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Electric DNA arrays for determination of pathogenic Bacillus cereus
KTH, School of Biotechnology (BIO).
2007 (English)Licentiate thesis, comprehensive summary (Other scientific)
Abstract [en]

Silicon-based electric chip arrays were developed for characterization of Bacillus

cereus with respect to the capacity to produce toxins involved in food poisoning and foodborne infections. Bacteria of the B. cereus group contain different sets of four toxins encoded by eight genes. The purpose of this work was to develop a fast method for determination of the presence of these genes in colonies from primary enrichment cultures. The specific DNA detection was based on immobilization of DNA capture probes, which hybridize to specific sites on the target genes. Biotin-labeled detection probes were designed to hybridize with the target DNA adjacent to the capture probes. An extravidin - alkaline phosphatase complex was subsequently bound to the hybridized detection probes. Finally, p-aminophenyl phosphate was added as substrate for the enzyme, and the product p-aminophenol was brought in contact with the interdigitated gold electrode on the silicon chips surface. The p-aminophenol was oxidized at the anode to quinoneimine, which was then reduced back to paminophenol at the cathode. This redox recycling generates a current that was used as the DNA-chip response to the target DNA. Two versions of the assay were used. In the first version the capture probes were immobilized on magnetic beads and all

chemical reactions until and including the enzymatic reaction took place in an

eppendorf tube while the redox recycling was used to measure the amount of paminophenol produced after transfer from the tube to the silicon chip surface. In the second version a silicon chip array was used with 16 parallel electrode positions, each activated by immobilization of one type of capture probes on the gold electrodes. With this system all chemical reactions took place at the chip surface. The kinetics of cell disruption and DNA fragmentation from B. cereus by ultrasonication was determined. Maximum cell disruption was achieved within 5 min and the chip response increased in proportion to the ultrasonic time. Further ultrasonication up to 10 min resulted in further increasing current although no further cell disruption was observed. If the sonication time was extended above 10 min the signal declined. Based on analysis of the DNA size distribution by early end-point PCR and gel electrophoresis, it is suggested that the first 5 min ultrasonication increased the signal by increasing the release of target DNA molecules. Thereafter the signal was increased by fragmentation of target DNA which increases the diffusion rate and also the accessibility of the hybridization site. Finally, the DNA fragment sizes approached that of the hybridization site (51-bp) which may reduce the signal because of cleavage of the target DNA in the hybridization region. These studies were performed with the bead-based hybridization assay. The assay was highly specific to the target gene (hblC) of both B. cereus and B. thuringiensis with no response from negative control

cells of B. subtilis. The 16 positions of the silicon chip array were activated by

immobilization of all known toxin-coding genes of B. cereus and also included both a positive control and a negative control electrode positions. When these chips were exposed to ultrasonicated B. cereus, the gold electrodes were fouled by some component in DNA cell lysates. To circumvent this, the released large DNA was first extracted and then ultrasonicated again, since the extract mainly contains large molecular weight DNA. This DNA extract was applied to characterize one “diarrheal” and one “emetic” strain of B. cereus with the DNA chip arrays. The results agreed with PCR control analysis which means that these electric DNA chip arrays can be used to characterize bacterial colonies with respect to the genes coding of all known toxins of B. cereus: haemolysin (hblA, hblC, hblD), non-haemolytic enterotoxin (nheA, nheB, nheC), cytotoxin K-2 (cytK-2), and cereulide (ces). The chip assay required about 30 min after application of DNA samples. Due to the generic properties of the chips, this technique should also be applicable for characterization of the pathogenicity potential of many other organisms. Keywords: Bacillus cereus, haemolysin, non-haemolytic enterotoxin, cytotoxin K-2, cereulide, toxin-coding genes, bacterial colony, electric DNA chip, ultrasonication, DNA fragmentation.

Place, publisher, year, edition, pages
Stockholm: KTH , 2007. , 32 p.
Series
Trita-BIO-Report, ISSN 1654-2312 ; 2007:4
Keyword [en]
Bacillus cereus, haemolysin, non-haemolytic enterotoxin, cytotoxin K-2, cereulide, toxin-coding genes, bacterial colony, electric DNA chip, ultrasonication, DNA fragmentation
National Category
Dentistry
Identifiers
URN: urn:nbn:se:kth:diva-4403ISBN: 978-91-7178-655-5 (print)OAI: oai:DiVA.org:kth-4403DiVA: diva2:12161
Presentation
2007-05-24, FA31, KTH, AlbaNova, Stockholm, 10:00
Opponent
Supervisors
Note
QC 20101111Available from: 2007-05-29 Created: 2007-05-29 Last updated: 2010-11-11Bibliographically 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

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