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Molecular characterisation of verocytoxigenic Escherichia coli O157:H7 by random amplification of polymorphic DNA (RAPD) typing

For the past two decades, polymerase chain reaction (PCR)based genotyping methods have played an important role in bacterial typing schemes. One such PCR-based method, random amplification of polymorphic DNA (RAPD)-PCR, also known as arbitrarily primed-PCR (AP-PCR), has proved useful on account of its simplicity and utility for analysis of large-throughput samples. This technique utilises a variable short-length arbitrary primer and does not require previous knowledge of the target DNA sequence data. The primer is amplified arbitrarily at low stringency, where the oligonucleotide binds at complementary and partially mismatched sites and generates bands that differ in length and nucleotide composition. The RAPD-PCR methodology has been applied successfully for typing a wide variety of Gram-positive and Gram-negative bacteria. Various genus-specific and universal primers have been used, including the enterobacterial repetitive intergenic consensus (ERIC) sequence and M13 primers, and have successfully typed Gram-positive bacteria (e.g., methicillin-resistant Staphylococcus aureus [MRSA]) and Gram-negative organisms (e.g., Pseudomonas aeruginosa). Therefore, the aim of this study is to examine use of the arbitrary primer M13 to genotype verocytotoxigenic Escherichia coli clinical isolates from Northern Ireland as a rapid means of strain differentiation and as an alternative to pulsed-field gel electrophoresis (PFGE). Isolates of E. coli O157:H7 (n=48) were used in the study, all of which were from clinical faecal specimens collected by the Northern Ireland Public Health Laboratory over a twoyear period All E. coli O157 isolates were cultured on Columbia blood agar at 37 ̊C overnight and passaged at least twice before DNA extraction. All DNA isolation procedures were carried out in a class II biological safety cabinet (MicroFlow, England) in a room physically separate from that used to set up nucleic acid amplification reaction mixes and also from the ‘post-PCR’ room, in accordance with the good molecular diagnostic procedure (GMDP) guidelines of Millar et al., in order to minimise contamination and hence the possibility of false-positive results. Genomic DNA from a single overnight colony of all E. coli isolates was extracted using the High Pure PCR Template Preparation Kit (Roche, England), in accordance with the manufacturer’s instructions, and was quantified using the Ultrospec 2100 pro UV/visible spectrometer (Amersham Pharmacia Biotech, UK). All genomic DNA was within the concentration range 112–366 ng/μL and the purity coefficient of 2.01–2.13. Extracted DNA samples were visualised on 1% (w/v) agarose gel to access qualitatively the quality of the DNA. BIOMEDICAL SCIENCE IN BRIEF 161