Genomic typing of Escherichia coli O157:H7 by semi-automated fluorescent AFLP analysis

• Published in: Microbes and infection Vol. 2; no. 2; pp. 107 - 113
• Main Authors: Zhao, Shaohua, Mitchell, Sharon E., Meng, Jianghong, Kresovich, Stephen, Doyle, Michael P., Dean, Rob E., Casa, Alexandra M., Weller, Jennifer W.
• Format: Journal Article
• Language: English
• Published: Lausanne Elsevier SAS 01.02.2000; Amsterdam Elsevier; Paris
• Subjects: AFLP; amplified fragment length polymorphism; Animals; Automation; Bacterial Typing Techniques; Bacteriological methods and techniques used in bacteriology; Bacteriology; Biological and medical sciences; Cattle; DNA fingerprinting; DNA Fingerprinting - methods; DNA Primers; DNA Restriction Enzymes; DNA, Bacterial - analysis; DNA, Bacterial - genetics; Electrophoresis, Gel, Pulsed-Field; Escherichia coli; Escherichia coli O157 - classification; Escherichia coli O157 - genetics; Escherichia coli O157 - isolation & purification; Escherichia coli O157:H7; Fluorescent Dyes; Fundamental and applied biological sciences. Psychology; Genome, Bacterial; Humans; Microbiology; Polymerase Chain Reaction; DNA fingerprinting; Escherichia coli O157:H7; AFLP; Human; Pulsed field electrophoresis; Typing; Animal; Escherichia coli; Fingerprint method; Bacteria; Genotype; Isolate; Method; Enterobacteriaceae
• ISSN: 1286-4579; 1769-714X
• DOI: 10.1016/S1286-4579(00)00278-1

Escherichia coli serotype O157:H7 isolates were analyzed using a relatively new DNA fingerprinting method, amplified fragment length polymorphism (AFLP). Total genomic DNA was digested with two restriction endonucleases ( EcoRI and MseI), and compatible oligonucleotide adapters were ligated to the ends of the resulting DNA fragments. Subsets of fragments from the total pool of cleaved DNA were then amplified by the polymerase chain reaction (PCR) using selective primers that extended beyond the adapter and restriction site sequences. One of the primers from each set was labeled with a fluorescent dye, which enabled amplified fragments to be detected and sized automatically on an automated DNA sequencer. Three AFLP primer sets generated a total of thirty-seven unique genotypes among the 48 E. coli O157:H7 isolates tested. Prior fingerprinting analysis of large restriction fragments from these same isolates by pulsed-field gel electrophoresis (PFGE) resulted in only 21 unique DNA profiles. Also, AFLP fingerprinting was successful for one DNA sample that was not typable by PFGE, presumably because of template degradation. AFLP analysis, therefore, provided greater genetic resolution and was less sensitive to DNA quality than PFGE. Consequently, this DNA typing technology should be very useful for genetic subtyping of bacterial pathogens in epidemiologic studies.