Untersuchung zur Einsetzbarkeit der Psoralen-PEO-Biotin Markierung in der DNA-Micorarray Technologie für die Analyse von Starterkulturbakterien und ihren Phagenresistenzgenen

Abstract

Subject of this work was the investigation of the Psoralen-PEO-Biotin labelling method for the direct detection of starter culture bacteria and their phage-resistance genes with the DNA-Microarray-Technology. The use of DNA-Microarray-Technology in industry was up to now limited through different methodological limitations. In this work, the direct detection of genes was established by the combination of DNA-extraction, fragmentation, labelling and hybridization of genomic DNA in an easy to handle and inexpensive protocol.The Psoralen-PEO-Biotin labelling method was established and optimized for genomic DNA in a model system. Various parameters affecting the hybridization efficiency were investigated in this system. Genomic DNA was fragmented with a chemical fragmentation reaction to increase probe accessibility. 73 probes were generated and validated to detect milk bacteria and all phage resistance genes that occur in Lactococcus lactis. The DNA-Microarray was able to detect milk bacteria and phage resistance genes. Phage-resistance genes can be lost and acquired due to the fact that they are plasmid-coded. Only those phage-resistance genes could be directly detected who occurred in more than 16% of the examined starter culture isolates. To increase the sensitivity, two different strategies for signal- and target amplification were embedded into the current protocol. With the Multiplex-PCR in the combination with the created DNA-Microarray all genes were identified correctly. The signal amplification with Tyramide-Cy5 led to signal enhancement for the direct detection of genomic DNA. However, the unambiguous detection of the phage- resistance did not succeed since unspecific signal amplification could not be avoided. The correct proof can certainly be corrected by the optimization of TSA on DNA-Microarrys. So in the near future, it should be possible, to detect low-abundance genes with DNA-Microarrys by the hybridization of genomic DNA without target-amplification.