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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

EHEC DNA chip array assessment. Four individual assays, A1, A2, A3 and A4, were serially conducted with four various products of simplex PCR, etpC, stx1, stx2 and eae, respectively, used as targets, and with corresponding detection probes (10 nM). 0.4 nM of each purified PCR amplicon was sequentially applied onto the chip array functionalized with capture probes. Each column is an average of three independent determinations. NC is non-biotinylated negative control capture probe of acoC of B. subtilis.
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Figure 5: EHEC DNA chip array assessment. Four individual assays, A1, A2, A3 and A4, were serially conducted with four various products of simplex PCR, etpC, stx1, stx2 and eae, respectively, used as targets, and with corresponding detection probes (10 nM). 0.4 nM of each purified PCR amplicon was sequentially applied onto the chip array functionalized with capture probes. Each column is an average of three independent determinations. NC is non-biotinylated negative control capture probe of acoC of B. subtilis.

Mentions: Capture oligonucleotides, i.e. O157 cp, Stx1 cp, Stx2 cp, and Eae cp (see Table 1) were designed and afterwards immobilized on randomly chosen positions of the chip array. Detection probes, i.e. O157 dp, Stx1 dp, Stx2 dp, and Eae dp (see Table 1) labeled with a biotin at the 3' end were selected to bind adjacent to the capture region of the target. Additionally, four array positions with negative control probe relevant for target sequence of B. subtilis were used for validation of the probes' specificity and assay performance. The EHEC chip arrays were used for hybridization assays, A1, A2, A3 and A4, with corresponding amplicons of etpC, stx1, stx2 and eae, products of simplex PCR. 0.4 nM of each purified PCR amplicon was sequentially applied to the chip test (Fig. 5). Basically, in each particular assay only specific signals were generated from the target corresponding positions. None of the spots resulted in a signal after exposure to the non-relevant PCR amplicon, indicating that no significant unspecific binding occurred. Also, no cross-reactions were observed for the negative control positions.


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)

EHEC DNA chip array assessment. Four individual assays, A1, A2, A3 and A4, were serially conducted with four various products of simplex PCR, etpC, stx1, stx2 and eae, respectively, used as targets, and with corresponding detection probes (10 nM). 0.4 nM of each purified PCR amplicon was sequentially applied onto the chip array functionalized with capture probes. Each column is an average of three independent determinations. NC is non-biotinylated negative control capture probe of acoC of B. subtilis.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 5: EHEC DNA chip array assessment. Four individual assays, A1, A2, A3 and A4, were serially conducted with four various products of simplex PCR, etpC, stx1, stx2 and eae, respectively, used as targets, and with corresponding detection probes (10 nM). 0.4 nM of each purified PCR amplicon was sequentially applied onto the chip array functionalized with capture probes. Each column is an average of three independent determinations. NC is non-biotinylated negative control capture probe of acoC of B. subtilis.
Mentions: Capture oligonucleotides, i.e. O157 cp, Stx1 cp, Stx2 cp, and Eae cp (see Table 1) were designed and afterwards immobilized on randomly chosen positions of the chip array. Detection probes, i.e. O157 dp, Stx1 dp, Stx2 dp, and Eae dp (see Table 1) labeled with a biotin at the 3' end were selected to bind adjacent to the capture region of the target. Additionally, four array positions with negative control probe relevant for target sequence of B. subtilis were used for validation of the probes' specificity and assay performance. The EHEC chip arrays were used for hybridization assays, A1, A2, A3 and A4, with corresponding amplicons of etpC, stx1, stx2 and eae, products of simplex PCR. 0.4 nM of each purified PCR amplicon was sequentially applied to the chip test (Fig. 5). Basically, in each particular assay only specific signals were generated from the target corresponding positions. None of the spots resulted in a signal after exposure to the non-relevant PCR amplicon, indicating that no significant unspecific binding occurred. Also, no cross-reactions were observed for the negative control positions.

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