Computational prediction and experimental validation of novel markers for detection of STEC O157:H7

Table 1 Characteristics and genotypes of Shiga toxin producing Escherichia coli O157:H7 isolates

Isolate No.	Source	Sorbitol fermentation	stx1	stx2
1	Raw milk	Negative	+	+
2	Raw milk	Negative	+	+
3	Meat	Negative	+	+
4	Meat	Negative	+	+
5	Meat	Negative	+	+
6	Cattle	Negative	+	+
7	Human	Negative	+	+
8	Human	Negative	+	+
9	Human	Negative	+	+
10	Human	Negative	+	+
11	Cattle	Negative	+	+
12	Human	Negative	+	+
13	Human	Negative	+	+
14	Human	Negative	+	+
15	Human	Negative	+	+
16	Human	Negative	+	+
17	Human	Negative	+	+
18	Human	Negative	+	+

Table 2 Oligonucleotide primer sequences and parameters used for detection Shiga toxin producing Escherichia coli O157:H7

Gene	sequence (5' to 3' )	Expected	PCR condition
		length (bp)	Ta/ Te (°C)
eaeA	F-AAGCGACTGAGGTCACT	45	55/60
	R-ACGCTGCTCACTAGATGT
wzy	F-GAACGATTTCTTTCCGACACC	276	50/60
	R-GCGCAATTTATCGAGCTATG
Z037	F-AGAATCTCATCCTCGCATTT	342	52/60
	R-TCTCGCAGTTTCGCATCTTAT
Z0344	F-ATTGTCAGGGAAATTAGCGTG	121	51/60
	R-TGCTGTTAATGGTTGAACCGA

F: Forward; R: Reverse; Ta: Annealing temperature; Te: Elongation time; PCR: Polymerase chain reaction.

Table 3 Candidate marker genes for Shiga toxin producing Escherichia coli O157:H7

EDL933	Sakai	EC4115	3000 bp flanking regions				Function
			Ident %	tRNA	Tpase	phage
wzy	ECs2844	ECH74115_2973	78	-	-	-	O antigen polymerase
Z0372	ECs0334	ECH74115_0348	56	-	-	-	Hypothetical protein
Z0344	ECs0307	ECH74115_0324	50	-	-	-	Hypothetical protein
Z1539	ECs1281	ECH74115_1278	50	-	-	-	Hypothetical protein
Z3621	ECs3239	ECH74115_3589	13	-	-	-	Hypothetical protein
Z3271	ECs2909	ECH74115_3086	0	-	-	-	Hypothetical protein
Z0948	ECs0804	ECH74115_0880	9	-	-	+	Hypothetical protein
Z1328	ECs1061	ECH74115_1143	32	-	-	+	Hypothetical protein
Z1430	ECs1165	ECH74115_3572	0	-	-	+	Hypothetical protein
Z3348	ECs2979	ECH74115_3543	0	+	-	+	Hypothetical protein
Z3118	ECs2755	ECH74115_2802	0	-	-	+	Hypothetical protein
Z0244	ECs0212	ECH74115_0230	38	-	+	+	Hypothetical protein
Z1153/Z1592	ECs5413	ECH74115_1331	50	-	-	-	Hypothetical protein
Z2107/Z2378/ Z6055/Z3108	ECs1960/ ECs2748	ECH74115_2190/ ECH74115_2260/ ECH74115_2792	0	+	+	+	Hypothetical protein
Z1782/Z6064	ECs2271	ECH74115_3158/ ECH74115_1841/ ECH74115_2270/ ECH74115_1520	2	-	-	+	Hypothetical protein

“Ident %” is the identity percentage of 3000-bp flanking regions among the three genomes. “+” and “-“ in column “tRNA”, “Tpase” and “phage” represent if the 3000-bp flanking regions contain any tRNA, transposase or phage. The gene names and their functions were retrieved from the NCBI Prokaryotic Genome Database, as described in materials and methods.