Agarose gel electrophoresis

Total genomic DNA was extracted from each pooled faecal sample from four piglets per treatment per timepoint using the Quick-DNA/soil microbe microprep kit (Zymoresearch, CA, USA) according to the manufacturer’s recommendation. The extracted DNA was checked for purity by A₂₆₀/A₂₈₀ comparison using the OneDrop TOUCH lite micro-volume spectrophotometer (Biometrics Technologies, Wilmington, DE, USA). DNA degradation was checked by 2% agarose gel electrophoresis (Vivantis, Selangor Darul Ehsan, Malaysia) and visualized under UV in the Syngene™ Ingenius 3 Manual Gel Documentation System (SynGene InGenius, Cambridge, UK). In addition, the total DNA concentration was measured using a Qubit™ 4 fluorometer with the dsDNA broad-range assay kit (Invitrogen™, Thermo Fisher Scientific, Waltham, USA). Shotgun metagenomic sequencing was undertaken using the Illumina Novaseq 6000 on the Illumina HiSeq-PE150 platform at 10-GB data output according to the manufacturer’s instructions (Novogene Bioinformatics Technology Co. Ltd., Beijing, China).

PCR detection was performed in a total volume of 25 μl containing 1 μl purified genomic DNA, 0.2 mM of dNTPs, 1× of Q5 reaction buffer, 1× of Q5 high GC enhancer, 0.02 units of Q5 high-fidelity DNA polymerase (New England Biolabs, MA, US), and 0.4 μM of each BimA_Bth-2F and BimA_Bth-R primers. The DNA amplification involved initial denaturation at 98°C for 3 min, and 35 cycles at 98°C for 30 s, 57°C for 30 s, and 72°C for 30 s, followed by a final extension for 10 min. PCR products were visualized following 1.5% agarose gel electrophoresis (Vivantis Technologies Sdn. Bhd., Malaysia).

B. pseudomallei (K96243), B. thailandensis (E264), and B. mallei (NCTC12938) genomic DNA were used as DNA templates for optimization of PCR conditions. Multiplex PCR detection was performed in a total volume of 25 μl containing 1 μl of bacterial lysates or purified genomic DNA, 0.2 mM of dNTPs, 1× of Q5 reaction buffer, 1× of Q5 high GC enhancer, 0.02 units of Q5 high-fidelity DNA polymerase (New England Biolabs, Inc., MA, USA), five PCR primers including 0.92 μM of BimA_com-R primer, 0.72 μM of BimA_Bps-F primer, 0.10 μM of BimA_BPBM-F primer and 0.30 μM each of BimA_Bth-F and BimA_Bth-R primers.
The DNA amplification involved initial denaturation at 98°C for 3 min, and 35 cycles at 98°C for 30 s, 68°C for 45 s, and 72°C for 30 s, followed by a final extension for 10 min. The PCR products were verified by 1.5% agarose gel electrophoresis (Vivantis Technologies Sdn. Bhd., Selangor Darul Ehsan, Malaysia) and visualized using a UV transilluminator (Syngene, Cambridge, UK). A GeneRuler 100 bp Plus DNA Ladder (Thermo Fisher Scientific, Inc., MA, USA) was included as a DNA marker.