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.