Miljøprojekt, 1135 – Påvisning af patogene bakterier i vand ved anvendelse af DNA-chip-prototype og mikrobiologiske dyrkningsmetoder

Påvisning af patogene bakterier i vand ved anvendelse af DNA-chip-prototype og mikrobiologiske dyrkningsmetoder

Summary and conclusions

The background for this preliminary investigation is that Bioneer A/S has developed a molecular method based on DNA chip technology for rapid and simultaneous detection of pathogenic microorganisms. This rapid method makes is possible to detect and at the same time identify the various pathogenic bacteria that are relevant in relation to water. Proof-of-concept for use of a DNA chip prototype for detection of pathogenic bacteria is established, and the next step is testing the applicability of the concept.

The aim is to establish the potential and applicability of the DNA chip method as a rapid screening analysis for selected pathogenic microorganisms in water. In the project, the DNA chip prototype in its present form and design (with the prototype's limits concerning specificity and sensitivity) is compared to usual microbiological cultivation methods for detection and identification of pathogenic bacteria.

For this investigation 5 model organisms have been selected because of their capability of causing water-borne diseases and their occurrence in drinking water as well as in bathing water. The organisms are Campylobacter jejuni, Aeromonas hydrophila, Pseudomonas aeruginosa, Legionella pneumophila, and Salmonella spp.

The experimental plan run in two parallel blocks; the first covering direct detection of pathogenic bacteria in suspension samples, the second covering detection of pathogens spiked in sterile water.

The DNA chip analysis is based on an enzymatic polymerase chain reaction (PCR) amplification of DNA by means of a so-called consensus 16S rDNA-PCR reaction with end-labelled ribosomal primers.

The result of DNA chip analysis of suspension samples and spiked water samples containing mixtures of test organisms shows that the expected (correct) signals for all pathogenic bacteria in tests of 10⁸and 10⁶ cells per pathogen per sample are obtained. Following the microbiological analysis results it is conclusive that the growing methods generally retrieve the expected pathogenic bacteria in suspension samples and spiked water samples containing 10⁸, 10⁶, 10⁴ and 10² cells per sample.

Generally, the results obtained from the DNA chip prototype are in accordance with the results from the microbiological growing methods for the bacterial mixtures containing 10⁸and 10⁶ cells per sample. In its present design and assay size, the DNA chip method cannot detect specific bacteria as few as 10² cells per sample, but in some cases it was possible to detect 10⁴ specific bacteria.