Detecting and genotyping Escherichia coli O157 : H7 using multiplexed PCR and nucleic acid microarrays

Rapid detection and characterization of food borne pathogens such as Escher ichia coli O157:H7 is crucial for epidemiological investigations and food s afety surveillance. As an alternative to conventional technologies, we exam ined the sensitivity and specificity of nucleic acid microarrays for detect ing and genotyping E. coli O157:H7. The array was composed of oligonucleoti de probes (25-30 mer) complementary to four virulence loci (intimin, Shiga- like toxins I and Il, and hemolysin A). Target DNA was amplified from whole cells or from purified DNA via single or multiplexed polymerase chain reac tion (PCR), and PCR products were hybridized to the array without further m odification or purification. The array was 32-fold more sensitive than gel electrophoresis and capable of detecting amplification products from < 1 ce ll equivalent of genomic DNA (1 fg). Immunomagnetic capture, PCR and a micr oarray were subsequently used to detect 55 CFU ml(-1) (E. coli O157:H7) fro m chicken rinsate without the aid of pre-enrichment. Four isolates of E, co li O157:H7 and one isolate of O91:H2, for which genotypic data were availab le, were unambiguously genotyped with this array. Glass-based microarrays a re relatively simple to construct and provide a rapid and sensitive means t o detect multiplexed PCR products; the system is amenable to automation. (C ) 2001 SOCIETY. Published by Elsevier Science B.V. All rights reserved.