TABLE 3.

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

Target	Method	Amplicon size (bp)	Primer or probe (sequence)	Reference(s)
Detection
SSU rDNA	PCR	887	DF400 (5′-TATCGGAGGTGGTAATGACC-3′)	130, 459, 460
			DF1250 (5′-CATCTTCCTCCTGCTTAGACG-3′)
SSU rDNA	Real-time (TaqMan) PCR	77	DF3 (5′-GTTGAATACGTCCCTGCCCTTT-3′)	127, 138, 451, 460, 461
			DF4 (5′-TGATCCAATGATTTCACCGAGTCA-3′)
			Probe (5′-FAM-CACACCGCCCGTCGCTCCTACCG-TAMRA-3′)
rDNA (5.8S)	Real-time (TaqMan) PCR	98	Df-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 rDNA	PCR-RFLP	887	DF400 (5′-TATCGGAGGTGGTAATGACC-3′)	130, 459, 460
			DF1250 (5′-CATCTTCCTCCTGCTTAGACG-3′)
ITS1-5.8S-ITS2	PCR-sequencing	≈440	TFR1 (5′-TGCTTCAGTTCAGCGGGTCTTCC-3′)	133
			TFR2 (5′-CGGTAGGTGAACCTGCCGTTGG-3′)
SSU rDNA	PCR	1,700	TRD5 (5′-GATACTTGGTTGATCCTGCCAAGG-3′)	110, 129–131
			TRD3 (5′-GATCCAACGGCAGGTTCACCTACC-3′)
SSU rDNA	PCR-RFLP	662	DF1 (5′-CTCATAATCTACTTGGAACCAATT-3′)	85, 90, 131, 463
			DF4 (5′-CCCCGATTATTCTCTTTGATATT-3′)
SSU rDNA	PCR-HRM	662	DF1 (5′-CTCATAATCTACTTGGAACCAATT-3′)	85
			DF4 (5′-CCCCGATTATTCTCTTTGATATT-3′)
SSU rDNA	Nested PCR-sequencing	366	DF1 (5′-CTCATAATCTACTTGGAACCAATT-3′)	90
			DF4 (5′-CCCCGATTATTCTCTTTGATATT-3′)
			DF322For (5′-GAGAAGGCGCCTGAGAGATA-3′)
			DF687Rev (5′-TTCATACTGCGCTAAATCATT-3′)
ITS1-5.8S-ITS2	PCR-sequencing	500	ssu2 (5′-GGAATCCCTTGTAAATGCGT-3′)	134
			lsu1 (5′-AGTTCAGCGGGTCTTCCTG-3′)
ITS1	PCR-sequencing	≈100	ssu2 (5′-GGAATCCCTTGTAAATGCGT-3′)	134
			5.8s1 (5′-TGTGAGGAGCCAAGACATCC-3′)
ITS1	Nested PCR-sequencing	366	SSU2 (5′-GGAATCCCTTGTAAATGCGT-3′)	90
			Df-ITSRev (5′-GCGGGTCTTCCTATATAAACAAGAACC-3′)
			Df-ITSnesFor (5′-ATACGTCCCTGCCCTTTGTA-3′)
			Df-ITSnesRev (5′-GCAATGTGCATTCAAAGATCGAAC-3′)
SSU rDNA	PCR-pyrosequencing	129	D.FRAGILISpyroF (5′-CGGAGGTGGTAATGACCAGTTAT-3′)	132
			D.FRAGILISpyroR (5′-[biotin-C6]-TTGCAGAGCTGGAATTACCG-3′)
			D.FRAGILISpyroS (5′-TGGTAATGACCAGTTATAA-3′)
SSU rDNA	PCR-sequencing	364	DFpn_1f (5′-GCCAAGGAAGCACACTATGG-3′)	136
			DFpn_364r (5′-GTAAGTTTCGCGCCTGCT-3′)
Actin	PCR-sequencing	134–840	DF 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-1α	PCR-sequencing	99–836	DF_EF_1f (5′-CTCACTTTGGAAGTTCGAATC-3′)	136
			DF_EF_265f (5′-TCAAAGGCTCGTTATGATGAAATC-3′)
			DF_EF_364r (5′-GAAACCTGAGATTGGAACAAAC-3′)
			DF_EF_836r (5′-CTGTGTGGCAATCGAAAAC-3′)

^aAbbreviations: 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.