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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

Accessibility of B. cereus cell lysate obtained by two-cycle French Press disintegration for the electric chip assay of the hblC gene. The lysate was heat treated (95°C/10 min) and solid particles were removed by centrifugation (5000 g/10 min). The hblC DNA targets were detected in an equivalent of 1010, 109, and 108 bacterial cells, using 2 × 107 capturing beads and 10 nM HblC D2 probe (1 h hybridization at 40°C, 1 h enzyme binding, and 2 h enzymatic reaction at 30°C). Control I: capturing beads and target without detection probe; Control II: capturing beads and detection probe without target; Control III: capturing beads without target and detection probe.
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Figure 2: Accessibility of B. cereus cell lysate obtained by two-cycle French Press disintegration for the electric chip assay of the hblC gene. The lysate was heat treated (95°C/10 min) and solid particles were removed by centrifugation (5000 g/10 min). The hblC DNA targets were detected in an equivalent of 1010, 109, and 108 bacterial cells, using 2 × 107 capturing beads and 10 nM HblC D2 probe (1 h hybridization at 40°C, 1 h enzyme binding, and 2 h enzymatic reaction at 30°C). Control I: capturing beads and target without detection probe; Control II: capturing beads and detection probe without target; Control III: capturing beads without target and detection probe.

Mentions: The results from the previous experiments were applied to set up a procedure for analysis of the hblC gene in assays containing genomic B. cereus DNA. To establish that the assay was able to detect DNA from biological samples, B. cereus cells were disrupted mechanically by two-cycle French Press disintegration and the crude lysate from 1010, 109 and 108 cells was applied directly to the assay after heat treatment and centrifugation (Figure 2). For this experiment a new detection probe (HblC D2) was employed in the assay (Figure 1). This probe hybridizes in the target sequence adjacent to the capture probe. The reason for the use of new detection probe was the consideration that due to mechanical degradation of the DNA in cell homogenization the target sequences might be too short for successful detection with HblC D1. The assay was compared with three different controls and it was found that the components of the lysate did not generate any background signal. When using crude cell lysates, the sensitivity of this assay was comparable to results from artificial DNA assays (i.e. assays with PCR product). 108 target cells, approximately 200 amol, resulted in a significant signal.


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)

Accessibility of B. cereus cell lysate obtained by two-cycle French Press disintegration for the electric chip assay of the hblC gene. The lysate was heat treated (95°C/10 min) and solid particles were removed by centrifugation (5000 g/10 min). The hblC DNA targets were detected in an equivalent of 1010, 109, and 108 bacterial cells, using 2 × 107 capturing beads and 10 nM HblC D2 probe (1 h hybridization at 40°C, 1 h enzyme binding, and 2 h enzymatic reaction at 30°C). Control I: capturing beads and target without detection probe; Control II: capturing beads and detection probe without target; Control III: capturing beads without target and detection probe.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 2: Accessibility of B. cereus cell lysate obtained by two-cycle French Press disintegration for the electric chip assay of the hblC gene. The lysate was heat treated (95°C/10 min) and solid particles were removed by centrifugation (5000 g/10 min). The hblC DNA targets were detected in an equivalent of 1010, 109, and 108 bacterial cells, using 2 × 107 capturing beads and 10 nM HblC D2 probe (1 h hybridization at 40°C, 1 h enzyme binding, and 2 h enzymatic reaction at 30°C). Control I: capturing beads and target without detection probe; Control II: capturing beads and detection probe without target; Control III: capturing beads without target and detection probe.
Mentions: The results from the previous experiments were applied to set up a procedure for analysis of the hblC gene in assays containing genomic B. cereus DNA. To establish that the assay was able to detect DNA from biological samples, B. cereus cells were disrupted mechanically by two-cycle French Press disintegration and the crude lysate from 1010, 109 and 108 cells was applied directly to the assay after heat treatment and centrifugation (Figure 2). For this experiment a new detection probe (HblC D2) was employed in the assay (Figure 1). This probe hybridizes in the target sequence adjacent to the capture probe. The reason for the use of new detection probe was the consideration that due to mechanical degradation of the DNA in cell homogenization the target sequences might be too short for successful detection with HblC D1. The assay was compared with three different controls and it was found that the components of the lysate did not generate any background signal. When using crude cell lysates, the sensitivity of this assay was comparable to results from artificial DNA assays (i.e. assays with PCR product). 108 target cells, approximately 200 amol, resulted in a significant signal.

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