Final Report Summary - EBIOSENSE (Electrical Bio Sensor Arrays for Analyses of Harmful Micro Organisms and Microbial Toxins)
During the first project year the main activities were put on developing the chip arrays and instrumentation in which all the steps of the assay are automatised. Large inputs were also made on design and evaluation of suitable DNA probes and antibodies, and on the immobilisation of the capturing molecules on the chip surface. During the second project year silicon chip arrays with 16 positions have been designed and manufactured for analysis of several pathogens and toxins:
1) a DNA chip for simultaneous detection of the genes involved in synthesis of the four known toxins produced by many strains of the common food pathogens of the Bacillus cereus group;
2) a DNA chip for analysis of the pathogenic E. coli strains called EHEC;
3) an RNA-chip for analysis of Legionella;
4) a DNA chip for analysis of Salmonella and fumonisin producing Fusarium;
5) an antibody based chips for analysis of several staphylococcal toxins and for the O157 antigen of EHEC bacteria.
For DNA analysis different primary enrichment methods are used depending on the type of sample to be analysed. Analysis of a single colony of suspected pathogenic Bacillus cereus provides in 45 min the full picture of which of the 8 known toxinencoding genes that are present in the bacterial strain. The chip for quantitative analysis of staphylococcal enterotoxin B, currently detects 15 ng toxin in a 250 µL sample.
The electric biochips will improve the risk assessment of food and feed products in several ways. The quality of the risk assessment will be improved by the introduction of DNA analyses. They will give information of the real potential for toxin production and other pathogenicity factors, rather than the taxonomic information provided by traditional tests. This taxonomic information is often poorly related to the real pathogenicity of a micro-organism. The EHEC and the Bacillus chips can illustrate this: EHEC is traditionally identified with the serological analysis for presence of the antigen O157:H7, but there are many other serological types of E. coli that produce the shiga-like toxin that cause the serious disease of EHEC. Many but not all Bacillus cereus strains produce haemolysins, and other toxins but some of these toxins are also produced by other Bacillus species. Thus, only a DNA based analysis gives the information of the health hazard related to these organisms. DNA based analysis can also be made with PCR that is rapidly gaining use in food analysis. Compared to the PCR technology, the silicon based chips offer the potential of faster and simultaneous analysis of a large number of genes in a sample with disposable chips operated by a portable alarm-type detector. The advantage offered by the hybridisation based form of analysis with these chips include also full automation of the detection processes once the sample is provided. The electric chips can also be used as rapid and sensitive detectors of the PCR products.
The third project year included an evaluation of the performance of an antibody based chip and a DNA-chip in comparison with conventional analyses. The organisms/toxins employed in the EBIOSENSE project are specifically associated with food and water born health hazards. However, the technology is generic and a wide range of analytical applications, e.g. rapid determination of antibiotics resistance of bacteria, are expected based on the results of the project.
1) a DNA chip for simultaneous detection of the genes involved in synthesis of the four known toxins produced by many strains of the common food pathogens of the Bacillus cereus group;
2) a DNA chip for analysis of the pathogenic E. coli strains called EHEC;
3) an RNA-chip for analysis of Legionella;
4) a DNA chip for analysis of Salmonella and fumonisin producing Fusarium;
5) an antibody based chips for analysis of several staphylococcal toxins and for the O157 antigen of EHEC bacteria.
For DNA analysis different primary enrichment methods are used depending on the type of sample to be analysed. Analysis of a single colony of suspected pathogenic Bacillus cereus provides in 45 min the full picture of which of the 8 known toxinencoding genes that are present in the bacterial strain. The chip for quantitative analysis of staphylococcal enterotoxin B, currently detects 15 ng toxin in a 250 µL sample.
The electric biochips will improve the risk assessment of food and feed products in several ways. The quality of the risk assessment will be improved by the introduction of DNA analyses. They will give information of the real potential for toxin production and other pathogenicity factors, rather than the taxonomic information provided by traditional tests. This taxonomic information is often poorly related to the real pathogenicity of a micro-organism. The EHEC and the Bacillus chips can illustrate this: EHEC is traditionally identified with the serological analysis for presence of the antigen O157:H7, but there are many other serological types of E. coli that produce the shiga-like toxin that cause the serious disease of EHEC. Many but not all Bacillus cereus strains produce haemolysins, and other toxins but some of these toxins are also produced by other Bacillus species. Thus, only a DNA based analysis gives the information of the health hazard related to these organisms. DNA based analysis can also be made with PCR that is rapidly gaining use in food analysis. Compared to the PCR technology, the silicon based chips offer the potential of faster and simultaneous analysis of a large number of genes in a sample with disposable chips operated by a portable alarm-type detector. The advantage offered by the hybridisation based form of analysis with these chips include also full automation of the detection processes once the sample is provided. The electric chips can also be used as rapid and sensitive detectors of the PCR products.
The third project year included an evaluation of the performance of an antibody based chip and a DNA-chip in comparison with conventional analyses. The organisms/toxins employed in the EBIOSENSE project are specifically associated with food and water born health hazards. However, the technology is generic and a wide range of analytical applications, e.g. rapid determination of antibiotics resistance of bacteria, are expected based on the results of the project.
