Bacteria genomic dna kit

Genomic DNA from the three strains was prepared using Bacteria Genomic DNA kit (Geneaid, Taiwan). Nuclei were isolated according to the manufacturer’s instructions. Purity and quantity of DNA samples were estimated using the Qubit dsDNA HS Assay Kit (Thermo-Fisher Scientific) and Agilent BioAnalyzer 2100 High Sensitivity DNA Kit (Agilent). Sequencing libraries were prepared using Nextera DNA Flex Library Prep Kit (Illumina). Whole genome shotgun sequencing was performed on an Illumina MiSeq instrument using MiSeq Reagent Kit v.3 to generate 2 × 300 bp paired-end reads with an average of 2.8–3.1 million paired-end Illumina reads per genome. On average, the final coverage of the assembled genomes exceeded 200 × of the Illumina reads (Supplementary Table 1).

The bacterial genomic DNA was extracted using the Bacteria Genomic DNA Kit (Geneaid, Taiwan). The antibiotic resistance determinants and biofilm-related genes were detected by PCR using specific primers. PCR assays were performed using Phusion™ Plus PCR Master Mix (Thermo Scientific), and PCR amplicons were analyzed by 1.5% agarose gel electrophoresis, visualized by health view nucleic acid stain and photographed under UV light (Tang et al., 2021 (link)).

SYBR 202 real-time PCR assays to detect scrub typhus were also used for comparison with the ICT AgTK. DNA was extracted from individual plasma samples of Nan Provincial hospital using Bacteria Genomic DNA Kit (Presto™ Mini gDNA Bacteria Kit, No. GBB100, Geneaid Biotech, Shilr District, New Taipei City, Taiwan) following the manufacturer’s instructions. Real-time PCR could not be used for analyses of the first or third sample sets. SYBR real-time PCR assays were used for the specific detection of the gene coding for the GroEL of O. tsutsugamushi. The primer pair were: F: TAA TTG CTA GTG CAA TGT CTG CGT T and R: CCA AAG TCA CGA TCA GCT ATA CT. The PCR reaction mixture contained 200 nM each primer, 1 µL of DNA template, 10 µL of master mix (KAPA SYBR FAST qPCR Master Mix) containing SYBR green, Taq polymerase, 4 mM MgCl₂, dNTPs and distilled water to a final volume of 20 µL. The PCR reactions were carried out and analyzed using real-time thermocycler (CFX96 Touch Real-Time PCR Detection System, BioRad, Hercules, CA, USA) with an initial temperature of 95 °C for 3 min, followed by 40 cycles at 95 °C for 3 s, 55 °C for 30 s and 72 °C for 20 s, with fluorescence monitoring at the 55 °C annealing step on a predetermined SYBR channel. Melting curve analysis was performed with increment of 1 °C/step (72–95 °C) to determine the change in peak fluorescence over time (dF/dT).