Table 3. Primers and probes used in this study.

A, Primers prnAF (top) and prnAR (bottom) [14] were used for prn sequencing. B, C-1, ARMS-qPCR primers for the determination of the ptxA, ptxB, fimD, bvgS and tcfA alleles (B) as well as for the discrimination between the ptxP1-like and ptxP3 alleles (C-1). For each gene, the forward primer is shown on top, then the reverse primer corresponding to the Tohama I sequence and the reverse primer corresponding to the alternative allele, with the base at the SNP site underlined, and finally the labelled hydrolysis probe. Lower case letters, artificial base replacement with both sequences at the SNP site; FAM, 6-carboxyfluorescein; TET, 4,5,6,7-tetrachlorofluorescein; BHQ1, Black Hole Quencher-1; Cy5, red sulfoindocyanine dye. C-2, oligonucleotides for the fimD non-discriminatory control assay.

Gene	5’ Nucleotide position 1	Sequence	Length [bp]	Tm [°C]	Amplicon length [bp]
A: PCR primers for sequence analysis of prn alleles [14]
prn	55187	GCCAATGTCACGGTCCAA	18	57	586
	55772	GCAAGGTGATCGACAGGG	18	56
B: Oligonucleotides for typing the ptxA, ptxB, fimD, and tcfA alleles by ARMS-qPCR
ptxA	160397	AACCCCTACACATCGCGAAGGT	22	63	73
	160469	CGCCTATCACCGGCcCC	17	63
	160469	CGCCTATCACCGGCcCT	17	60
	160445	Cy5-CCAATGTGCCGACGATCGACGC TACG-BHQ1	26	74
ptxB	160878	CCATCGTAGAGCGCAAATATTG	22	58	148(+1) 2
	160731	AACAGATTACCCAGCATGtCC	21	53
	160730	GAACAGATTACCCAGCATGtCA	22	56
	160765	FAM-CTGCGCGAACAAGACCCGTG CC-BHQ1	22	72
fimD	250435	CGGCCGCAGTCCTATGG	17	60	65(+1) 2
	250500	GGTCTCCTCCGTGGAGCaGA	20	61
	250499	GTCTCCTCCGTGGAGCaGG	19	59
	250453	FAM-CATCCGGGCATCGTGGTCGA CTTGC-BHQ1	25	74
tcfA	221437	GGTTGCCCGGTATAGGAAAGGT	22	61	134
	221570	ATCCTTGCCCGCCCaAC	17	60
	221570	ATCCTTGCCCGCCCaAT	17	60
	221513	FAM-CATGGCGTCCGGAGCGG-BHQ1	17	66
bvgS	232055	CGACTCGCTGGGCGAACT	18	61	92
	231964	CCTTGGCGTCGTGCAGaTT	19	61
	231964	CCTTGGCGTCGTGCAGaTC	19	61
	232025	FAM-CGGCGGCTGGATCGACATCA CCG-BHQ1	23	75
C: Duplex qPCR for typing of ptxP3 and ptxP1-like alleles
1 –ARMS-qPCR assay
ptxP	159628	CTACTGCAATCCAACACGGC	20	58	68(+1) 2
ptxP1-like	159695	GACGGTGACCGGTtCCA	17	57
ptxP3	159696	GGACGGTGACCGGTtCTA	18	55
	159655	TET-CTCCTTCGGCGCAAAGTCG CG-BHQ1	21	70
2 –Consensus (non-discriminatory) control assay
fimD	250434	CCGGCCGCAGTCCTATG	17	60	113
	250546	CCTTCTGCATCGGCGAACT	19	60
	250453	FAM-CATCCGGGCATCGTGGTCGA CTTGC-BHQ1	25	74

¹The nucleotide numbering refers to the numbering from the Tohama I genome [19]. Segment accession numbers: BX640414 (prn), BX640422 (ptxP, ptxA, ptxB), BX640416 (fimD and bvgS) and BX640414 (tcfA).

²The “colder”primer forming only two hydrogen bonds at the specific base has a one base extension at its 5´-end to compensate the reduced T_m.