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. 2004 Mar;72(3):1812–1816. doi: 10.1128/IAI.72.3.1812-1816.2004

TABLE 2.

PCR primers, conditions, and control strains used in this study

Primer Sequence Target PCR conditions (°C, s)a
PCR product (bp) Source or reference Positive controlb (reference)
Denaturing Annealing Extension
cdtI-fc 5′-TGG TGA GAA TCG GAA CTG-3′ cdt-IA 94, 30 51, 60 72, 60 418 This study 6468/62 (22)
cdtI-rc 5′-CAT TCC ATC AGG TTT GTC-3′
cdtII-fd 5′-AAT CCC TAT CCC TGA ACC-3′ cdt-IIA 94, 30 52, 60 72, 60 542 This study 9142/88 (20)
cdtII-rd 5′-GTT CTA TTG GCT GTG GTG-3′
cdtIII-f 5′-AAACAGGACGGTAATAATGACTAATA-3′ cdt-III 94, 30 54, 60 72, 180e 2,230 4 1404 (19)
cdtIII-r 5′-GTGATCTCCTTCCATGAAAATATAGT-3′ Complete sequence
CDT-IVs 5′-CCTGATGGTTCAGGAGGCTGGTTC-3′ cdt-IVB 94, 60 55, 60 72, 60e 350 24 M375/95 (this study)
CDT-IVas 5′-TTGCTCCAGAATCTATACCT-3′
c338f 5′-AGC ATT AAA TAA AAG CAC GA-3′ cdt-VA 94, 30 52, 60 72, 60 1,329 11 493/89 (11)
c2135r 5′-TAC TTG CTG TGG TCT GCT AT-3′
c1309f 5′-AGC ACC CGC AGT ATC TTT GA-3′ cdt-VB 94, 30 52, 60 72, 60 1,363 11 493/89 (11)
c2166r 5′-AGC CTC TTT TAT CGT CTG GA-3′
P105 5′-GTC AAC GAA CAT TAG ATT AT-3′ cdt-VC 94, 30 49, 60 72, 60 748 11 493/89 (11)
c2767r 5′-ATG GTC ATG CTT TGT TAT AT-3′
c338f 5′-AGC ATT AAA TAA AAG CAC GA-3′ cdt-V 94, 30 51, 60 72, 180e 2,449 11 493/89 (11)
c2767r 5′-ATG GTC ATG CTT TGT TAT AT-3′ Complete sequence
a

PCRs were performed with 25-μl volumes as previously described (8) and included 30 cycles followed by a final extension of 5 min at 72°C unless otherwise stated.

b

The E. coli control strains 6468/62 (serotype O86:H34, allele cdt-I+), 9142/88 (O128:H−, cdt-II+), and 1404 (O78:H?, cdt-III+) were provided by D. A. Scott (University of Maryland School of Medicine, Baltimore, Md.), C. L. Pickett (University of Kentucky Medical Center, Lexington, Ky.) and E. Oswald (Ecole Nationale Veterinaire, Toulouse, France), respectively. E. coli strain M375/95 (O75, cdt-IV+) was isolated from a patient with a urinary tract infection (M. Bielaszewska, unpublished data), and the presence of cdt-IVB was verified by sequencing. E. coli strain C600 was a negative control in all PCRs.

c

The primers for the detection of cdt-IA were designed from the published cdt-I sequence (GenBank accession number U03293) (22).

d

The primers for the detection of cdt-IIA were designed from the published cdt-II sequence (GenBank accession number U04208) (20).

e

The final extension was performed at 72°C for 10 min.