Table 2. Primers used for PCR and sequence and fragment analyses for hmbR and hpuA.

Target	PCR/Seq/fraga	Primer ID (direction)	Primer sequence (5′ to 3′)	Reference	Approx size (bases)
hmbR	PCR/Seq/fragb	hmbR-RF3 (fwd)	TGCCAACCTCTTTTACGAATGG	[25]	400
		hmbR-RF4 (rev)	GCTACTGAACACGTCGTTCC	[35]
	PCRc	hmbRzF (fwd)	CCACA(A/G)CTT(C/T)TTGGGTAAGATTGC	This study	1000
		hmbRzR (rev)	GACGCTACTTTGTCCACATTCAGACG	This study
	PCRd	hmbReF (fwd)	AAAT(C/T)AACGA(C/T)AACCACCGCATCG	This study	600
		hmbRyR (rev)	GGCATTCAATTCCTGAGGCGTCA	This study
	PCRe	exl3-seqF (fwd)	GGCGGAGTGCAAAATGATGC	[15]	600
		exl3-seqR (rev)	GCCATCTTTTAATTTAGCCGC	[15]
hpuA	PCR/Seq/fragb	hpuAC (fwd)	ATGCGATGAAATACAAAGCCC	[25]	350
		hpuA350Rev (rev)	GGATGAAAGGGCGTATTGCGC	[25]
	PCRf	hpuAFnest2 (fwd)	CAAATCCGCCAACGAAGCGAT(C/T)AA	This study	2000
		P26.85 (rev)	GGGAAACGCTTGGGCGATGG	[36]
	PCRg	Hpu-pmt (fwd)	CCGATTTTTGCACCGACCCAC	This study	600
		hpuR-Seq3 (rev)	GAGGTCGATTTCGCCGTTGG	This study
	PCRh	hpuA-for1 (fwd)	GCAACAATGCCTTGTCATCC	[16]	1000–3000
		hpuA-rev13 (rev)	TGATCGAAATGGGCGTACTC	[16]

^aPurpose; conditions

^bCharacterisation of homopolymeric tract and closely flanking regions; PCR - [MgCl₂] = 2.5 mM, 25 (up to 45 for direct non-culture fragment analysis) cycles of (95°C – 30 seconds, 53°C – 30 seconds, 72°C – 60 seconds). Sequence analysis annealing temperature  = 53°C. For fragment analysis, primers hmbR-RF3 and hpuA350Rev were FAM-labeled.

^c‘Round 1’ nested PCR for non-culture characterization of homopolymeric tract and closely flanking regions; [MgCl₂] = 3 mM, 45 cycles of (95°C – 30 seconds, 56°C – 30 seconds, 72°C – 60 seconds).

d‘Round 2’ nested PCR for non-culture characterization of homopolymeric tract and closely flanking regions; [MgCl₂] = 3 mM, 25 cycles of (95°C – 30 seconds, 56°C – 30 seconds, 72°C – 60 seconds).

eConfirmation of presence of exl3 (that replaces hmbR); [MgCl₂] = 3 mM, 35 cycles of (95°C for 30 seconds, 56°C for 30 seconds, 72°C for 60 seconds).

f’Round 1’ nested PCR for non-culture characterization of homopolymeric tract and closely flanking regions; [MgCl₂] = 3 mM, 45 cycles of (95°C – 30 seconds, 57°C – 30 seconds, 72°C – 150 seconds).

g‘Round 2’ nested PCR for non-culture characterization of homopolymeric tract and closely flanking regions; [MgCl₂] = 3 mM, 25 cycles of (95°C – 30 seconds, 59°C – 30 seconds, 72°C – 60 seconds).

^hConfirmation of absence of hpuA; [MgCl₂] = 3 mM, 35 cycles (95°C – 30 seconds, 53°C – 30 seconds, 72°C – 180 seconds).

Seq =  sequence analysis

Frag =  fragment analysis