TABLE 2.

Primers^a for PCR-based RFLP analysis and DNA sequencing, fragment sizes, and assay conditions for four genes

Gene	Method	Primers (sequences)	Size (bp)	Annealing temp (°C) (time)	Elongation temp (°C) (time)	Published sequencesb (reference)	Deposited sequences (this study)
cspA	Sequencing	F2391 (5′-CGAAGTTCCTGGTTCAGAAGATT-3′)	574	53 (30 s)	60 (2 min)		FJ752028-52115
		R2964 (5′-TACTGCAGGACGAGCTTTGAAG-3′)
gbs2018	PCRc	O1a (5′-AAAATAAACGTGGTCCTATCCTAATAAA-3′)	2,000-4,000	54 (1 min)	72 (4 min)	AM051290-94 (9); AM183357-67, -72-86 (35)	FJ752117-52155
		O2a (5′-AAAGGCAAAGTTCTGATGAGGTT-3′)
	Sequencing	Same forward as PCR (O1a)	600	54 (30 s)	60 (2 min)
scpB	PCR	F284 (5′-CAGCAACCTCAAAAGCGACTATTA-3′)	1,411	54 (1 min)	72 (1 min)	AF189002 (7)	FJ752116
		R1994 (5′-ACGGTGACTTGTTTGCTGCTATT-3′)
	Sequencingd	F907 (5′-TGATAGTAGCTTTGGGGGCAAG-3′)	1,411	56 (20 s)	60 (2 min)
		R1373 (5′-CTTGCCCCCAAAGCTACTATCA-3′)
sip	PCR	F48 (5′-GCTATTATCAGTCGCAAGTGTTCA-3′)	1,218	54 (1 min)	72 (1 min)	AF151357, -59-62 (10);	DQ914240-42, -44-51, -53-56, -59, -61-63, -65, -67-69, FJ752156-61
		R1265 (5′-GCAGTAA CGCCACCACGAT-3′)
	Sequencinge	F464 (5′-TTGTTTCGCCAATGAAGACATA-3′)	1,920	54 (30 s)	60 (2 min)
		R485 (5′-TATGTCTTCATTGGCGAAACAA-3′)
		F1133 (5′-ACTCTACACAAAATATGGCAGCAA-3′)
		R1156 (5′-TTGCTGCCATATTTTGTGTAGAGT-3′)
		F541 (5′-AGTCAAGCAGCAGCTAATGAACAG-3′)
		R564 (5′-CTGTTCATTAGCTGCTGCTTGACT-3′)

^aPrimers (except gbs2018) are labeled by the gene or locus name, F or R for forward and reverse, and location within the gene in bp from the start codon and were developed based on the NEM316 genome sequence (AL732656) (22).

^bPublished sequences from the genome strains (NEM316 [AL732656] [22], A909 [CP000114] [55], and 2603V/R [AE009948] [56]), and incomplete genome strains (H36B [AAJS00000000] [55], COH1 [AAJR00000000] [55], CJB111 [AAJQ00000000] [55], 18RS21 [AAJO00000000] [55], and 515 [AAJP00000000] [55]) were used, as were additional sequences.

^cSlightly modified primers developed in a prior study (35) were utilized to amplify a gbs2018 fragment between 2,000 and 4,000 bp depending on the number of repeats. Sequence analysis was conducted on the first 600 bp (5′ end); however, for strains sequenced in this study, only single-stranded DNA was examined.

^dSequencing of scpB utilized three primer sets: the original PCR primers, F907 and R1373, and the reverse complement of F907 and R1373. The one strain chosen for scpB sequencing yielded a PCR fragment smaller than the expected size using the original primer set; the sequenced fragment was between F284 and R1994, as there is a 128-bp deletion within the original primer site.

^esip sequencing used three primer sets; sip allele 3d required the use of a different primer set (F541 and R564) because of a deletion within the F464 and R485 binding sites.