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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

Kinetics of cell disruption by ultrasonication as shown by a histogram of forward scatter values from E. coli EDL933 cells subjected to 0 – 300 sec ultrasonication.
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Figure 1: Kinetics of cell disruption by ultrasonication as shown by a histogram of forward scatter values from E. coli EDL933 cells subjected to 0 – 300 sec ultrasonication.

Mentions: To evaluate the cell disruption during ultrasonication, samples containing 1 × 108 cells (corresponding to one agar colony) were subjected to ultrasonic disintegration followed by flow cytometry analysis (Fig. 1). The forward scatter profiles obtained for each sample are shown. At first, one broad peak with a strong signal representing non-disrupted cells was visible. With increasing ultrasonication time, this signal progressively became weaker and most of the main peak corresponding to the undisrupted cells disappeared after 150 sec sonication. Thus, the 2.5-minute sonicated sample was selected for further handling.


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)

Kinetics of cell disruption by ultrasonication as shown by a histogram of forward scatter values from E. coli EDL933 cells subjected to 0 – 300 sec ultrasonication.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 1: Kinetics of cell disruption by ultrasonication as shown by a histogram of forward scatter values from E. coli EDL933 cells subjected to 0 – 300 sec ultrasonication.
Mentions: To evaluate the cell disruption during ultrasonication, samples containing 1 × 108 cells (corresponding to one agar colony) were subjected to ultrasonic disintegration followed by flow cytometry analysis (Fig. 1). The forward scatter profiles obtained for each sample are shown. At first, one broad peak with a strong signal representing non-disrupted cells was visible. With increasing ultrasonication time, this signal progressively became weaker and most of the main peak corresponding to the undisrupted cells disappeared after 150 sec sonication. Thus, the 2.5-minute sonicated sample was selected for further handling.

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