Quantifying Bacterial Burden in Plasma

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).