For detection of broad-range bacterial 16S rRNA and panfungal 18S rRNA from blood and surgical materials when accessible, followed by sequencing for identification of positive cases, DNA was extracted from blood and surgical materials using the QIAamp DNA Mini Kit (Qiagen, Hilden, Germany).
16SrRNA bacterial gene amplification (matching to regions 8 to 806 of the Escherichia coli 16S rRNA gene) was performed using the eubacterial 16SrRNA primer set 536F 5′-CAGCAGCCGCGGTAATAC-3′ and RP2 5′-ACGGCACCTTGTTACGACTT-3′ (Bioneer, AccuOligo®, Oakland, CA, USA) [7 (link)], and the conserved region of the 18S (ITS-1) rRNA of fungi was amplified using the oligonucleotide panfungal primer sequences CUF 5′-TCCGTAGGTGAACCTGCGGG-3′ and CUR 5′-GCTGCGTTCTTCATCGATGC-3′ mainly targeting the V4 and V5 portions of the 18S rRNA gene, as previously designated [12 ].
Human β-globin housekeeping gene was amplified as an internal control by adding the primers BGF (5′-CAACTTCATCCACGTTCACC-3′) and BGR (5′ GAAGAGCCAAGGACAGGTAC-3′) to each PCR vial. Staphylococcus aureus (ATCC 25923), E.coli (ATCC 25922), and Candida albicans (ATCC 10231) were included as a positive control in each run, as was a negative control of PCR-grade water without DNA.
Amplified DNA fragments were purified from the PCR products using the Thermo Scientific™ GeneJET PCR Purification Kit according to the manufacturer’s protocol.
16SrRNA bacterial gene amplification (matching to regions 8 to 806 of the Escherichia coli 16S rRNA gene) was performed using the eubacterial 16SrRNA primer set 536F 5′-CAGCAGCCGCGGTAATAC-3′ and RP2 5′-ACGGCACCTTGTTACGACTT-3′ (Bioneer, AccuOligo®, Oakland, CA, USA) [7 (link)], and the conserved region of the 18S (ITS-1) rRNA of fungi was amplified using the oligonucleotide panfungal primer sequences CUF 5′-TCCGTAGGTGAACCTGCGGG-3′ and CUR 5′-GCTGCGTTCTTCATCGATGC-3′ mainly targeting the V4 and V5 portions of the 18S rRNA gene, as previously designated [12 ].
Human β-globin housekeeping gene was amplified as an internal control by adding the primers BGF (5′-CAACTTCATCCACGTTCACC-3′) and BGR (5′ GAAGAGCCAAGGACAGGTAC-3′) to each PCR vial. Staphylococcus aureus (ATCC 25923), E.coli (ATCC 25922), and Candida albicans (ATCC 10231) were included as a positive control in each run, as was a negative control of PCR-grade water without DNA.
Amplified DNA fragments were purified from the PCR products using the Thermo Scientific™ GeneJET PCR Purification Kit according to the manufacturer’s protocol.