| Importance of fingerprinting foodborne microorgansims | - differentiate strains (serotypes or serovars) of microorgansims
- track the source of pathogen that caused a disease outbreak |
| Species | - collection of strains that have a similar guanine + cytosine (G + C) composition
- 70% or greater similarity based on DNA hybridization experiments |
| Strain | - an organism that is distinguishable from other organisms within the same species |
| Serotyping | - differentiates various serovars in a species
- specific antibodies are used to identify homologous antigens
- a match between specific antibody and antigen on bacteria results in agglutination (visible clumping) |
| Bacteriophage typing | - method based on high specificity of a phage for its bacterial host
- phage surface receptors specific for cell's surface receptors |
| Bacteriophage typing method | 1. inoculate surface of agar medium with bacterial culture
2. mark 15-20 squares on the back side of the agar plate
3. inoculate agar surface in each square with a drop of phage suspension (using different phage for each square)
4. incubate inoculated agar plates at an appropriate temperature for 24 hours
5. observe plates for plaques (clear zones from cell lysis)
6. record phage type information |
| Electrophoresis | a technique for separating substances based on differences in their migration rate in an electric field |
| Gel electrophoresis | - differences in migration rate attributed to variations in charge and size of substances
- DNA fragments from various bacterial isolates inserted into wells in gel
- gel is 1-3% agarose or 3-20% polyacrylamide
- electric current applied to gel
- DNA fragments (neg charge) migrate through gel to the anode (positive charge)
- smallest fragments migrate faster |
| Pulsed Field Gel Electrophoresis (PFGE) basics | - used to determine relatedness among strains of the same species
- gold standard for bacteria subtyping (fingerprinting)
- primary subtyping method used by PulseNet
1. genomic DNA from isolated pathogen digested by one or more pathogens
2. digested DNA added to well in agarose gel
3. application of pulsed electric fields (pulse times ramped from 1 to 100 seconds over various time periods)
4. electrophoretic profile of the DNA sample is produced and used to fingerprint the foodborne outbreak strain |
| Restriction endonucleases | - cut DNA at specific sites
- produce DNA fragments
- mixture of fragments is unique to each organism |
| Pulsed Field Gel Electrophoresis (PFGE) methods | - subtyping (fingerprinting) pathogen isolated from suspect food and from clinical sample to establish an epidemiological link |
| Ribosomes | - occur in cytoplasm of living cells
- consist of RNA and protein
- translate mRNA into proteins
- bind the mRNA and tRNA to make proteins
- bacterial ribosome (50S and 30S subunits) |
| 16S rRNA of bacterial ribosome | - part of the 30S subunit of the ribosome
- highly conserved: very slow rate of evolution of this region
- can be sequenced to determine precise phylogenetic relationships among bacteria
- genes for ribosomal RNA are scattered in several locations on the bacterial chromosome |
| Ribotyping | 1. Determine the nucleotide sequence of the 16S rRNA
2. Use the 16S rRNA nucleotide sequence to make a cDNA (copy DNA) probe
3. Extract DNA from pathogenic isolate linked to foodborne disease outbreak
4. DNA digested with restriction endonucleases
5. Fragmented DNA separated by agarose gel electrophoresis
6. Transfer to nylon membrane and hybridize with labeled cDNA probe
7. Record chemiluminescent pattern and compare to known isolates |
| Whole Genome Sequencing | - provides the order of bases in the whole genome of an organism
- gives a very precise DNA fingerprint
- allows outbreaks to be detected and solved quicker
- shows genetic variations between and within species
- high precision differentiation between organisms
- better than PFGE |
| Whole Genome Sequencing Methods | 1. DNA extraction (culture bacterial cells, lyse cells to release DNA, purify DNA)
2. DNA cutting (enzymatically cut the DNA with restriction endonucleases, producing short fragments. can use mechanical shearing)
3. preparation of DNA library (DNA fragments placed in PCR thermocycler, PCR amplifies DNA fragments to form copies)
4. DNA sequencing (DNA library placed into sequencer, nuclotides of each DNA fragment are read, yielding millions of DNA reads)
5. DNA sequence analysis (computer-aided analysis of millions of DNA reads, program puts reads together in the correct order) |
| Food safety application of Whole Genome Sequencing | - identify pathogens isolated from food or the environment
- compare clinical isolate (from patients) with food isolate
- allows reliable epidemiological links to be made
- can match pathogen with a geographical location |
