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

Effect of target length and probes positions on the assay of hblC and hblA. Assays were performed using 0.4 nM PCR product (transparent bar) or 1.4 × 109 lysed cells (solid bar) as targets, 2 × 107 capturing beads, and 10 nM of each detection probe (1 h hybridization at 40°C, 1 h enzyme binding, and 30 min enzymatic reaction at 40°C). 100% signal corresponds to 109.9 nA in case of PCR product, and 7.4 nA in case of DNA in crude cell lysate.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC411050&req=5

Figure 3: Effect of target length and probes positions on the assay of hblC and hblA. Assays were performed using 0.4 nM PCR product (transparent bar) or 1.4 × 109 lysed cells (solid bar) as targets, 2 × 107 capturing beads, and 10 nM of each detection probe (1 h hybridization at 40°C, 1 h enzyme binding, and 30 min enzymatic reaction at 40°C). 100% signal corresponds to 109.9 nA in case of PCR product, and 7.4 nA in case of DNA in crude cell lysate.

Mentions: To study the influence of the distance between the capture and detection probe in the target sequence on the electric signal, two alternative detection probe placements in the target sequence were investigated. Two different detection probes were available and ready to use in bead-based hybridization assay. Detection probe number 1, i.e. HblC D1, and HblA D1, complementary in sequence to the lower PCR primer, thus hybridizing distinct from the capturing probe at the 3' end of PCR product, and detection probe number 2, i.e. HblC D2, and HblA D2, which were designed to hybridize closely to the capturing probe (Figure 1). The experiments were aimed to detect the sequences of two selected genes, hblC and hblA, by employing either PCR amplicons or DNA in cell lysates as analyte material. Assay responses are presented as a percentage of the maximum signal that was recorded from the detection of either PCR target or cell lysate (Figure 3). The results showed that there is a strong correlation between the signal and the relative position of the two hybridizing sequences that probably influences the accessibility of the probes to the complementary region. It was concluded, that the primary influence of the target length on the assay is based on the exposition of the detection probe hybridization site in the target molecule. The terminal sequence gives better exposition and more efficient hybridization. At the same time, the effectiveness of the association reaction was reduced when the hybridization sequences were located within the target molecule. In this way, the hybridization efficiency in the assay with PCR amplicons utilizing probes D1, was approximately 95% higher when compared to the use of detection probes D2. Thus, assays targeting PCR products should employ probes which mimic the PCR primers.


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)

Effect of target length and probes positions on the assay of hblC and hblA. Assays were performed using 0.4 nM PCR product (transparent bar) or 1.4 × 109 lysed cells (solid bar) as targets, 2 × 107 capturing beads, and 10 nM of each detection probe (1 h hybridization at 40°C, 1 h enzyme binding, and 30 min enzymatic reaction at 40°C). 100% signal corresponds to 109.9 nA in case of PCR product, and 7.4 nA in case of DNA in crude cell lysate.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 3: Effect of target length and probes positions on the assay of hblC and hblA. Assays were performed using 0.4 nM PCR product (transparent bar) or 1.4 × 109 lysed cells (solid bar) as targets, 2 × 107 capturing beads, and 10 nM of each detection probe (1 h hybridization at 40°C, 1 h enzyme binding, and 30 min enzymatic reaction at 40°C). 100% signal corresponds to 109.9 nA in case of PCR product, and 7.4 nA in case of DNA in crude cell lysate.
Mentions: To study the influence of the distance between the capture and detection probe in the target sequence on the electric signal, two alternative detection probe placements in the target sequence were investigated. Two different detection probes were available and ready to use in bead-based hybridization assay. Detection probe number 1, i.e. HblC D1, and HblA D1, complementary in sequence to the lower PCR primer, thus hybridizing distinct from the capturing probe at the 3' end of PCR product, and detection probe number 2, i.e. HblC D2, and HblA D2, which were designed to hybridize closely to the capturing probe (Figure 1). The experiments were aimed to detect the sequences of two selected genes, hblC and hblA, by employing either PCR amplicons or DNA in cell lysates as analyte material. Assay responses are presented as a percentage of the maximum signal that was recorded from the detection of either PCR target or cell lysate (Figure 3). The results showed that there is a strong correlation between the signal and the relative position of the two hybridizing sequences that probably influences the accessibility of the probes to the complementary region. It was concluded, that the primary influence of the target length on the assay is based on the exposition of the detection probe hybridization site in the target molecule. The terminal sequence gives better exposition and more efficient hybridization. At the same time, the effectiveness of the association reaction was reduced when the hybridization sequences were located within the target molecule. In this way, the hybridization efficiency in the assay with PCR amplicons utilizing probes D1, was approximately 95% higher when compared to the use of detection probes D2. Thus, assays targeting PCR products should employ probes which mimic the PCR primers.

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