Microarray analysis of Escherichia coli O157:H7

Table 1 PCR primers and oligonucleotide probes for O157:H7 microarray

Targetgene	Primer sequences forPCR amplification	Labeledprimer	PCR productsize (bp)	Oligonucleotides targetadsorbed by array chip	GenBank No. ofBLAST search
O157	L5’AACGGTTGCTCTTCATTTAG3’	R	678	GGTGGAATGGTTGTCACGAATGACAAA	AE005429, AP002559, AB008676
(rfbE)	R5’GAGACCATCCAATAAGTGTG3’				AF061251 S83460
H7	L5’TACCACCAAATCTACTGCTG3’	R	560	GACGATGCAGGCAACTTGACGACTAA	AE005415, AF228496, U47614
(fliC)	R5’TACCACCTTTATCATCCACA3’				AP002559, AB028474, L07388
SLT1	L5’TGTAACTGGAAAGGTGGAGTATAC3’	R	210	GGATGACGTAACCATTAAAACTAATGCC	AY123842, AF461172,AJ251754,
	R5’GCTATTCTGAGTCAACGAAAAATAAC3’				AB071624, AE005442,X07903
SLT2	L5’TTTACGATAGACTTCTCGAC3’	L	228	AGATGGTCAAAACGCGCCTGATAGAC	AF329817, AE005296, AJ272135
	R5’CACATATAAATTATTTCGCTC3’				AB052227, AP002554, Y10775

Table 2 Microchip design and spotting map

16 3×SSC	○	○	○	○	●	○	○	○	○	●	8 Vibrio cholerae ompW
15 3×SSC	○	○	○	○	●	○	○	○	○	●	7 Schistosoma japonicum
14 random probe	○	○	○	○	●	○	○	○	○	●	6 Plasmodium V
13 SLT1 gene	○	○	○	○	●	○	○	○	○	●	5 Plasmodium F
12 SLT2 gene	○	○	○	○	●	○	○	○	○	●	4 Leptospira interrogans
11 fliC gene (H7)	○	○	○	○	●	○	○	○	○	●	3 Rickettsia tsutsugamushi
10 rfbE gene (O157)	○	○	○	○	●	○	○	○	○	●	2 HFRSV 1
9 Vibrio cholerae ctx	○	○	○	○	●	○	○	○	○	●	1 HFRSV 2

○: Detect probe, ●: Quality control probe.

Table 3 Results of multiplex PCR and microchip analysis by four primers and probes

Strain	PCR				Chipping
	rfbE	fliC	SLT1	SLT2	rfbE	fliC	SLT1	SLT2
O157:H7 933	+	+	+		+	+	+	+
O157:H7 882364	+	+	+		+	+	+	+
O157:H19 44752	+	-	-	-	+	-	-	-
O1:H7 44766	-	+	-	-	-	+	-	-
O119:H6 44146	-	-	-	-	-	-	-	-
Vibrio cholerae16017	-	-	-	-	-	-	-	-
Shigella dysenteriae51252	-	-	-	-	-	-	-	-
Shigella flexneri51302	-	-	-	-	-	-	-	-
Shigella boydii51315	-	--	-	-	-	-	-	-
Shigella sonnei51334	-	-	-	-	-	-	-	-
Salmonella 50087	-	-	-	-	-	-	-	-
DH5a	-	-	-	-	-	-	-	-