TABLE 2.
PCR primers and probes used in conventional, nested, and real-time PCR assays for amplification of D. fragilis DNAd
| Assay and gene target | Amplicon size | Primer or probe | Sequence | Reference |
|---|---|---|---|---|
| Conventional PCR | ||||
| 18S rRNA | 1.7 kb | TRD5a | 5′-GATACTTGGTTGATCCTGCCAAGG-3′ | 30 |
| TRD3a | 5′-GATCCAACGGCAGGTTCACCTACC-3′ | |||
| 18S rRNA | 850 bp | DF400 | 5′-TATCGGAGGTGGTAATGACC-3′ | 30 |
| DF1250 | 5′-CATCTTCCTCCTGCTTAGACG-3′ | |||
| 18S rRNA | 364 bp | DFpn_1f | 5′-GCCAAGGAAGCACACTATGG-3′ | 155 |
| DFpn_364r | 5′-GTAAGTTTCGCGCCTGCT-3′ | |||
| 18S rRNA | 662 bp | DF1 | 5′-CTCATAATCTACTTGGAACCAATT-3′ | 123 |
| DF4 | 5′-CCCCGATTATTCTCTTTGATATT-3′ | |||
| ITS1-5.8S | 300 bp | ssu2 | 5′-GGAATCCCTTGTAAATGCGT-3′ | 35 |
| 1su1 | 5′-AGTTCAGCGGGTCTTCCTG-3′ | |||
| ITS1 | 300 bp | ssu2 | 5′-GGAATCCCTTGTAAATGCGT-3′ | 35 |
| 5.8s1 | 5′-TGTGAGGAGCCAAGACATCC-3′ | |||
| Nested PCR | ||||
| 18S rRNA | 662 bp | DF1b | 5′-CTCATAATCTACTTGGAACCAATT-3′ | 38 |
| DF4Ib | 5′-CCCCGATTATTCTCTTTGATATT-3′ | |||
| 18S rRNA | 366 bp | DF322Forc | 5′-GAGAAGGCGCCTGAGAGATA-3′ | 38 |
| DF687Revc | 5′-TTCATACTGCGCTAAATCATT-3′ | |||
| 18S rRNA | 850 bp | DF400b | 5′-TATCGGAGGTGGTAATGACC-3′ | 98 |
| DF1250b | 5′-CATCTTCCTCCTGCTTAGACG-3′ | |||
| 18S rRNA | 403 bp | DFF2c | 5′-CGGGGATAGATCTATTTCATGGC-3′ | 98 |
| DFR2c | 5′-CCAACGGCCATGCACCACC-3′ | |||
| ITS1 | ~540 bpe | Ssu2b | 5′-GGAATCCCTTGTAAATGCGT-3′ | 38 |
| DF-ITSREVb | 5′-GCGGGTCTTCCTATATAAACAAGAACC-3′ | |||
| ITS1 | 380 bp | Df-ITSnesForc | 5′-ATACGTCCCTGCCCTTTGTA-3′ | 38 |
| ITSnesRevc | 5′-GCAATGTGCATTCAAAGATCGAAC-3′ | |||
| Real-time PCR | ||||
| 18S rRNA | 78 bp | DF3 | 5′-GTTGAATACGTCCCTGCCCTTT-3′ | 95 |
| DF4 | 5′-TGATCCAATGATTTCACCGAGTCA-3′ | |||
| TaqMan probe | 5′-FAM-CACACCGCCCGTCGCTCCTACCG-TAMRA-3′ | |||
| 18S rRNA | 101 bp | 5DMB | 5′-GGCGAAAGCATCTATCAAGTGTAAT-3′ | 96 |
| 3DMB | 5′-CGGCATCGTTTAAGGTAGGAAC-3′ | |||
| TaqMan probe | 5′-FAM-ACCCGGGTCTCTGATCCGGTTGG-TAMRA-3′ | |||
| 18S rRNA | 662 bp | DF1 | 5′-CTCATAATCTACTTGGAACCAATT-3′ | 37 |
| DF4 | 5′-CCCCGATTATTCTCTTTGATATT-3′ | |||
| 5.8S rRNA | 98 bp | Df-124F | 5′-CAACTTGGCTCTTTA-3′ | 51 |
| Df-221R | 5′-TGCATTCAAAGATCGAACTTATCAC-3′ | |||
| Df-172revT | 5′-FAM-CAATTCTAGCCGCTTAT-3′-MGB |
a
Generic trichomonad primer.
b
Outer nested primer.
c
Inner nested primer.
d
NA, not applicable; FAM, 6-carboxyfluorescein; TAMRA, 6-carboxytetramethylrhodamine.
e
The amplicon size for ITS1 is variable.