Abstract
BackgroundExploitation 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.ResultsA 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.ConclusionIn 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.
Highlights
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
Enterohemorrhagic Escherichia coli (EHEC) strains comprise a subset of Shiga toxin (Verocytotoxin) – producing E. coli associated with serious endemic outbreaks [1,2,3]
It is believed that for the assessment of E. coli pathogenicity, a DNA chip array with the capacity to detect the presence of the etpC gene, the two stx genes and the eae gene should be more efficient and rapid than the ISO method
Summary
Cell number count of colony The E. coli strains, EDL933, CB571, 86–24, and DH5α were cultured on agar plates at 37°C for colony formation. Considerable work on the development of a procedure for bacterial sample processing for a confirmative analysis of EHEC, via simultaneous screening of virulence genes, on a single colony with DNA chip array was made. The use of ultrasound itself to extract DNA from cells is not a new issue, in this work, the procedure comprising ultrasonication implemented for the DNA fragmentation was developed and optimized with the intention to prepare samples for DNA hybridization on a chip array. The sample processing for the performance of DNA chip array-based confirmative analysis of enterohemorrhagic Escherichia coli (EHEC) on a single colony was demonstrated. The sample processing method will be implemented and tested in utilization of EHEC DNA chip array-based analysis performed directly on bacterial cells. It is worth mentioning that this sample preparation mode might be applied to other types of EHEC DNA-based sensing systems
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