Supplementary file 2:
DNA extraction and qPCR protocol
All specimens were subjected to 3 freeze-thawing cycles and followed by 37°C
incubation for 1 h with Mutanolysin (200 U/mL, Sigma, St Louis, MO) and Lysostaphin (4
g/mL, Sigma). Genomic DNA was extracted from digested specimens by the QIAamp Mini
DNA kit (Qiagen Inc., Valencia, CA) according to the manufacturer’s instruction. Prior to
DNA extraction, all buffers and reagents from the QIAamp DNA mini kit (Qiagen) were
sterilized with 0.22 m filtering (Millipore, Bedford, MA) to minimize false positive signals.
Control samples using sterile water were also subjected to parallel DNA extraction.
qPCR was performed in duplicate assays using the TaqMan Universal Master Mix
(Applied Biosystems, Foster City, CA) according to the manufacturer’s protocol (Applied
Biosystems). A pair of universal primers and 2 specific Minor Groove Binder (MGB) probes
labelled with either 6-carboxyfluorescein (FAM) or 2’-chloro-7’-phenyl-1, 4-dichloro-6carboxyfluorescein (VIC) fluorescent dye were designed for qPCR detection and differentiation
of Gram-negative and Gram-positive bacterial DNA. The sequences of the primer pair are 5'GGAGGAAGGTGGGGAYGAC-3' (300 nM final concentration) and 5'CCATTGTAGCACGTGTGTAGCC-3' (300 nM final concentration), and the sequences of
the MGB probes are 5' 6-FAM-TCAAATCATCATGCCCC-3’ and 5’ VICTCAAGTCATCATGGCCC-3' (200 nM final concentration). Ten microliters of DNA
template were used for each reaction and the final volume was 25 L. The two-step PCR
conditions were set as 2 min at 50°C, 10 min at 95°C, 40 cycles with 30 sec at 95°C, and 1
min at 61°C. The emission intensity was detected by the 7300 real time PCR system
(Applied Biosystems). A no template negative control was included in each experiment.
Absolute quantification values expressed as threshold cycle (Ct) were averaged and
subsequently used to determine the absolute copy number according to the standard curves.