. 2023 May 18;9(5):587. doi: 10.3390/jof9050587

Table 5.

Primers and corresponding assays for diagnosis of Fusarium spp. on soybean.

Target Gene	Target Species (Specificity)	Primer/Probe (Combination)	Sequence (5′-3′)	T_m (°C)	Fragment Length (bp)	Assay	Ref.
ITS1	F. solani f. sp. phaseoli	FspF	ACCCCCTAACTCTTGTTATATCC	60	957–958	PCR	[82]
ITS1	F. solani f. sp. phaseoli	FspR	GCGCAATACCCTGAGGCG	60	957–958	PCR	[82]
TEF1	F. solani f. sp. phaseoli	Effp-1	AACCCCGCCCGAGGACTCA	72	562	PCR/qPCR	[84]
TEF1	F. solani f. sp. phaseoli	Effp-2	AGACATGAGCGATGAGAGGCA	72	562	PCR/qPCR	[84]
TEF1	F. solani f. sp. glycines	FsgEF1	GAGTCGGTTAGCTTCTGTC	66 ^a/56	237	PCR ^b	[90]
TEF1	F. solani f. sp. glycines	FsgEF2	GCGCGCCTTGCTATTCTCC	66 ^a/56	237	PCR ^b	[90]
mtSSU	F. solani f. sp. glycines	Fsg1	GTCTTCTAGGATGGGCTGGT	66 ^b/56	438	PCR ^c	[90]
mtSSU	F. solani f. sp. glycines	Fsg2	CATTTAATGCCTAGTCCCCTATCA	66 ^b/56	438	PCR ^c	[90]
mtSSU	F. solani f. sp. glycines	Fsg-q-1F	GATACCCAAGTAGTCTTTGCAGTAAATG	60		qPCR	[91]
		Fsg-q-1R	TTAATGCCTAGTCCCCTATCAACAT
		Fsg-q-1P	6FAM-TGAATGCCATAGGTCAGAT-MGBNFQ
mtSSU	F. solani f. sp. glycines	FSGq1	AACCCTTTGTGAACATACC	60		qPCR	[92]
		FSGq2	CCCTCCGGATCCCAG
		FSG-MGB probe	6FAM-TCTTCTAGGATGGGCTGGT-MGBNFQ
FvTox1	F. virguliforme	FV-F	GCAGGCCATGTTGGTTCTGTA	60	200	qPCR	[96]
		FV-R	GCACGTAAAGTGAGTCGTCTCATC
		FV-MGB probe	6FAM-ACTCAGCGCCCAGGA-MGBNFQ
IGS	F. virguliforme	FvIGS-F1	GGTGGTGCGGAAGGTCT	66		qPCR	[95]
		FvIGS-R3	CCCTACACCTTTCGTACCAT
		FvIGS-Probe2	6FAM-ATAGGGTAGGCGGATCTGACTTGGCG-TAMRA
IGS	F. virguliforme	F6-3	GTAAGTGAGATTTAGTCTAGGGTAGGTGAC	60		qPCR	[97]
		R6	GGGACCACCTACCCTACACCTACT
		FvPrb-3	6FAM-TTTGGTCTAGGGTAGGCCG-MGBNFQ
IGS	F. brasiliense	Fb_F2	AGGTCAGATTTGGTATAGGGTAGGTGAGA	67 ^f	130	qPCR ^d	[98]
		Fb_R2	CGGACCATCCGTCTGGGAATTT	66 ^f
		Fb_Prb1	5HEX-TGGGATGCCCT+AATTTTT+ACGG-3IABkFQ ^e	65 ^f
TEF1	F. acuminatum	FacuF	TCGCGCACTACATGTCTT	54 ^g	142	qPCR	[99]
		FacuR	AGAGAGCGATATCAATGGTGA	53 ^g
		FacuP	FAM-AACCACTGG/ZEN/ACAATAGGAAGCCGC	61 ^g
TEF1	F. graminearum	FgraF	CTCTTCCCACAAACCATTCC	53 ^g	104	qPCR	[99]
		FgraR	TACTTGAAGGAACCCTTACC	51 ^g
		FgraP	FAM-ACCACCTGT/ZEN/CAATAGGAAGCCGCC	63 ^g
TEF1	F. proliferatum	FproF	GCGTTTTTGCCCTTTCCTGT	57 ^g	123	qPCR	[99]
		FproR	AACCCAGGCGTACTTGAAGG	57 ^g
		FproP	FAM-AGGAAGCCG/ZEN/CTGAGCTCGGT	64 ^g
TEF1	F. solani	FsolF	AAACCCTCATCGCGATCTG	55 ^g	108	qPCE	[99]
		FsolR	AGTGACCGGTCTGTAGATGA	55 ^g
		FsolP	FAM-CCTGGTATC/ZEN/TCGGGCGGG	60 ^g
IGS	F. equiseti	FequiF	TGTTGGGACTCGCGGTAA	56 ^g	94	qPCR	[99]
		FequiR	GATTACCAGTAACGAGGTGTA	51 ^g
		FequiP	FAM-CACGTCGAG/ZEN/CTTCCATAGCGTAGT	60 ^g
IGS	F. oxysporum	FoxyF	CCGTCGATAGGAGTTCCGTC	57	80	qPCR	[99]
		FoxyR	TCGAACCGACCATCTCCAAG	57
		FoxyP	FAM-TGGACGGTG/ZEN/CAGGGTAGG	64

^a First is the first melting temperature in the touchdown program, the other is the last. ^b For optimal specificity, a protocol for touchdown PCR is used. For optimal sensitivity, a nested PCR is described using additional general TEF1 primers for the first round. ^c For optimal specificity, a protocol for touchdown PCR is used. For optimal sensitivity, a nested PCR is described using additional general mtDNA primers (NMS) for the first round. ^d The assay was designed for duplexing with the primer/probe set for F. virguliforme above. ^e The + sign indicates that the next base is a locked nucleic acid (LNA) residue. These are necessary because the probe covers only two nucleotides that differ between F. brasiliense and other Fusarium spp., i.e., F. virguliforme. Do not be discouraged when checking Figure 1 in [98]; there the probe sequence is given wrongly and the position is off by one base, but the sequence given here is correct. ^f Calculated primer melting temperatures. The actual annealing temperature used was not reported. ^g Calculated primer melting temperatures. All these assays were run with an annealing temperature of 60 °C.