Detecting and genotyping Escherichia coli O157:H7 using multiplexed PCR and nucleic acid microarrays.
Rapid detection and characterization of food borne pathogens such as Escherichia coli O157:H7 is crucial for epidemiological investigations and food safety surveillance. As an alternative to conventional technologies, we examined the sensitivity and specificity of nucleic acid microarrays for detecting and genotyping E. coli O157:H7. The array was composed of oligonucleotide probes (25-30 mer) complementary to four virulence loci (intimin, Shiga-like toxins I and II, and hemolysin A). Target DNA was amplified from whole cells or from purified DNA via single or multiplexed polymerase chain reaction (PCR), and PCR products were hybridized to the array without further modification or purification. The array was 32-fold more sensitive than gel electrophoresis and capable of detecting amplification products from < 1 cell equivalent of genomic DNA (1 fg). Immunomagnetic capture, PCR and a microarray were subsequently used to detect 55 CFU ml(-1) (E. coli O157:H7) from chicken rinsate without the aid of pre-enrichment. Four isolates of E. coli O157:H7 and one isolate of O91:H2, for which genotypic data were available, were unambiguously genotyped with this array. Glass-based microarrays are relatively simple to construct and provide a rapid and sensitive means to detect multiplexed PCR products; the system is amenable to automation.
Call
D R
DR
Environmental Microbiology, Pacific Northwest National Laboratory, Richland, WA 99352, USA. drcall@wsu.edu
Brockman
F J
FJ
Chandler
D P
DP
D000818:Animals; D002645:Chickens / Q000382:microbiology*; D015169:Colony Count, Microbial; D004269:DNA, Bacterial / Q000032:analysis*; D004587:Electrophoresis, Agar Gel; D019453:Escherichia coli O157 / Q000145:classification* / Q000235:genetics / Q000302:isolation & purification*; D005516:Food Microbiology; D005784:Gene Amplification; D005838:Genotype; D020411:Oligonucleotide Array Sequence Analysis / Q000379:methods; D016133:Polymerase Chain Reaction / Q000379:methods; D012680:Sensitivity and Specificity
D004269:DNA, Bacterial