Quantstudio 3d digital polymerase chain reaction system

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).

Circulating bacterial fragment was represented by plasma endotoxin and bacterial DNA levels. Plasma endotoxin level was measured by a commercially available Limulus Amebocyte Lysate assay (Cambrex, Verviers, Belgium) as described previously [21 (link)]. All samples were diluted to 20% with endotoxin-free water and then heated to 70°C for 10 min to inactivate plasma proteins. The detection limit of the assay was 0.01 EU/mL. Plasma bacterial DNA level was measured by the QuantStudio 3D Digital Polymerase Chain Reaction (PCR) System (Life Technologies, Carlsbad, CA, USA) as described previously [22 (link)]. In essence, PCR amplification was performed by the ProFlex µPCR system, the result captured by the QuantStudio 3D Digital PCR Instrument, and analyzed by the QuantStudio Analysis Suite Software (all from Life Technologies).