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Identification of pathogenic microbial cells and spores by electrochemical detection on a biochip.

Gabig-Ciminska M, Andresen H, Albers J, Hintsche R, Enfors SO - Microb. Cell Fact. (2004)

Bottom Line: Despite the recent development of different detection methods, new effective control measures and better diagnostic tools are required for quick and reliable detection of pathogenic micro-organisms.The method was also successful when applied directly to unpurified spore and cell extract samples.The assay for the haemolytic enterotoxin genes resulted in reproducible signals from B. cereus and B. thuringiensis while haemolysin-negative B. subtilis strain did not yield any signal.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Biotechnology, Royal Institute of Technology KTH, S-10691 Stockholm, Sweden. gabig@biotech.univ.gda.pl

ABSTRACT
BACKGROUND: Bacillus cereus constitutes a significant cause of acute food poisoning in humans. Despite the recent development of different detection methods, new effective control measures and better diagnostic tools are required for quick and reliable detection of pathogenic micro-organisms. Thus, the objective of this study was to determine a simple method for rapid identification of enterotoxic Bacillus strains. Here, a special attention is given to an electrochemical biosensor since it meets the requirements of minimal size, lower costs and decreased power consumption. RESULTS: A bead-based sandwich hybridization system was employed in conjugation with electric chips for detection of vegetative cells and spores of Bacillus strains based on their toxin-encoding genes. The system consists of a silicon chip based potentiometric cell, and utilizes paramagnetic beads as solid carriers of the DNA probes. The specific signals from 20 amol of bacterial cell or spore DNA were achieved in less than 4 h. The method was also successful when applied directly to unpurified spore and cell extract samples. The assay for the haemolytic enterotoxin genes resulted in reproducible signals from B. cereus and B. thuringiensis while haemolysin-negative B. subtilis strain did not yield any signal. CONCLUSIONS: The sensitivity, convenience and specificity of the system have shown its potential. In this respect an electrochemical detection on a chip enabling a fast characterization and monitoring of pathogens in food is of interest. This system can offer a contribution in the rapid identification of bacteria based on the presence of specific genes without preceding nucleic acid amplification.

No MeSH data available.


Related in: MedlinePlus

Assay time in relation to detection sensitivity. 2 × 107 capturing beads and 10 nM HblC D2 probe were used in each assay. 50 min assay (transparent bar): 20 min hybridization at 40°C, 10 min enzyme binding, and 30 min enzymatic reaction at 40°C; 4 h assay (grey bar): 2 h hybridization at 40°C, 10 min enzyme binding, and 2 h enzymatic reaction at 30°C; 8 h assay (black bar): 6 h hybridization at 40°C, 10 min enzyme binding, and 2 h enzymatic reaction at 30°C.
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Figure 4: Assay time in relation to detection sensitivity. 2 × 107 capturing beads and 10 nM HblC D2 probe were used in each assay. 50 min assay (transparent bar): 20 min hybridization at 40°C, 10 min enzyme binding, and 30 min enzymatic reaction at 40°C; 4 h assay (grey bar): 2 h hybridization at 40°C, 10 min enzyme binding, and 2 h enzymatic reaction at 30°C; 8 h assay (black bar): 6 h hybridization at 40°C, 10 min enzyme binding, and 2 h enzymatic reaction at 30°C.

Mentions: The predominant parts of the assay time are the hybridization and the enzymatic reaction. By extending these reaction times, higher sensitivity is achieved. The relationship between sensitivity and assay time for analysis of vegetative B. cereus cells is shown in Figure 4. The detection limit here was 107 target cells (16.7 amol DNA) which required 4 hours. A 50 minutes assay resulted in a significant signal for 109 cells.


Identification of pathogenic microbial cells and spores by electrochemical detection on a biochip.

Gabig-Ciminska M, Andresen H, Albers J, Hintsche R, Enfors SO - Microb. Cell Fact. (2004)

Assay time in relation to detection sensitivity. 2 × 107 capturing beads and 10 nM HblC D2 probe were used in each assay. 50 min assay (transparent bar): 20 min hybridization at 40°C, 10 min enzyme binding, and 30 min enzymatic reaction at 40°C; 4 h assay (grey bar): 2 h hybridization at 40°C, 10 min enzyme binding, and 2 h enzymatic reaction at 30°C; 8 h assay (black bar): 6 h hybridization at 40°C, 10 min enzyme binding, and 2 h enzymatic reaction at 30°C.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC411050&req=5

Figure 4: Assay time in relation to detection sensitivity. 2 × 107 capturing beads and 10 nM HblC D2 probe were used in each assay. 50 min assay (transparent bar): 20 min hybridization at 40°C, 10 min enzyme binding, and 30 min enzymatic reaction at 40°C; 4 h assay (grey bar): 2 h hybridization at 40°C, 10 min enzyme binding, and 2 h enzymatic reaction at 30°C; 8 h assay (black bar): 6 h hybridization at 40°C, 10 min enzyme binding, and 2 h enzymatic reaction at 30°C.
Mentions: The predominant parts of the assay time are the hybridization and the enzymatic reaction. By extending these reaction times, higher sensitivity is achieved. The relationship between sensitivity and assay time for analysis of vegetative B. cereus cells is shown in Figure 4. The detection limit here was 107 target cells (16.7 amol DNA) which required 4 hours. A 50 minutes assay resulted in a significant signal for 109 cells.

Bottom Line: Despite the recent development of different detection methods, new effective control measures and better diagnostic tools are required for quick and reliable detection of pathogenic micro-organisms.The method was also successful when applied directly to unpurified spore and cell extract samples.The assay for the haemolytic enterotoxin genes resulted in reproducible signals from B. cereus and B. thuringiensis while haemolysin-negative B. subtilis strain did not yield any signal.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Biotechnology, Royal Institute of Technology KTH, S-10691 Stockholm, Sweden. gabig@biotech.univ.gda.pl

ABSTRACT
BACKGROUND: Bacillus cereus constitutes a significant cause of acute food poisoning in humans. Despite the recent development of different detection methods, new effective control measures and better diagnostic tools are required for quick and reliable detection of pathogenic micro-organisms. Thus, the objective of this study was to determine a simple method for rapid identification of enterotoxic Bacillus strains. Here, a special attention is given to an electrochemical biosensor since it meets the requirements of minimal size, lower costs and decreased power consumption. RESULTS: A bead-based sandwich hybridization system was employed in conjugation with electric chips for detection of vegetative cells and spores of Bacillus strains based on their toxin-encoding genes. The system consists of a silicon chip based potentiometric cell, and utilizes paramagnetic beads as solid carriers of the DNA probes. The specific signals from 20 amol of bacterial cell or spore DNA were achieved in less than 4 h. The method was also successful when applied directly to unpurified spore and cell extract samples. The assay for the haemolytic enterotoxin genes resulted in reproducible signals from B. cereus and B. thuringiensis while haemolysin-negative B. subtilis strain did not yield any signal. CONCLUSIONS: The sensitivity, convenience and specificity of the system have shown its potential. In this respect an electrochemical detection on a chip enabling a fast characterization and monitoring of pathogens in food is of interest. This system can offer a contribution in the rapid identification of bacteria based on the presence of specific genes without preceding nucleic acid amplification.

No MeSH data available.


Related in: MedlinePlus