The Acinetobacter species PCR identification protocol was modified from previous studies (19 (link)). Briefly, a clinical isolate from each patient was separately grown on LB agar at 37°C overnight. Genomic DNA was extracted by using boiling method (35 (link)); approximately three to five isolate colonies were resuspended in sterile deionized water and then boiled at 95°C for 10 min. After centrifugation at 12,000 × g for 10 min, supernatants were collected and estimated for DNA concentration by measuring the absorbance at 260 nm. PCR was performed according to the GoTaq Flexi DNA polymerase (Promega) manufacturers' instructions with GeneAmp PCR System 2700 (Applied Biosystems) thermocycle settings as follows: 94°C for 5 min, followed by 45 cycles of 94°C for 1 min, 60°C for 1 min, 72°C for 1 min, and a final extension at 72°C for 10 min. The primers used in this study and their interpretation are shown in Table S1.
The PCR amplicons were separated by electrophoresis (100 V, 80 min) in 1.5% agarose gel in 40 mM Tris, 20 mM boric acid, and 1 mM EDTA (TBE) buffer pH 8.3 containing DNA Gel Loading Dye (Thermo Fisher Scientific). The gels were visualized and captured using a gel image analysis system (UVitec, Cambridge, United Kingdom). The multiplex PCR results were validated for the first 18 isolates using 16S rRNA sequencing (Macrogen, Inc., South Korea) (Fig. S1).
The PCR amplicons were separated by electrophoresis (100 V, 80 min) in 1.5% agarose gel in 40 mM Tris, 20 mM boric acid, and 1 mM EDTA (TBE) buffer pH 8.3 containing DNA Gel Loading Dye (Thermo Fisher Scientific). The gels were visualized and captured using a gel image analysis system (UVitec, Cambridge, United Kingdom). The multiplex PCR results were validated for the first 18 isolates using 16S rRNA sequencing (Macrogen, Inc., South Korea) (Fig. S1).
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