DNA microarray for discrimination between pathogenic 0157:H7 EDL933 and non-pathogenic Escherichia coli strains.
The primary technique currently used to detect biological agents is based on immunoassays. Although sensitive and specific, currently employed immunoassays generally rely on the detection of a single epitope, and therefore often cannot discriminate subtle strain-specific differences. Since DNA microarrays can hybridize hundreds to thousands of genomic targets simultaneously and do not rely on phenotypic expression of these genetic features for identification purposes, they have enormous potential to provide inexpensive, flexible and specific strain-specific detection and identification of pathogens. In this study, pathogenic Escherichia coli O157:H7-specific genes, non-pathogenic K12-specific genes, common E. coli genes, and negative control genes were polymerase chain reaction-amplified and spotted onto the surface of treated glass slides. After labeled bacterial cDNA samples were hybridized with probes on the microarray, specific fluorescence patterns were obtained, enabling identification of pathogenic E. coli O157:H7 and non-pathogenic E. coli K12. To test the utility of this microarray device to detect genetically engineered bacteria, E. coli BL21 (a B strain derivative with antibiotic resistance gene, ampR) and E. coli JM107 (a K12 strain derivative lacking the gene ompT) were also employed. The array successfully confirmed the strain genotypes and demonstrated that antibiotic resistance can also be detected. The ability to assess multiple data points makes this array method more efficient and accurate than a typical immunoassay, which detects a single protein product.
Wu
Chi-Fang
CF
US Army Edgewood Chemical Biological Center, Aberdeen Proving Ground, MD 21010, USA.
Valdes
James J
JJ
Bentley
William E
WE
Sekowski
Jennifer W
JW
D015169:Colony Count, Microbial / Q000379:methods; D004867:Equipment Design; D019544:Equipment Failure Analysis; D004926:Escherichia coli / Q000235:genetics* / Q000302:isolation & purification* / Q000472:pathogenicity; D019453:Escherichia coli O157 / Q000235:genetics / Q000302:isolation & purification / Q000472:pathogenicity; D005819:Genetic Markers / Q000235:genetics; D020411:Oligonucleotide Array Sequence Analysis / Q000295:instrumentation* / Q000379:methods*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity
D005819:Genetic Markers