Microarray analysis of Escherichia coli O157:H7.
OBJECTIVE
To establish the rapid, specific, and sensitive method for detecting O157:H7 with DNA microchips.


METHODS
Specific oligonucleotide probes (26-28 nt) of bacterial antigenic and virulent genes of E. coli O157:H7 and other related pathogen genes were pre-synthesized and immobilized on a solid support to make microchips. The four genes encoding O157 somatic antigen (rfbE), H7 flagellar antigen (fliC) and toxins (SLT1, SLT2) were monitored by multiplex PCR with four pairs of specific primers. Fluorescence-Cy3 labeled samples for hybridization were generated by PCR with Cy3-labeled single prime. Hybridization was performed for 60 min at 45 degrees. Microchip images were taken using a confocal fluorescent scanner.


RESULTS
Twelve different bacterial strains were detected with various combinations of four virulent genes. All the O157:H7 strains yielded positive results by multiplex PCR. The size of the PCR products generated with these primers varied from 210 to 678 bp. All the rfbE/fliC/SLT1/SLT2 probes specifically recognized Cy3-labeled fluorescent samples from O157:H7 strains, or strains containing O157 and H7 genes. No cross hybridization of O157:H7 fluorescent samples occurred in other probes. Non-O157:H7 pathogens failed to yield any signal under comparable conditions. If the Cy3-labeled fluorescent product of O157 single PCR was diluted 50-fold, no signal was found in agarose gel electrophoresis, but a positive signal was found in microarray hybridization.


CONCLUSIONS
Microarray analysis of O157:H7 is a rapid, specific, and efficient method for identification and detection of bacterial pathogens.
Jin
Hui-Ying
HY
Huadong Research Institute for Medicine and Biotechnics, Nanjing 210002, Jiangsu Province, China. laoyingj@hotmail.com
Tao
Kai-Hua
KH
Li
Yue-Xi
YX
Li
Fa-Qing
FQ
Li
Su-Qin
SQ
D004927:Escherichia coli Infections / Q000175:diagnosis*; D019453:Escherichia coli O157 / Q000235:genetics* / Q000302:isolation & purification* / Q000472:pathogenicity; D029968:Escherichia coli Proteins / Q000032:analysis / Q000235:genetics; D005456:Fluorescent Dyes / Q000378:metabolism; D006801:Humans; D025202:Molecular Diagnostic Techniques; D020411:Oligonucleotide Array Sequence Analysis; D012680:Sensitivity and Specificity
D029968:Escherichia coli Proteins; D005456:Fluorescent Dyes