8/30/2016 g<,HiH亚 PCR-most widely used method Three-steps to amplify a specific DNA segment 1.Denaturation-template DNA is denatured into single strands 2.Annealing-primers are annealed to the template DNA 之 annealing temp provides stringency 3.Extension-polymerization of DNA from the primers using a thermostable polymerase .Within 2 h,a single copy of DNA may be amplified a million-fold DNA is visualized using an agarose gel stained PCR Ribotyping Few kits available for identification of Fingerprinting of genomic DNA restriction foodborne pathogens fragments that contain all or part of the genes ·Assay is coding for the 16S and 23S rRNA very sensitive -The genes are cut at specific sites using expensive to run -not technically simple-expertise needed restriction enzymes generating pieces of affected by food components DNA of different lengths -Results within 16 h Enrichment may be needed to dilute food components and increase cell numbers Used for characterizing and fingerprinting Alternatives to conventional PCR may be strains and for epidemiological useful investigations 8/30/2016 8 PCR - most widely used method Three-steps to amplify a specific DNA segment 1. Denaturation -template DNA is denatured into single strands 2. Annealing - primers are annealed to the template DNA annealing temp provides stringency 3. Extension -p y ol merization of DNA from the primers using a thermostable polymerase • Within 2 h, a single copy of DNA may be amplified a million-fold • DNA is visualized using an agarose gel stained PCR • Few kits available for identification of foodborne pathogens • A issay s - very sensitive - expensive to run - not technically simple – expertise needed - affected by p food components • Enrichment may be needed to dilute food components and increase cell numbers • Alternatives to conventional PCR may be useful Ribotyping Fingerprinting of genomic DNA restriction fragments that contain all or part of the genes codi f th 16S d 23S RNA ding for the 16S and 23S rRNA - The genes are cut at specific sites using restriction enzymes generating pieces of DNA of different lengths - R lt ithi 16 h Results within 16 h - Used for characterizing and fingerprinting strains and for epidemiological investigations