TABLE 3

Primer and probe sequences for PCR-based assays for detection and molecular characterization of Dientamoeba fragilis a

TargetMethodAmplicon size (bp)Primer or probe (sequence)Reference(s)
Detection
    SSU rDNAPCR887DF400 (5′-TATCGGAGGTGGTAATGACC-3′) 130, 459, 460
DF1250 (5′-CATCTTCCTCCTGCTTAGACG-3′)
    SSU rDNAReal-time (TaqMan) PCR77DF3 (5′-GTTGAATACGTCCCTGCCCTTT-3′) 127, 138, 451, 460, 461
DF4 (5′-TGATCCAATGATTTCACCGAGTCA-3′)
Probe (5′-FAM-CACACCGCCCGTCGCTCCTACCG-TAMRA-3′)
    rDNA (5.8S)Real-time (TaqMan) PCR98Df-124F (5′-CAACGGATGTCTTGGCTCTTTA-3′) 27, 128, 135, 162, 462
Df-221R (5′-TGCATTCAAAGATCGAACTTATCAC-3′)
Df-172revT (5′-FAM-CAATTCTAGCCGCTTAT-MGB-3′)
Molecular characterization/genotyping
    SSU rDNAPCR-RFLP887DF400 (5′-TATCGGAGGTGGTAATGACC-3′) 130, 459, 460
DF1250 (5′-CATCTTCCTCCTGCTTAGACG-3′)
    ITS1-5.8S-ITS2PCR-sequencing≈440TFR1 (5′-TGCTTCAGTTCAGCGGGTCTTCC-3′) 133
TFR2 (5′-CGGTAGGTGAACCTGCCGTTGG-3′)
    SSU rDNAPCR1,700TRD5 (5′-GATACTTGGTTGATCCTGCCAAGG-3′) 110, 129 131
TRD3 (5′-GATCCAACGGCAGGTTCACCTACC-3′)
    SSU rDNAPCR-RFLP662DF1 (5′-CTCATAATCTACTTGGAACCAATT-3′) 85, 90, 131, 463
DF4 (5′-CCCCGATTATTCTCTTTGATATT-3′)
    SSU rDNAPCR-HRM662DF1 (5′-CTCATAATCTACTTGGAACCAATT-3′) 85
DF4 (5′-CCCCGATTATTCTCTTTGATATT-3′)
    SSU rDNANested PCR-sequencing366DF1 (5′-CTCATAATCTACTTGGAACCAATT-3′) 90
DF4 (5′-CCCCGATTATTCTCTTTGATATT-3′)
DF322For (5′-GAGAAGGCGCCTGAGAGATA-3′)
DF687Rev (5′-TTCATACTGCGCTAAATCATT-3′)
    ITS1-5.8S-ITS2PCR-sequencing500ssu2 (5′-GGAATCCCTTGTAAATGCGT-3′) 134
lsu1 (5′-AGTTCAGCGGGTCTTCCTG-3′)
    ITS1PCR-sequencing≈100ssu2 (5′-GGAATCCCTTGTAAATGCGT-3′) 134
5.8s1 (5′-TGTGAGGAGCCAAGACATCC-3′)
    ITS1Nested PCR-sequencing366SSU2 (5′-GGAATCCCTTGTAAATGCGT-3′) 90
Df-ITSRev (5′-GCGGGTCTTCCTATATAAACAAGAACC-3′)
Df-ITSnesFor (5′-ATACGTCCCTGCCCTTTGTA-3′)
Df-ITSnesRev (5′-GCAATGTGCATTCAAAGATCGAAC-3′)
    SSU rDNAPCR-pyrosequencing129D.FRAGILISpyroF (5′-CGGAGGTGGTAATGACCAGTTAT-3′) 132
D.FRAGILISpyroR (5′-[biotin-C6]-TTGCAGAGCTGGAATTACCG-3′)
D.FRAGILISpyroS (5′-TGGTAATGACCAGTTATAA-3′)
    SSU rDNAPCR-sequencing364DFpn_1f (5′-GCCAAGGAAGCACACTATGG-3′) 136
DFpn_364r (5′-GTAAGTTTCGCGCCTGCT-3′)
    ActinPCR-sequencing134–840DF ACTIN_3f (5′-CCACACATTCTACAACGAATTAC-3′) 136
DF_ACTIN_157f (5′-TTCTTTCACTTTACTCATCAGGTC-3′)
DF_ACTIN_291r (5′-GACCAGCAAGGTTGAGTCTC-3′)
DF_ACTIN_843r (5′-TGGACCAGCTTCATTGTATTC-3′)
    EF-1αPCR-sequencing99–836DF_EF_1f (5′-CTCACTTTGGAAGTTCGAATC-3′) 136
DF_EF_265f (5′-TCAAAGGCTCGTTATGATGAAATC-3′)
DF_EF_364r (5′-GAAACCTGAGATTGGAACAAAC-3′)
DF_EF_836r (5′-CTGTGTGGCAATCGAAAAC-3′)
  • a Abbreviations: SSU, small subunit; ITS, internal transcribed spacer; EF-1α, elongation factor 1α; RFLP, restriction fragment length polymorphism; HRM, high-resolution melting; TAMRA, 6-carboxytetramethylrhodamine; FAM, 6-carboxyfluorescein; MGB, minor groove binder.