A 20-mL specimen of PD effluent was collected on randomization (i.e. 5 days before antibiotic completion, according to the ISPD guideline) for the measurement of bacterial DNA fragment levels. For the extended group, a second PD effluent sample was collected 5 days before the completion of the extended treatment. DNA was extracted using the EZ1 DNA tissue kit and BioRobot EZ1 with the EZ1 bacteria card (Qiagen, Germantown, MD, USA), according to the manufacturer’s instructions. Purified DNA was eluted in 50 µL of elution buffer before amplification. The bacterial DNA fragment level in PD effluent was measured by the QuantStudio 3D Digital Polymerase Chain Reaction (PCR) System (Life Technologies, Carlsbad, CA, USA). Briefly, the PCR mixture was prepared and loaded into the chip according to the manufacturer’s protocol. PCR amplification was performed by the ProFlex µPCR system (Life Technologies). The result was captured by the QuantStudio 3D Digital PCR Instrument and analyzed by the QuantStudio AnalysisSuite Software (both from Life Technologies).
Ez1 bacteria card
Manufactured by Qiagen
Sourced in United States
The EZ1 bacteria card is a lab equipment product manufactured by Qiagen. It is designed to facilitate the extraction and purification of bacterial DNA samples. The core function of the EZ1 bacteria card is to automate the DNA extraction process, providing a streamlined and efficient solution for researchers and laboratories.
Automatically generated - may contain errors
Lab products found in correlation
Biorobot ez1, by Qiagen (2 mentions)
Ez1 dna tissue kit, by Qiagen (2 mentions)
Proflex pcr system, by Thermo Fisher Scientific (1 mentions)
Quantstudio 3d digital pcr instrument, by Thermo Fisher Scientific (1 mentions)
Quantstudio 3d digital polymerase chain reaction system, by Thermo Fisher Scientific (1 mentions)
Quantstudio analysissuite software, by Thermo Fisher Scientific (1 mentions)
2 protocols using ez1 bacteria card
1
Bacterial DNA Quantification in PD Effluent
2
Amplification of Bacterial 16S rRNA
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The method of bacterial DNA amplification has been described previously [19 (link),20 (link)]. Briefly, DNA from 200 μl aliquot of EDTA-treated whole blood was extracted using the EZ1 DNA tissue kit and BioRobot EZ1 with the EZ1 bacteria card (Qiagen), according to the manufacturer’s instructions. Purified DNA was eluted in 50 μl of elution buffer before amplification. Universal primers used for polymerase chain reaction (PCR) amplification of the bacterial 16S rRNA gene were p16SrRNA+ and p16SrRNA-, which are able to amplify DNA from either Gram positive or Gram negative bacteria. Aliquots of 20-μl DNA samples were used for amplification in a 50-μl PCR reaction mixture. All samples were run in triplicates. Since plasma was directly used as the template and there is no intrinsic housekeeping gene for comparison, the number of PCR cycles at which bacterial DNA could be detected is reported.
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