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

Journal Article · Thu Jul 05 00:00:00 EDT 2001 · International Journal of Food Microbiology, 67(1-2):71-80

DOI:https://doi.org/10.1016/S0168-1605(01)00437-8· OSTI ID:993682

Call, Douglas R; Brockman, Fred J; Chandler, Darrell P

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 CFUs 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 and the system is amenable to automation.

• Research Organization: Pacific Northwest National Lab. (PNNL), Richland, WA (United States)
• Sponsoring Organization: USDOE
• DOE Contract Number: AC05-76RL01830
• OSTI ID: 993682
• Report Number(s): PNNL-SA-35552; TRN: US201023%%565
• Journal Information: International Journal of Food Microbiology, 67(1-2):71-80, Vol. 67, Issue 1-2
• Country of Publication: United States
• Language: English

Related Subjects

59 BASIC BIOLOGICAL SCIENCES
99 GENERAL AND MISCELLANEOUS//MATHEMATICS, COMPUTING, AND INFORMATION SCIENCE
AMPLIFICATION
AUTOMATION
DETECTION
DNA
ELECTROPHORESIS
ESCHERICHIA COLI
FOOD
HEMOLYSINS
MODIFICATIONS
NUCLEIC ACIDS
OLIGONUCLEOTIDES
PATHOGENS
POLYMERASE CHAIN REACTION
PROBES
PURIFICATION
SAFETY
TARGETS
TOXINS
VIRULENCE