Entwicklung von DNA-Markierungs- und Detektionsverfahren für die Mikroarray-Untersuchung von Milchprodukten und Starterkulturen unter dem Aspekt der Praxistauglichkeit

Abstract

Subject of this work is the development of DNA-labelling and detection methods for PCR-free microarray analyses of milk products and starter cultures. For direct detection of genes an improved labelling with Psoralen-PEO-Biotin was combined with an easy to handle DNA-extraction, fragmentation, and streptavidin-Cy5 detection protocol. The combination of these methods allowed an identification of bacteria and Lactococcus-phages without a PCR-amplification of the target molecules up to 10E5 � 10E6 DNA-copies. To further improve the sensitivity of the system, the tyramide signal amplification was embedded in the current protocol. The signal amplification with tyramide-Cy5 led to an 8-fold increase of the signal intensity and by this to a better discrimination of the signal from the background. The use of a thrombin-aptamer and thrombin to increase the binding of tyramide-Cy5 molecules on the microarray surface leads to an additional 2-fold increase. For a cheaper detection of microarray hybridizations a streptavidin-gold method was established to detect the Psoralen-PEO-Biotin labelled DNA. With this protocol it was possible to identify organisms by analysing the microarrays with a normal flat bed scanner. The detection limit of the method was as good as the fluorescent one and enabled an identification with 10E6 DNA copies.The complete streptavidin-Cy5 protocol was tested by analysing samples from a dairy plant. Twenty five specimens from an ice-cream production, four milk powder samples, one whey sample and four starter cultures could be analysed by microarray hybridization. Due to the analysis with conventional microbiological methods the composition or the contaminating organisms were known and made it possible to compare it with the results of the microarray hybridization. The microarray analysis of the samples demonstrated the great advantage of this technique. It was possible to detect all known contaminating organisms and also some that were not found with conventional microbiological methods. Due to the faster and more parallel analysis of specimen the developed protocol could be used as a supportive method in dairy plants.