Droplet digital PCR
ddPCR was carried out using a ddPCR platform (Pilot Gene Technology, Hangzhou, China). A duplex ddPCR assay was performed to detect A. baumannii and K. pneumonia simultaneously in one chip. ddPCR analysis was performed using a Pilot Gene Droplet Digital PCR System following the manufacturer’s protocol. Briefly, the ddPCR master mix for each testing panel had a final volume of 15 μL and contained 1 × ddPCR premix, 1 μM forward and reverse primers, 300 nM each probe, 5 μL of isolated plasma DNA, and DNase-free water. The mixture was loaded on a ready-to-use disposable plastic chip. About 20,000 droplets were generated using a droplet generator (DG32; Pilot Gene Technology), followed by amplification on a TC1 thermal cycler (Pilot Gene Technology). The thermal cycling parameters were 95 oC for 5 min followed by 40 cycles of 95 oC for 15 s and 60 oC for 60 s. After PCR amplification, droplets were analysed using an iScanner 5 chip scanner. Data analysis for droplet counts and amplitudes was performed with 30 min of hands-on time using GenePMS software version v2.0.01.20011.