Limits...
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

Spore DNA analysis. B. cereus spores were harvested from a 4-day-culture; the spores were washed three times and resuspended (both in PBS buffer, pH 7.4). Disruption was performed with an ultrasonic disruptor, using a microtip and 50 W power output at approx. 20 000 Hz for 5, 10 and 15 min. The assay included an equivalent of 1 × 107 disrupted spores, 3 × 107 capturing beads and 10 nM HblC D2 probe (2 h hybridization at 40°C, 10 min enzyme binding, and 2 h enzymatic reaction at 30°C).
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC411050&req=5

Figure 6: Spore DNA analysis. B. cereus spores were harvested from a 4-day-culture; the spores were washed three times and resuspended (both in PBS buffer, pH 7.4). Disruption was performed with an ultrasonic disruptor, using a microtip and 50 W power output at approx. 20 000 Hz for 5, 10 and 15 min. The assay included an equivalent of 1 × 107 disrupted spores, 3 × 107 capturing beads and 10 nM HblC D2 probe (2 h hybridization at 40°C, 10 min enzyme binding, and 2 h enzymatic reaction at 30°C).

Mentions: The following experiments were directed towards integration of ultrasonic cell disruption into the analytical method. Thus, a two-cycle French Press disintegration was compared with 5 min ultrasonication for analysis of 107 to 109 B. cereus cells (Figure 5). In these assays the ultrasonication resulted in a slightly higher signal. Therefore this method was optimized with respect to time (data not shown). Microscopic analysis revealed that almost all cells were disrupted after 30 s of ultrasonication. Using shorter time, the disruption stays incomplete and causes loss of potential DNA target material which remains in the cells. The electric chip signal continued to increase with further ultrasonication time up to 5 min. This may be due to an increased hybridization efficiency caused by the DNA fragmentation, as discussed above. The electric chip was also used to identify spore DNA of B. cereus. Prior to the assay endospores of microbial pathogens were disrupted by ultrasonication. By using an assay with 2 × 107 capturing beads and 10 nM HblC D2 probe, as few as 107 spores resulted in significant signals (Figure 6). The negative control containing undisrupted spores gave a signal close to zero, indicating that the recorded signals were exclusively based on the detection of released endosporal DNA. For the spore assay, optimal sonication time was about 10 minutes.


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)

Spore DNA analysis. B. cereus spores were harvested from a 4-day-culture; the spores were washed three times and resuspended (both in PBS buffer, pH 7.4). Disruption was performed with an ultrasonic disruptor, using a microtip and 50 W power output at approx. 20 000 Hz for 5, 10 and 15 min. The assay included an equivalent of 1 × 107 disrupted spores, 3 × 107 capturing beads and 10 nM HblC D2 probe (2 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 6: Spore DNA analysis. B. cereus spores were harvested from a 4-day-culture; the spores were washed three times and resuspended (both in PBS buffer, pH 7.4). Disruption was performed with an ultrasonic disruptor, using a microtip and 50 W power output at approx. 20 000 Hz for 5, 10 and 15 min. The assay included an equivalent of 1 × 107 disrupted spores, 3 × 107 capturing beads and 10 nM HblC D2 probe (2 h hybridization at 40°C, 10 min enzyme binding, and 2 h enzymatic reaction at 30°C).
Mentions: The following experiments were directed towards integration of ultrasonic cell disruption into the analytical method. Thus, a two-cycle French Press disintegration was compared with 5 min ultrasonication for analysis of 107 to 109 B. cereus cells (Figure 5). In these assays the ultrasonication resulted in a slightly higher signal. Therefore this method was optimized with respect to time (data not shown). Microscopic analysis revealed that almost all cells were disrupted after 30 s of ultrasonication. Using shorter time, the disruption stays incomplete and causes loss of potential DNA target material which remains in the cells. The electric chip signal continued to increase with further ultrasonication time up to 5 min. This may be due to an increased hybridization efficiency caused by the DNA fragmentation, as discussed above. The electric chip was also used to identify spore DNA of B. cereus. Prior to the assay endospores of microbial pathogens were disrupted by ultrasonication. By using an assay with 2 × 107 capturing beads and 10 nM HblC D2 probe, as few as 107 spores resulted in significant signals (Figure 6). The negative control containing undisrupted spores gave a signal close to zero, indicating that the recorded signals were exclusively based on the detection of released endosporal DNA. For the spore assay, optimal sonication time was about 10 minutes.

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