Table 10 PCR Oligonucleotides Used for the Molecular Characterization of the Samples [7, 13, 62, 63]
Pan-fungal PCR 1 (variable 18S rRNA gene region) | |
Forward primer RTP_1 | 5′-ATT-GGA-GGG-CAA-GTC-TGG-TG-3′ |
Reverse primer RTP_2 | 5′-CCG-ATC-CCT-AGT-CGG-CAT-AG-3′ |
Probe 1 RTP_p1 | 5′-3FL-TTC-AAC-TAC-gAg-CTT-TTT-AAC-Tg-3′ |
Probe 2 RTP_p2 | 5′-LC-Red640-AAC-AAC-TTT-AAT-ATA-CgC-TAT-Tgg A-3′ |
Pan-fungal PCR 2 (variable ITS-2 region of the fungal rRNA operon) | |
Forward primer 5.8S | 5′-GTG-AAT-CAT-CGA-RTC-TTT-GAA-C-3′ |
Reverse primer 28S1-rev | 5′-TAT-GCT-TAA-GTT-CAG-CGG-GTA-3′ |
Pan-fungal PCR 3 (variable 28S rRNA gene region) | |
Forward primer 28S-10 | 5′-GAC-ATG-GGT-TAG-TCG-ATC-CTA-3′ |
Reverse primer 28S-12-rev | 5′-CCT-TAT-CTA-CAT-TRT-TCT-ATC-AAC-3′ |
Pan-fungal PCR 4 (variable ITS-2 region of the fungal rRNA operon) | |
Forward primer ITS-3 | 5′-GCA-TCG-ATG-AAG-AAC-GCA-GC-3′ |
Reverse primer ITS-4 | 5′-TCC-TCC-GCT-TAT-TGA-TAT-GC-3′ |
Pan fungal PCR 5 (variable ITS-1 region of the fungal rRNA operon) | |
Forward primer ITS-1 | 5′-TCC-GTA-GGT-GAA-CCT-GCG-G-3′ |
Reverse primer ITS-2 | 5′-GCT-GCG-TTC-TTC-ATC-GAT-GC-3′ |
Histoplasma capsulatum PCR (gene encoding the unique fungal 100-kDa-like protein) | |
Forward primer 1 Hc I | 5′-GCG-TTC-CGA-GCC-TTC-CAC-CTC-AAC-3’ |
Forward primer 2 Hc III | 5′-GAG-ATC-TAG-TCG-CGG-CCA-GGT-TCA-3’ |
Reverse primer 1 Hc IV | 5′-AGG-AGA-GAA-CTG-TAT-CGG-TGG-CTT-G-3’ |
Reverse primer 3 Hc II | 5′-ATG-TCC-CAT-CGG-GCG-CCG-TGT-AGT-3’ |
Mucorales PCR (18S rRNA gene) | |
Forward primer ZM1 | 5′-ATT-ACC-ATG-AGC-AAA-TCA-GA-3’ |
Reverse primer 1 ZM3 | 5′-CAA-TCC-AAG-AAT-TTC-ACC-TCT-AG-3‘ |
Reverse primer 2 ZM2 | 5′-TCC-GTC-AAT-TCC-TTT-AAG-TTT-C-3’ |
Duplex PCR for Entamoeba histolytica and Entamoeba dispar (fragment of the ribosomal RNA gene located on an episomal plasmid) | |
Forward primer Ehd-239F | 5′-ATT-GTC-GTG-GCA-TCC-TAA-CTC-A-3′ |
Reverse primer Ehd-88R | 5′-GCG-GAC-GGC-TCA-TTA-TAA-CA-3′ |
Probe 1 histolytica-96 T (specific for E. histolytica) | 5′-JOE-TCA-TTG-AAT-GAA-TTG-GCC-ATT-T-BHQ1–3′ |
Probe 2 dispar-96 T (specific for E. dispar) | 5′-Cy5-TTA-CTT-ACA-TAA-ATT-GGC-CAC-TTT-G-BHQ2–3′ |
Inhibition control PCR (Phocid herpesvirus 1 (PhHV-1) 1 gB gene) | |
Forward primer PhHV-267 s | 5′-GGG-CGA-ATC-ACA-GAT-TGA-ATC-3′ |
Reverse primer PhHV-337as | 5′-GCG-GTT-CCA-AAC-GTA-CCA-A-3′ |
Probe PhHV-305tq | 5′-Cy55-TTT-TTA-TGT-GTC-CGC-CAC-CAT-CTG-GAT-C-BBQ-3′ |
Sample quality assessment PCR (human 18S rRNA gene) | |
Forward primer 18S-f | 5′-CTC-TTA-GCT-GAG-TGT-CCC-GC-3′ |
Reverse primer 18S-r | 5′-CTT-AAT-CAT-GGC-CTC-AGT-TCC-GA-3′ |
Probe 18S-p | 5′-FAM-CCG-AGC-CGC-CTG-GAT-ACC-GCA-GCT-A-TAMRA-3′ |