, by Covaris (1 mentions)
Ez1 advanced xl robot, by Qiagen (1 mentions)
Multiplex pcr kit, by Qiagen (1 mentions)
Ariamx real time pcr system, by Agilent Technologies (1 mentions)
7 protocols using ez1 dna extraction kit
1
Detecting Antibiotic Resistance Genes
2
Molecular Detection of Resistance Genes
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Isolates that showed cefoxitin resistance or a keyhole effect were further studied using real-time PCR as detailed in Dandachi et al. (2018) (link). Brie y, DNA was extracted from isolates using EZ1 DNA extraction kits (Qiagen, France) and real-time PCR was used for the detection of CTX-M, TEM, SHV, and other resistance genes (Roschanski et al., 2014) .
3
Carbapenem Resistance Profiling in Isolates
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DNA extraction from all isolates showing non-susceptibility to carbapenems was performed using EZ1 DNA extraction kits (Qiagen NV, Venlo, the Netherlands) with the EZ1 Advanced XL biorobot according to the manufacturer’s instructions.
Real-time PCR and standard PCR were performed to screen for the presence of carbapenem hydrolyzing enzyme-encoding genes: blaOXA-23, blaOXA-24, blaOXA-58, blaOXA-48, blaKPC, blaIMP, blaVIM, and blaNDM genes,2 (link) and ESBL genes (blaCTX-M, blaTEM, blaSHV, blaGES, blaVEB, and blaPER).13 (link) The positive PCR products were purified and sequenced using the Big Dye terminator chemistry on an ABI 3130XL automated sequencer (Thermo Fisher Scientific, Waltham, MA, USA). OprD mutations were investigated on imipenem-resistant P. aeruginosa isolates. The obtained sequences were analyzed using Codon Code Aligner software and then examined using the BlastN and BlastP compared against the NCBI database (www.ncbi.nlm.nih.gov ) and ARG-ANNOT (Antibiotic Resistance Gene-ANNOTation) (http://en.mediterranee-infection.com/article.php?laref=283&titre=arg-annot- ).
Real-time PCR and standard PCR were performed to screen for the presence of carbapenem hydrolyzing enzyme-encoding genes: blaOXA-23, blaOXA-24, blaOXA-58, blaOXA-48, blaKPC, blaIMP, blaVIM, and blaNDM genes,2 (link) and ESBL genes (blaCTX-M, blaTEM, blaSHV, blaGES, blaVEB, and blaPER).13 (link) The positive PCR products were purified and sequenced using the Big Dye terminator chemistry on an ABI 3130XL automated sequencer (Thermo Fisher Scientific, Waltham, MA, USA). OprD mutations were investigated on imipenem-resistant P. aeruginosa isolates. The obtained sequences were analyzed using Codon Code Aligner software and then examined using the BlastN and BlastP compared against the NCBI database (
4
16S rRNA Gene Sequencing Protocol
5
Microsporidian SSU rRNA Gene Amplification
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DNA was extracted from ethanol-fixed samples of hepatopancreas using an automatic EZ1 DNA extraction kit (Qiagen). Primers: MF1 (5 0 -CCGGAGAGGGAGCCTGAGA-3 0 ) and MR1 (5 0 -GACGGGCGG TGTGTACAAA-3 0 ) (Tourtip et al., 2009) , were used to amplify a fragment of the microsporidian SSU rRNA gene using a GoTaq flexi PCR reaction [1.25U of Taq polymerase, 2.5 mM MgCl 2 , 0.25 mM of each dNTP, 100pMol of each primer and 2.5 ll of DNA template (10-30 ng/ll) in a 50 ll reaction volume]. Thermocycler settings were as follows: 94 °C (1 min) followed by 30 cycles of 94 °C (1 min), 55 °C (1 min), 72 °C (1 min) and then a final 72 °C (10 min) step. Electrophoresis through a 2% Agarose gel (120 V, 45 min) was used to separate and visualise a resulting 939 bp amplicon. Amplicons were purified from the gel and sent for forward and reverse DNA sequencing (Eurofins genomics sequencing services: https://www.eurofinsgenomics.eu/).
6
DNA Extraction from Blood and Eschar
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For each patient, 200 μl of whole blood was used for DNA extraction and the remaining blood was stored at -20°C. Each eschar swab was immersed in 400 μL of G2 Buffer—a lysis buffer for use with EZ1 genomic DNA procedures—for 1 hour to release eschar materials and 200 μL of the supernatant was collected after a quick spin for DNA extraction. Genomic DNA was individually extracted from both the whole blood and eschar swab suspension using the DNA EZ1 extraction kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. The DNA was then eluted in 100 μL of Tris EDTA (TE) buffer using the DNA-extracting EZ1 Advanced XL Robot (Qiagen). DNA was either immediately used or stored at -20°C until molecular analysis. A disinfection of the DNA-extracting EZI Advanced XL Robot was performed after each batch of extraction as per manufacturer’s recommendations to avoid cross-contamination.
7
Multiplex qPCR for Ricketsiaceae Detection
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To differentiate Ricketsiaceae species, the primers and probes were designed to target specific sequences of O. tsutsugamushi (47 kDa gene encoding outer membrane protein), Rickettsia spp. (17 kDa gene encoding outer membrane proteins) and OmpB protein-coding gene of R.typhi (The sequence of primers and probes is provided in Table S1
Whole blood was collected in EDTA tubes, then processed it with blood genomic DNA isolation Mini Kit (https://norgenbiotek.com/product/blood-dna-isolation-mini-kit https://www.qiagen.com
For each qPCR assay, we extracted 5 μL of bacteria DNA sample and added it to a final reaction volume of 25 μL, using QIAGEN Multiplex PCR Kit (https://www.qiagen.com Table S2 3 Figure S1
Whole blood was collected in EDTA tubes, then processed it with blood genomic DNA isolation Mini Kit (
For each qPCR assay, we extracted 5 μL of bacteria DNA sample and added it to a final reaction volume of 25 μL, using QIAGEN Multiplex PCR Kit (
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