Sample processing for DNA chip array-based analysis of enterohemorrhagic Escherichia coli (EHEC)
1 School of Biotechnology, Royal Institute of Technology (KTH), S-10691 Stockholm, Sweden
2 Department of Molecular Biology, University of Gdansk, PL-80822 Gdansk, Poland
3 Laboratory of Molecular Biology (affiliated with the University of Gdansk), Institute of Biochemistry and Biophysics, Polish Academy of Sciences, PL-80822 Gdansk, Poland
Microbial Cell Factories 2008, 7:29 doi:10.1186/1475-2859-7-29Published: 13 October 2008
Exploitation of DNA-based analyses of microbial pathogens, and especially simultaneous typing of several virulence-related genes in bacteria is becoming an important objective of public health these days.
A procedure for sample processing for a confirmative analysis of enterohemorrhagic Escherichia coli (EHEC) on a single colony with DNA chip array was developed and is reported here. The protocol includes application of fragmented genomic DNA from ultrasonicated colonies. The sample processing comprises first 2.5 min of ultrasonic treatment, DNA extraction (2×), and afterwards additional 5 min ultrasonication. Thus, the total sample preparation time for a confirmative analysis of EHEC is nearly 10 min. Additionally, bioinformatic revisions were performed in order to design PCR primers and array probes specific to most conservative regions of the EHEC-associated genes. Six strains with distinct pathogenic properties were selected for this study. At last, the EHEC chip array for a parallel and simultaneous detection of genes etpC-stx1-stx2-eae was designed and examined. This should permit to sense all currently accessible variants of the selected sequences in EHEC types and subtypes.
In order to implement the DNA chip array-based analysis for direct EHEC detection the sample processing was established in course of this work. However, this sample preparation mode may also be applied to other types of EHEC DNA-based sensing systems.