Presto mini gdna bacteria kit

After clinical testing was complete, deoxyribonucleic acid (DNA) was extracted from isolates using the Geneaid Presto MinigDNA Bacteria Kit and stored at −80 until sequencing was performed. Whole genome sequencing libraries were prepared as described elsewhere [18 (link)]. Sequencing was performed on the Illumina NextSeq platform using 300 cycle chemistries to a minimum average read depth of at least 20× per isolate. After sequencing, de novo draft genomes were assembled using AbySS v2.0.2 [19 (link)]. To ensure that the conventional microbiological species identification was concordant with the genomic classification, pairwise ANIb analysis was performed against a representative collection of publicly available Klebsiella genomes using the Pyani package (https://github.com/widdowquinn/pyani). Of the 70 total genomes analyzed, 10 matched Klebsiella species that were not K pneumoniae and 2 others evidenced polymicrobial contamination, resulting in a final set of 58 K pneumoniae genomes included in further analysis.

Bacillus pumilus group isolates were grown in nutrient broth for 24 to 48 h at 28°C. Twenty bacterial isolates of B. pumilus group extracted using a commercial genomic DNA isolation kit (Presto Mini gDNA Bacteria Kit, Geneaid Biotech Ltd., New Taipei City, Taiwan) following the protocol provided.

The CLs from the participants were obtained within 4 h of CL wear on the same day, received at the laboratory and aseptically transferred to a culture tube. The CLs were grown on brain heart infusion broth (BHI, HiMedia, India) at 37 °C for 48 h for recovery of bacterial cells. The culture was centrifuged at 10,000×g for 5 min at 4 °C, washed once with Tris-ethylenediaminetetraacetic acid (TE) buffer [10 mM Tris-HCl (pH 8.0), 1 mM ethylenediaminetetraacetic acid], and resuspended in 0.5 ml of TE buffer. An aliquot of 1 mL of all samples was centrifuged for 1 min at 15,000×g, and then the supernatant was discarded from the tubes for DNA extraction. Genomic DNA (gDNA) was extracted from the bacterial pellet in accordance with the manufacturer's protocol of the Presto™ Mini gDNA Bacteria Kit (Geneaid Biotech, Ltd., New Taipei City, Taiwan). The pellet was resuspended in 200 μL of lysozyme and incubated at 37 °C for 30 min. The supernatant was removed, 20 μL of proteinase K was added to the tube, and the tube was incubated at 60 °C for 10 min. DNA was lysed and bound to the GD column. The gDNA was washed and eluted in a collection tube. The purified gDNA was collected into one microcentrifuge tube and centrifuged at 15,000×g for 30 s. The concentration of gDNA was determined spectrophotometrically in a Nanodrop instrument and kept at -20 °C until library construction.