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Electric DNA chips for determination of pathogenic microorganisms
KTH, School of Biotechnology (BIO), Bioprocess Technology.
2008 (English)Doctoral thesis, comprehensive summary (Other scientific)
Abstract [en]

Silicon-based electric DNA chip arrays were utilized to fast identify pathogenic microorganisms with respect to the capacity to produce toxins involved in foodborne poisoning and infections. Bacteria of the B. cereus and the enterohemorrhagic E. coli (EHEC) groups contain different set-ups of various virulence factors that are encoded by the corresponding genes. The purpose of this work was to develop a fast and simple method for determination of the presence of these virulence genes in a colony from primary enrichment cultures. A target gene is detected through hybridization to a surface-immobilized specific capture probe and biotin-labeled detection probe. Following binding of an enzyme conjugate to this sandwich hybrid complex, a current signal is generated by electronic redox recycling of the enzymatic product paminophenol (pAP). Two versions of the assay were developed. In the first version the capture probes were immobilized on magnetic beads, which carried out all reactions until the pAP generation, while the final electric signal was created by transferring pAP to a single-electrode chip surface. In the second version a silicon chip array with 16 parallel sensing electrode positions each of them functionalized by capture probes, carried out all assay steps on the chip surface. This instrument can realize automatic and multiplexed gene detection. The kinetics of bacterial cell disruption and impact of DNA fragmentation by ultrasound were determined. The experimental data suggested that the increased signal after first minutes of ultrasonication were due to the accumulation of released DNA amount, while the further signal increase resulted from the improved hybridization with the shortened target DNA strands. Studies on probe localization on the 16-electrode chip assays indicated that the probe-targeting site, which was located at the 5’-end of strands, gave rise to the highest signal level due to the efficient targetprobes hybridization and the following enzyme binding. When these functionalized chip arrays were exposed to the cell homogenates, the sensing electrodes were fouled by cellular proteins and therefore led to dramatically decreased redox-recycling current. To circumvent this, samples were treated by DNA extraction after the 1st sonication and then DNA fragmentation by a 2nd time sonication. The DNA extract removed most of the interfering components from bacterial cell. This sample treatment was applied to characterize one “diarrheal” and one “emetic” strain of B. cereus with the chip arrays functionalized by eight DNA probes. The signal patterns of eight virulence genes from chip assays agreed well with PCR control analyses for both strains. By simply adding the SDS detergent to cell homogenates, chip surface blocking effect can be significantly reduced even without DNA extraction treatment. After optimization of some critical factors, the 16-electrode DNA chips with the improved sensing performance can directly detect multiple virulence genes from a single E. coli colony in 25 min after the introduction of supernatant of ultrasonicated cell lysate.

Place, publisher, year, edition, pages
Stockholm: KTH , 2008. , 53 p.
Series
Trita-BIO-Report, ISSN 1654-2312 ; 2008:16
Keyword [en]
electric DNA chip array, fast determination, virulence genes, multiplexed gene detection, bacterial colony, ultrasonication, DNA fragmentation, Bacillus cereus
National Category
Biological Sciences Industrial Biotechnology
Identifiers
URN: urn:nbn:se:kth:diva-9187ISBN: 978-91-7415-106-0 (print)OAI: oai:DiVA.org:kth-9187DiVA: diva2:31693
Public defence
2008-10-09, FA31, AlbaNova, Roslagstullsbacken, Stockholm, 10:00 (English)
Opponent
Supervisors
Note
QC 20100824Available from: 2008-10-02 Created: 2008-10-02 Last updated: 2010-08-24Bibliographically approved
List of papers
1. Gene-based identification of bacterial colonies with an electric chip
Open this publication in new window or tab >>Gene-based identification of bacterial colonies with an electric chip
2005 (English)In: Analytical Biochemistry, ISSN 0003-2697, E-ISSN 1096-0309, Vol. 345, no 2, 270-276 p.Article in journal (Refereed) Published
Abstract [en]

A method for the identification of bacterial colonies based on their content of specific genes is presented. This method does not depend on DNA separation or DNA amplification. Bacillus cereus carrying one of the genes (hblC) coding for the enterotoxin hemolysin was identified with this method. It is based on target DNA hybridization to a capturing probe immobilized on magnetic beads, followed by enzymatic labeling and measurement of the enzyme product with a silicon-based chip. An hblC-positive colony containing 10(7) cells could be assayed in 30 min after ultrasonication and centrifugation. The importance of optimizing the ultrasonication is illustrated by analysis of cell disruption kinetics and DNA fragmentation. An early endpoint PCR analysis was used to characterize the DNA fragmentation as a function of ultrasonication time. The first minutes of sonication increased the signal due to both increased DNA release and increased DNA fragmentation. The latter is assumed to increase the signal due to improved diffusion and faster hybridization of the target DNA. Too long sonication decreased the signal, presumably due to loss of hybridization sites on the targets caused by extensive DNA fragmentation. The results form a basis for rational design of an ultrasound cell disruption system integrated with analysis on chip that will move nucleic acid-based detection through real-time analysis closer to reality.

Keyword
Bacillus cereus, enterotoxin, hemolysin, DNA fragmentation, ultrasonication, cereus diarrheal enterotoxin, bacillus-cereus, biochip technology, dna, cell, pcr, microarray, biosensors, pathogens, cytometry
National Category
Industrial Biotechnology
Identifiers
urn:nbn:se:kth:diva-15094 (URN)10.1016/j.ab.2005.07.024 (DOI)000232431900010 ()2-s2.0-25444529747 (Scopus ID)
Note
QC 20100525Available from: 2010-08-05 Created: 2010-08-05 Last updated: 2017-12-12Bibliographically approved
2. Confirmative electric DNA array-based test for food poisoning Bacillus cereus
Open this publication in new window or tab >>Confirmative electric DNA array-based test for food poisoning Bacillus cereus
2007 (English)In: Journal of Microbiological Methods, ISSN 0167-7012, E-ISSN 1872-8359, Vol. 70, no 1, 55-64 p.Article in journal (Refereed) Published
Abstract [en]

Detection of the full set of toxin encoding genes involved in gastrointestinal diseases caused by B. cereus was performed. Eight genes determining the B. cereus pathogenicity, which results in diarrhea or emesis, were simultaneously evaluated on a 16-position electrical chip microarray. The DNA analyte preparation procedure comprising first 5 min of ultrasonic treatment, DNA extraction, and afterwards an additional 10 min sonication, was established as the most effective way of sample processing. No DNA amplification step prior to the analysis was included. The programmed assay was carried out within 30 min, once the DNA analyte from 10(8) bacterial cells, corresponding to one agar colony, was subjected to the assay. In general, this work represents a mature analytical way for DNA review. It can be used under conditions that require almost immediate results.

Keyword
B. cereus; toxin genes; confirmative test; electrical DNA-array
National Category
Industrial Biotechnology
Identifiers
urn:nbn:se:kth:diva-7218 (URN)10.1016/j.mimet.2007.03.011 (DOI)000248109500007 ()2-s2.0-34250786075 (Scopus ID)
Note
QC 20100824Available from: 2007-05-29 Created: 2007-05-29 Last updated: 2010-08-24Bibliographically approved
3. Critical factors for the performance of chip array-based electrical detection of DNA for analysis of pathogenic bacteria
Open this publication in new window or tab >>Critical factors for the performance of chip array-based electrical detection of DNA for analysis of pathogenic bacteria
2008 (English)In: Analytical Biochemistry, ISSN 0003-2697, E-ISSN 1096-0309, Vol. 382, no 2, 77-86 p.Article in journal (Refereed) Published
Abstract [en]

Different factors influencing chip array-based electrical detection of DNA for analysis of pathogenic bacteria were examined. Both rehydration of capture probe layer of functionalized chip arrays and efficient hybridization of targets irrespective of their length resulted in signal enhancement when high-ionic phosphate-buffered saline (i.e., 600 mM sodium chloride and 40 mM disodium hydrogen phosphate) was used. Similarly, placement of two adjacent capture and detection probe-binding sites at a terminal part of the target strand resulted in significant signal increase. Moreover, 10-min ultrasonic fragmentation of targets amplified the signals Lip to twofold for longer DNA strands (i.e., >300 bp). No obvious effects on signals were visible for shorter than 400-bp PCR amplicons subjected to ultrasonication. For DNA strands of all sizes, more than 10 min ultrasonication diminished the specific electrical responses. Our results also demonstrate that target analytes are detected with discrimination against mismatches even for single nucleotide sequence alteration. The mismatch detection appeared in order of ease Of recognition as follows: triple random > quintuple middle > triple middle > single middle mismatch. Among the three variants of one-base mismatches, a sequence variation was most remarkable for adenine. On the other hand, no benefits in assay sensitivity were recognized by the use of longer capture probe linkers as the 6-C linker.

Keyword
electrical chip arrays; nucleic acid-based analysis; toxin-encoding genes; bacterial pathogenicity; Bacillus cereus
National Category
Industrial Biotechnology
Identifiers
urn:nbn:se:kth:diva-9181 (URN)10.1016/j.ab.2008.05.057 (DOI)000259787800001 ()2-s2.0-52049100571 (Scopus ID)
Note
QC 20100824. Uppdaterad från in press till published (20100824).Available from: 2008-09-30 Created: 2008-09-30 Last updated: 2017-12-13Bibliographically approved
4. Rapid determination of virulence factors in EHEC colonies with an electric DNA chip array
Open this publication in new window or tab >>Rapid determination of virulence factors in EHEC colonies with an electric DNA chip array
Show others...
(English)Article in journal (Other academic) Submitted
Keyword
EHEC; Shiga-like toxins; intimin; virulence genes; electric DNA chip array; agar colony; ultrasonication
National Category
Industrial Biotechnology
Identifiers
urn:nbn:se:kth:diva-9183 (URN)
Note
QS 20120328Available from: 2008-09-30 Created: 2008-09-30 Last updated: 2012-03-28Bibliographically approved

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