Table 1.
Primers, probes and artificial template used in the present study.
| Oligonucleotide | Specificity | Target gene | GenBank accession no. | Sequence (5′–3′)* |
|---|---|---|---|---|
| Primers | ||||
| ITS1/ITS1b† | Panfungal | 18S rRNA gene | HM802135 | 4 TCCGTAGGTGAACCTGCG 21 |
| ITS4 | Panfungal | 28S rRNA gene | HM802135 | 534 TCCTCCGCTTATTGATATGC 515 |
| F63 | Panfungal | 28S rRNA gene | EU882103 | 1 GCATATCAATAAGCGGAGGAAAAG 24 |
| R635/R635b† | Panfungal | 28S rRNA gene | EU882103 | 640 GGTCCGTGTTTCAAGACG 623 |
| RCA primer 1‡ | – | Padlock probe linker region | – | ATGGGCACCGAAGAAGCA |
| RCA primer 2‡ | – | Padlock probe linker region | – | CGCGCAGACACGATA |
| RLB probes† | ||||
| 12AP | Panfungal | ITS region | AF455524 | 211 CCAAGAGATCCGTTGTTGAAAG 190 |
| 23SP | Panfungal | ITS region | AF455524 | 271 GTGAATCATCGARTCTTTGAACG 293 |
| Tri-ITS-1 | T. asahii, T. domesticum and T. japonicum | ITS region | AF444473 | 31 GTGATTGCCTTAATTGGCTTATAAC 55 |
| Tri-ITS-2 | T. asahii, T. inkin and T. japonicum | ITS region | AF444473 | 93 ACGCAAGTCGAGTATTTTTACAAAC 117 |
| Tri-ITS-3 | T. asahii and T. japonicum | ITS region | AJ864867 | 10 GTGATTGCCTTTATAGGCTTATAAC 34 |
| Tri-ITS-4 | T. cutaneum | ITS region | AF444325 | 89 TTCGGTCAATTGATTTTACAAAC 111 |
| Tri-ITS-5 | T. dermatis and T. jirovecii | ITS region | AF444437 | 88 CTCCGGTCAATTACTTTACAAAC 110 |
| Tri-ITS-6 | T. jirovecii | ITS region | AF444437 | 373 GAGTTAGCGTGTTTAACTTGTCGAT 397 |
| Tri-ITS-7 | T. dermatis and T. jirovecii | ITS region | AF444437 | 385 TTAACTTGTCGATCTGGCGTA 405 |
| Tri-ITS-8 | T. domesticum | ITS region | AF444422 | 85 TTGAATCTTCGGATTCGATTTTATACAAA 113 |
| Tri-ITS-9 | T. domesticum and T. laibachii | ITS region | AF444422 | 377 AAAGAGTTAGCAAGTTGAACTATTGCTAT 405 |
| Tri-ITS-10 | T. inkin | ITS region | AF444420 | 31 GTGATTGCCTTTACAGGCTTAACTA 55 |
| Tri-ITS-11 | T. laibachii | ITS region | AF444421 | 84 TTGAATCTCTGATTCAATTTTACAAAC 110 |
| Tri-ITS-12 | T. laibachii | ITS region | AF444421 | 31 GTGATTGCCATCTTGGCTTAAAC 53 |
| Tri-D1D2-1 | T. cutaneum, T. dermatis and T. jirovecii | D1/D2 domain | AF105398 | 181 GCTTGATACGACGACCAGTGCTCT 204 |
| Tri-D1D2-2 | T. cutaneum, T. dermatis, T. domesticum and T. jirovecii | D1/D2 domain | AF105398 | 405 ATTCAGCTGGTTCTTCCAGTCTACT 429 |
| Tri-D1D2-3 | T. dermatis and T. domesticum | D1/D2 domain | EU559351 | 413 GATTCAGCTAGTTCTTCTAGTCTACTTCC 441 |
| Tri-D1D2-4 | T. domesticum | D1/D2 domain | AF189874 | 181 ACTTGACACAACAATCAGTGCTCT 204 |
| Tri-D1D2-5 | T. asahii | D1/D2 domain | AF337949 | 535 GGCCGGCCTTCGGGCACGTT 554 |
| Tri-D1D2-6 | T. asahii, T. inkin and T. japonicum | D1/D2 domain | AF308657 | 407 TCAGCCAGTTCTGCTGGTCTACT 429 |
| Tri-D1D2-7 | T. domesticum, T. inkin and T. laibachii | D1/D2 domain | AJ749822 | 566 GGCCGGGGTTCGCCCACGTT 585 |
| Tri-D1D2-8 | T. laibachii | D1/D2 domain | AJ507664 | 157 ACTTTACACAATCATCAGTGCTCT 180 |
| Tri-D1D2-9 | T. laibachii | D1/D2 domain | AJ507664 | 381 ATTCAGCCGGTCTTCGGTGTACT 403 |
| RCA padlock probes‡ | ||||
| Tri-jap | T. japonicum | ITS region | AF444473 | 51 TAAGCCAATTAAGGCAATCACTAATG 26 –linker-74 ACAGGTGTAAGTGGATATAGTTA 52 |
| Tri-cut | T. cutaneum | ITS region | AF444325 | 354 ACTGGCAGCGCCCAA 340 –linker-366 GCGAGCCAGGCT 355 |
| Tri-der-a | T. dermatis | ITS region | EU559351 | 441 GGAAGTAGACTAGAAGAACTAGCTGAATCC 412
–linker-457 TGTTGACCCGTTCAAT 442 |
| Tri-dom-a | T. domesticum | ITS region | AF444454 | 393 AACTTGCTAACTCTTTTAAGAGGAGCC 367
–linker-411 GCCAGATAGCAATAGTTC 394 |
| Tri-asa-a | T. asahii | D1/D2 domain | AF337949 | 540 CCGGCCATAAAGGCGA 525 –linker-552 CGTGCCCGAAGG 541 |
| Oligonucleotide | Specificity | Target gene | GenBank accession no. | Sequence (5′–3′)* |
| Tri-asa-b | T. asahii | D1/D2 domain | AF337949 | 547 CCGAAGGCCGGCCA 534 –linker-563 CTAAGCTCGAACGTGC 548 |
| Tri-der-b | T. dermatis | D1/D2 domain | EU559351 | 494 TACATTCCTACTATCTTTATCCACCGG 468
–linker-505 CCCGGGGAGC 495 |
| Tri-dom-b | T. domesticum | D1/D2 domain | AF189874 | 510 TAGGCTATAACACTCCCGAGGGA 488 –linker-528 ACCCAGTGTATGTGAACC 511 |
| Tri-ink | T. inkin | D1/D2 domain | AJ749822 | 519 CAGGCTATAACACTTCCGGAGAAG 496
–linker-537 ACCCAGTGTATGTGACAG 520 |
| Tri-jir-a | T. jirovecii | D1/D2 domain | AF105398 | 130 TCCAGCACGGAAAACACG 113 –linker-148 CAAGGGACTTAGATACGA 131 |
| Tri-jir-b | T. jirovecii | D1/D2 domain | AF105398 | 214 CGTGTATCACAGAGCACTGGTC 193 –linker-233 AACAACTCGACTCGTAGAA 215 |
| Tri-lai | T. laibachii | D1/D2 domain | EU559352 | 431 ACCGAAGACCGGCTGAAT 414 –linker-450 ACCCGTTTAAAGGAAGTAC 432 |
| Tri-ast§ | T. asteroides | D1/D2 domain | AF075513 | 490 GAAGTCACATTCCTACTACCTTTATCCAC 462
–linker-507 GCTATAACACTTCCGGG 491 |
| Artificial template§ | ||||
| T. asteroides-AT§ | T. asteroides | D1/D2 domain | AF075513 | 457 GTCCGGTGGATAAAGGTAGTAGGAATGT GACTTCCCCGGAAGTGTTATAGCCTATT 512 |
Abbreviations: D1/D2 domain, D1/D2 domain of 28S rRNA gene; ITS region, internal transcribed spacer region; RLB, reverse line blot; RCA, rolling cycle amplification. *Numbers represent the numbered base positions where the oligonucleotide sequences start or finish (commencing at point 1 of the corresponding gene GenBank sequence). †Primers ITS1b and R635b were based on previously published ITS1 and R635, respectively, with 5′ end biotin labeled for PCR in preparation for RLB. All RLB probes were 5′-hexylamine modified. ‡All RCA padlock probes were with 5′-end phosphorylation. 5′ and 3′ binding arms of the probes derived from reference GenBank sequence and their positions are showed. These are joined by the 64 bp linker region including (i) binding site of RCA primer 1 to the padlock probe (reverse compliment sequence of RCA primer 1), generating a long single-stranded DNA; (ii) binding site of RCA primer 2 (with the same sequence of RCA primer 2), primer binds to nascent single-stranded DNAs as their binding sites become available; and (iii) the non-specific linker region. §An additional probe Tri-ast was designed to identify T. asteroides, which has high sequence similarity with T. japonicum studied (1 bp difference each within the ITS region and D1/D2 domain, respectively). As no T. asteroides isolates were available for study, an artificial template ‘T. asteroides-AT’ was used along with the 48 Trichosporon isolates to test the sensitivity and specificity of the probe Tri-ast.