Limits...
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 multiplex PCR amplicons: etpC (219 pb); stx1 (302 bp); stx2 (519 bp); and eae (346 bp). A. Temperature gradient of annealing step in the multiplex PCR using E. coli EDL933 as DNA template. Line 1 presents the anealing temperature of 55°C; line 2: 55.3°C; line 3: 55.8°C; line 4: 56.7°C; line 5: 57.8°C; line 6: 59.3°C; line 7: 61°C; line 8: 62.4°C; line 9: 63.5°C; line 10: 64.3°C; and line 11: 65°C. For all and 3-minute elongation step was performed.B. 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: 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
getmorefigures.php?uid=PMC2572036&req=5

Figure 4: Electrophoretic analyses of multiplex PCR amplicons: etpC (219 pb); stx1 (302 bp); stx2 (519 bp); and eae (346 bp). A. Temperature gradient of annealing step in the multiplex PCR using E. coli EDL933 as DNA template. Line 1 presents the anealing temperature of 55°C; line 2: 55.3°C; line 3: 55.8°C; line 4: 56.7°C; line 5: 57.8°C; line 6: 59.3°C; line 7: 61°C; line 8: 62.4°C; line 9: 63.5°C; line 10: 64.3°C; and line 11: 65°C. For all and 3-minute elongation step was performed.B. 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: 2-minute primer annealing at 56°C, and 3-minute elongation step. NC is a blank PCR assay. L stands for DNA Ladder.

Mentions: In addition, amplification reaction conditions were determined to elaborate a multiplex PCR test allowing a quick typing of EHEC (Fig. 4). An annealing temperature gradient from 55 to 65°C was realized (Fig. 4A). No parasitic bands were seen with the increase of the annealing temperature. This analysis revealed that the 56°C as annealing temperature in the PCR assay was appropriate, so in consequence used to test all strains with the multiplex mix (Fig. 4B).


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 multiplex PCR amplicons: etpC (219 pb); stx1 (302 bp); stx2 (519 bp); and eae (346 bp). A. Temperature gradient of annealing step in the multiplex PCR using E. coli EDL933 as DNA template. Line 1 presents the anealing temperature of 55°C; line 2: 55.3°C; line 3: 55.8°C; line 4: 56.7°C; line 5: 57.8°C; line 6: 59.3°C; line 7: 61°C; line 8: 62.4°C; line 9: 63.5°C; line 10: 64.3°C; and line 11: 65°C. For all and 3-minute elongation step was performed.B. 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: 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 4: Electrophoretic analyses of multiplex PCR amplicons: etpC (219 pb); stx1 (302 bp); stx2 (519 bp); and eae (346 bp). A. Temperature gradient of annealing step in the multiplex PCR using E. coli EDL933 as DNA template. Line 1 presents the anealing temperature of 55°C; line 2: 55.3°C; line 3: 55.8°C; line 4: 56.7°C; line 5: 57.8°C; line 6: 59.3°C; line 7: 61°C; line 8: 62.4°C; line 9: 63.5°C; line 10: 64.3°C; and line 11: 65°C. For all and 3-minute elongation step was performed.B. 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: 2-minute primer annealing at 56°C, and 3-minute elongation step. NC is a blank PCR assay. L stands for DNA Ladder.
Mentions: In addition, amplification reaction conditions were determined to elaborate a multiplex PCR test allowing a quick typing of EHEC (Fig. 4). An annealing temperature gradient from 55 to 65°C was realized (Fig. 4A). No parasitic bands were seen with the increase of the annealing temperature. This analysis revealed that the 56°C as annealing temperature in the PCR assay was appropriate, so in consequence used to test all strains with the multiplex mix (Fig. 4B).

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