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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 PCR amplicons: etpC (219 pb); stx1 (302 bp); stx2 (519 bp); and eae (346 bp). A. Genomic DNAs from E. coli strains: (1) EDL933, (2) CB571, (3) 86–24, (4) DH5α, (5) MG1655, and (6) W3110, were applied as the PCR templates, respectively. Reaction conditions: 1-minute primer annealing at 57°C, and 2-minute elongation step. B. Genomic DNAs from E. coli strains: (1) EDL933, (2) CB571, (3) 86–24, (4) DH5α, were applied as the PCR templates, respectively. Reaction conditions: 2-minute primer annealing at 56°C, and 3-minute elongation step. NC is a blank PCR assay. L stands for DNA Ladder.
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Figure 3: Electrophoretic analyses of PCR amplicons: etpC (219 pb); stx1 (302 bp); stx2 (519 bp); and eae (346 bp). A. Genomic DNAs from E. coli strains: (1) EDL933, (2) CB571, (3) 86–24, (4) DH5α, (5) MG1655, and (6) W3110, were applied as the PCR templates, respectively. Reaction conditions: 1-minute primer annealing at 57°C, and 2-minute elongation step. B. Genomic DNAs from E. coli strains: (1) EDL933, (2) CB571, (3) 86–24, (4) DH5α, were applied as the PCR templates, respectively. Reaction conditions: 2-minute primer annealing at 56°C, and 3-minute elongation step. NC is a blank PCR assay. L stands for DNA Ladder.

Mentions: Genomic DNAs from all E. coli strains included in this study, EHEC and non-EHEC, were applied for PCR amplification of four virulence-related sequences. Fragments with expected sizes being amplicons of the etpC, stx1, stx2 and eae genes were obtained from the EDL933 and CB571 (Fig. 3A). When the E. coli 86–24 DNA was used as a template in the PCR, predicted fragments of etpC and eae genes were amplified (Fig. 3A). Additionally, the DH5α was found to be positive for stx2. However, some parasitic bands for the amplification of etpC and stx2 sequences were observed. In order to improve the hybridization of primers and the work of the Taq polymerase, a longer time for the annealing at slightly higher temperature, and prolonged elongation was experienced. In a consequence, 2-min at 57°C, instead of 1-min at 56°C primer annealing, as well as 3- instead of 2-min elongation allowed to appreciate the optimization's effects on the PCR program with the vision of clean bands (Fig. 3B).


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 PCR amplicons: etpC (219 pb); stx1 (302 bp); stx2 (519 bp); and eae (346 bp). A. Genomic DNAs from E. coli strains: (1) EDL933, (2) CB571, (3) 86–24, (4) DH5α, (5) MG1655, and (6) W3110, were applied as the PCR templates, respectively. Reaction conditions: 1-minute primer annealing at 57°C, and 2-minute elongation step. B. Genomic DNAs from E. coli strains: (1) EDL933, (2) CB571, (3) 86–24, (4) DH5α, were applied as the PCR templates, respectively. Reaction conditions: 2-minute primer annealing at 56°C, and 3-minute elongation step. NC is a blank PCR assay. L stands for DNA Ladder.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 3: Electrophoretic analyses of PCR amplicons: etpC (219 pb); stx1 (302 bp); stx2 (519 bp); and eae (346 bp). A. Genomic DNAs from E. coli strains: (1) EDL933, (2) CB571, (3) 86–24, (4) DH5α, (5) MG1655, and (6) W3110, were applied as the PCR templates, respectively. Reaction conditions: 1-minute primer annealing at 57°C, and 2-minute elongation step. B. Genomic DNAs from E. coli strains: (1) EDL933, (2) CB571, (3) 86–24, (4) DH5α, were applied as the PCR templates, respectively. Reaction conditions: 2-minute primer annealing at 56°C, and 3-minute elongation step. NC is a blank PCR assay. L stands for DNA Ladder.
Mentions: Genomic DNAs from all E. coli strains included in this study, EHEC and non-EHEC, were applied for PCR amplification of four virulence-related sequences. Fragments with expected sizes being amplicons of the etpC, stx1, stx2 and eae genes were obtained from the EDL933 and CB571 (Fig. 3A). When the E. coli 86–24 DNA was used as a template in the PCR, predicted fragments of etpC and eae genes were amplified (Fig. 3A). Additionally, the DH5α was found to be positive for stx2. However, some parasitic bands for the amplification of etpC and stx2 sequences were observed. In order to improve the hybridization of primers and the work of the Taq polymerase, a longer time for the annealing at slightly higher temperature, and prolonged elongation was experienced. In a consequence, 2-min at 57°C, instead of 1-min at 56°C primer annealing, as well as 3- instead of 2-min elongation allowed to appreciate the optimization's effects on the PCR program with the vision of clean bands (Fig. 3B).

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