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Sample processing for DNA chip array-based analysis of enterohemorrhagic Escherichia coli (EHEC).

Basselet P, Wegrzyn G, Enfors SO, Gabig-Ciminska M - Microb. Cell Fact. (2008)

Bottom Line: Additionally, bioinformatic revisions were performed in order to design PCR primers and array probes specific to most conservative regions of the EHEC-associated genes.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: School of Biotechnology, Royal Institute of Technology (KTH), S-10691 Stockholm, Sweden. gabig@biotech.kth.se.

ABSTRACT

Background: 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.

Results: 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 (2x), 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.

Conclusion: 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.

No MeSH data available.


Related in: MedlinePlus

Electrophoretic analyses of distribution of genomic DNA of E. coli EDL933 subjected to 0 – 15 min ultrasonication. L indicates the DNA Ladder.
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Figure 2: Electrophoretic analyses of distribution of genomic DNA of E. coli EDL933 subjected to 0 – 15 min ultrasonication. L indicates the DNA Ladder.

Mentions: In the next test, the samples sonicated previously for 2.5-min were subjected to an extraction step after heat treatment and centrifugation (see Methods). DNA released was extracted two times with a phenol:chloroform:isoamyl alcohol mix, and afterwards subjected to second sonication for 0 – 15 min. Agarose gel electrophoresis was used to determine the size distribution of DNA subjected to post-extraction ultrasonic fragmentation (Fig. 2). Highly fragmented DNA was evident from the presence of a DNA smear rather than high-molecular weight bands that were eliminated from samples sonicated for 2.5 min or longer. Longer sonication gradually reduced fragment lengths to approximately 150 – 600 bp, and sonication for 15 min further degraded these fragments, as can be seen mostly by the upper part of the smear. Thus, the average DNA fragment size gradually declined with ultrasonication time and the 5 min treatment allowed to obtain the sizes of DNA fragments most suitable for chip array assays [as proved previously by 22,23]. At last, the DNA analyte preparation procedure comprising first 2 min of ultrasonic treatment, DNA extraction (2×), and subsequent 5 min sonication, was established.


Sample processing for DNA chip array-based analysis of enterohemorrhagic Escherichia coli (EHEC).

Basselet P, Wegrzyn G, Enfors SO, Gabig-Ciminska M - Microb. Cell Fact. (2008)

Electrophoretic analyses of distribution of genomic DNA of E. coli EDL933 subjected to 0 – 15 min ultrasonication. L indicates the DNA Ladder.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2572036&req=5

Figure 2: Electrophoretic analyses of distribution of genomic DNA of E. coli EDL933 subjected to 0 – 15 min ultrasonication. L indicates the DNA Ladder.
Mentions: In the next test, the samples sonicated previously for 2.5-min were subjected to an extraction step after heat treatment and centrifugation (see Methods). DNA released was extracted two times with a phenol:chloroform:isoamyl alcohol mix, and afterwards subjected to second sonication for 0 – 15 min. Agarose gel electrophoresis was used to determine the size distribution of DNA subjected to post-extraction ultrasonic fragmentation (Fig. 2). Highly fragmented DNA was evident from the presence of a DNA smear rather than high-molecular weight bands that were eliminated from samples sonicated for 2.5 min or longer. Longer sonication gradually reduced fragment lengths to approximately 150 – 600 bp, and sonication for 15 min further degraded these fragments, as can be seen mostly by the upper part of the smear. Thus, the average DNA fragment size gradually declined with ultrasonication time and the 5 min treatment allowed to obtain the sizes of DNA fragments most suitable for chip array assays [as proved previously by 22,23]. At last, the DNA analyte preparation procedure comprising first 2 min of ultrasonic treatment, DNA extraction (2×), and subsequent 5 min sonication, was established.

Bottom Line: Additionally, bioinformatic revisions were performed in order to design PCR primers and array probes specific to most conservative regions of the EHEC-associated genes.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: School of Biotechnology, Royal Institute of Technology (KTH), S-10691 Stockholm, Sweden. gabig@biotech.kth.se.

ABSTRACT

Background: 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.

Results: 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 (2x), 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.

Conclusion: 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.

No MeSH data available.


Related in: MedlinePlus