Table 1.

Primers developed in this study, except primers for sequencing and detection of stx₂ (50)^a

Gene(s), primer use, and primer	Sequence (5′–3′)b	Position	Amplicon size (bp)	Comments
stx and stx1
Sequencing
stx1-seq-F1	ATGTCATTCGCTCTGCAATAGGTAC	119–143	1,020
stx1-seq-R1	GAAGAAGAGACTGAAGATTCCATCTG	1,113–1,138
Detection
stx1-det-F1	GTACGGGGATGCAGATAAATCGC	440–462	209
stx1-det-R1	AGCAGTCATTACATAAGAACGYCCACT	622–648
Subtyping
stx1a-F1	CCTTTCCAGGTACAACAGCGGTT	362–384	478	All 6 primers can be used in a triplex PCR for subtyping of stx/stx1c
stx1a-R2	GGAAACTCATCAGATGCCATTCTGG	815–839
stx1c-F1	CCTTTCCTGGTACAACTGCGGTT	362–384	252
stx1c-R1	CAAGTGTTGTACGAAATCCCCTCTGA	588–613
stx1d-F1	CAGTTAATGCGATTGCTAAGGAGTTTACC	50–78	203
stx1d-R2	CTCTTCCTCTGGTTCTAACCCCATGATA	225–252
stx2
Sequencing and detection
F4	GGCACTGTCTGAAACTGCTCCTGT	606–629	627	For detection, all 4 primers can be used in one reaction; for sequencing, use F4 and R1 for all subtypes except stx2e and stx2f, which are sequenced with F4-f and R1-e/f
R1	ATTAAACTGCACTTCAGCAAATCC	1,209–1,232
F4-f	CGCTGTCTGAGGCATCTCCGCT	606–629	625
R1-e/f	TAAACTTCACCTGGGCAAAGCC	1,209–1,230
Subtyping
stx2a-F2	GCGATACTGRGBACTGTGGCC	754–774
stx2a-R3	CCGKCAACCTTCACTGTAAATGTG	1,079–1,102	349
stx2a-R2	GCCACCTTCACTGTGAATGTG	1,079–1,100	347
stx2b-F1	AAATATGAAGAAGATATTTGTAGCGGC	968–994	251
stx2b-R1	CAGCAAATCCTGAACCTGACG	1,198–1,218
stx2c-F1	GAAAGTCACAGTTTTTATATACAACGGGTA	926–955	177
stx2c-R2	CCGGCCACYTTTACTGTGAATGTA	1,079–1,102
stx2d-F1	AAARTCACAGTCTTTATATACAACGGGTG	927–955
stx2d-R1	TTYCCGGCCACTTTTACTGTG	1,085–1,105	179d
stx2d-O55-R	TCAACCGAGCACTTTGCAGTAG	1,140–1,161	235
stx2d-R2	GCCTGATGCACAGGTACTGGAC	1,184–1,206	280
stx2e-F1	CGGAGTATCGGGGAGAGGC	695–713	411
stx2e-R2	CTTCCTGACACCTTCACAGTAAAGGT	1,080–1,105
stx2f-F1	TGGGCGTCATTCACTGGTTG	451–475	424
stx2f-R1	TAATGGCCGCCCTGTCTCC	856–874
stx2g-F1	CACCGGGTAGTTATATTTCTGTGGATATC	203–231	573
stx2g-R1	GATGGCAATTCAGAATAACCGCT	771–793

^aPCR conditions are as described in the text below, except annealing temperatures, which were 56°C for sequencing and detection and 64°C to 66°C for the subtyping of stx/stx₁ or stx₂. Especially, the resolution of stx_2a, stx_2c, and stx_2d may require individual calibration of thermocyclers. A well-defined single colony is inoculated in beef broth and incubated overnight at 37°C. One hundred microliters of broth is added to 900 μl of sterile H₂O, placed in a heating block at 100°C for 15 min, and centrifuged at 18,000 × g for 5 min. Upon transfer to a clean tube, the supernatant is used directly for PCR and stored at −18°C for further analyses. For PCR, a total volume of 20 μl contains 2.5 μl H₂O, 10 μl HotStarTaq Master Mix Kit (Qiagen), 1.25 μl of each of two primers (stock solution of primers is 5 μM) and 5 μl supernatant of boiled lysate (stock). The thermocycler conditions are 95°C for 15 min followed by 35 cycles of 94°C for 50 s, 56°C for sequencing and detection, and 64°C for subtyping for 40 s and 72°C for 60 s, ending with 72°C for 3 min. PCR amplicons are stored at 4°C. The final resolution of stx_2a, stx_2c, and stx_2d may require calibration of individual brands of thermocyclers by testing annealing temperatures from 64°C to 66°C on the test panel of reference strains. In our hands, an additional PCR using the stx_2d primers was run at an annealing temperature of 66°C. False-positive stx_2c fragments disappeared and true stx_2d-positive fragments persisted at this annealing temperature. A total volume (20 μl) for standard PCR contains 2.5 μl H₂O [if three primers are used (stx_2a), the H₂O volume is reduced to 1.25 μl; if four primers are used (stx_2d or detection of all stx₂ variants), H₂O is not added]; 10 μl Mastermix (HotStarTaq, Qiagen); 1.25 μl of each of two primers (stock solution of primers is 5 μM) [if three primers are used (stx_2a), the H₂O volume is reduced to 1.25 μl; if four primers are used (stx_2d or detection of all stx₂ variants), H₂O is not added]; and 5 μl supernatant of boiled lysate (stock).

^bWobble bases are shown in bold.

^cFor triplex PCR for subtyping of stx₁, a total volume of 25 μl contains 12 μl Mastermix (HotStarTaq, Qiagen), 1 μl of each of the four primers for stx_1c and stx_1d (stock solution of primers is 5 μM), 2 μl of each of two primers for stx_1a (stock solution of primers is 5 μM), and 5 μl supernatant of boiled lysate (stock).

^dAll three reverse primers in the same reaction will result in amplicons of 179 bp with nine stx_2d variants, 235 bp with variant stx_2d-O55-5905, 280 bp with five stx_2d variants, and finally two amplicons of 179 bp and 280 bp with variant stx_2d-O73-C165-02.