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Case 1 - totally unknown organism
- from an environmental sample
- from a new disease specimen
- from an industrial process that is going wrong in an unusual way
- 1) PCR 16S rRNA from organism and sequence
- 2) submit sequence to 16S database to find closest relatives
- - this will identify the genus and sometimes the species of your organism
- 3) PCR and sequence a set of 7-10 housekeeping genes
- - this will identify species and possibly strain, if your organism has been identified before (MLST)
- 4) still not a previously identified species?
- - characterize and submit description of a new species
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case 2 - have an unknown sample from a familiar source, can make an educated guess that organism is one you have seen before
- clinical samples from patients with various symptoms
- contaminants from processes
- bypass 16S rRNA sequencing and go to direct comparisons which can be faster or give information about strains
- 1) Ribotyping
- 2) FAME
- 3) MLST
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ribotyping
- 1) isolate DNA from your unknown organism and known candidates
- 2) Digest DNA with one or more restriction enzymes
- 3) Separate fragments by electrophoresis
- 4) visualize using a probe that hybridizes to rRNA sequences
- pattern of restriction fragments is based on the rRNA sequence, but no sequencing reactions are performed
- pattern is unique to species and sometimes to strains
- technique is useful for determining if your bug is one of a few known species
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FAME (fatty acid methyl ester) lipid analysis
- determine types and proportions of fatty acids present in phospholipids of the bacterial membranes
- fatty acids differ
- each species has a fatty acid fingerprint in a database and an algorithm picks the best matches to your organism
- good for identifying what species you have but not what strain
- many factors can influence the lipid composition - only useful when growth conditions can be strictly standardized
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multilocus sequence typing (MLST)
- PCR and sequence several housekeeping genes that are present in all of the organisms compared
- based on sequences, each organism gets a set of allele numbers
- compare the set of alleles in one organism to sets in the other organisms to determine relatedness
- best at distinguishing species and strains - has scientific, clinical and epidemiological utility
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