The 28 selected E. coli isolates from 2018 to 2020 were further tested using polymerase chain reaction (PCR) for the presence of blaTEM, blaSHV, blaCTX-M, mcr-1, qnrA, qnrB, papC, integron, and iss genes.
DNA was extracted from culture broth using a QIAamp DNA Mini Kit (Qiagen, Germany, GmbH Catalogue No. 51304). The extracted DNA was used in subsequent PCR assays for species confirmation and to detect genes responsible for virulence and antimicrobial agent resistance. The polymerase chain reaction was performed in a final volume of 25 μL that contained 12.5 μL of EmeraldAmp MAX PCR Master Mix (EmeraldAmp GT [2× premix], Japan), 1 μL of each primer (20 pmol), 4.5 μL of diethyl pyrocarbonate water, and 6 μL of the DNA template. The reaction was performed in a Biometra thermal cycler, T3000 (Germany). The oligonucleotide primers (Table-1 ) [39 (link)–45 (link)] were supplied by Metabion, Germany.
Polymerase chain reaction products were separated by electrophoresis [46 ] on a 1% agarose gel (AppliChem, Germany, GmbH) in 1× TBE buffer at room temperature (23°C to 27°C) using a gradient of 5 V/cm. Each well was loaded with 15 μL of the PCR product. A GelPilot 100 bp (Qiagen) ladder was used to determine the fragment sizes. The gel was photographed using a gel documentation system (Biometra BDA digital, Germany), and the data were analyzed using gel documentation (Alpha Innotech, Biometra, Germany) and specific software (automatic image capture software, Protein Simple, formerly Cell Bioscience, USA). The amplification conditions of the primers during PCR are shown inTable-2 .
The amplification efficiency was verified for positive field samples that may contain the tested genes, which were previously examined in a Veterinary Quality Control Reference Laboratory for Poultry Production, Animal Health Research Institute, Egypt.
DNA was extracted from culture broth using a QIAamp DNA Mini Kit (Qiagen, Germany, GmbH Catalogue No. 51304). The extracted DNA was used in subsequent PCR assays for species confirmation and to detect genes responsible for virulence and antimicrobial agent resistance. The polymerase chain reaction was performed in a final volume of 25 μL that contained 12.5 μL of EmeraldAmp MAX PCR Master Mix (EmeraldAmp GT [2× premix], Japan), 1 μL of each primer (20 pmol), 4.5 μL of diethyl pyrocarbonate water, and 6 μL of the DNA template. The reaction was performed in a Biometra thermal cycler, T3000 (Germany). The oligonucleotide primers (
Polymerase chain reaction products were separated by electrophoresis [46 ] on a 1% agarose gel (AppliChem, Germany, GmbH) in 1× TBE buffer at room temperature (23°C to 27°C) using a gradient of 5 V/cm. Each well was loaded with 15 μL of the PCR product. A GelPilot 100 bp (Qiagen) ladder was used to determine the fragment sizes. The gel was photographed using a gel documentation system (Biometra BDA digital, Germany), and the data were analyzed using gel documentation (Alpha Innotech, Biometra, Germany) and specific software (automatic image capture software, Protein Simple, formerly Cell Bioscience, USA). The amplification conditions of the primers during PCR are shown in
The amplification efficiency was verified for positive field samples that may contain the tested genes, which were previously examined in a Veterinary Quality Control Reference Laboratory for Poultry Production, Animal Health Research Institute, Egypt.
Free full text: Click here
