Diagnostic and Typing Methods Flashcards
What is a microbial culture (bacterial detection methods)?
Culture on suitable agar medium
Isolate bacteria
Identify by characterisation of enzyme activities, sugar fermentation tests
What are the advantages of microbial culture methods?
Yields bacterial isolates for future testing and study e.g. antibiotic sensitivities
What are the disadvantages of microbial culture methods?
Requires viable cells
Insensitive
Only small numbers of samples can be analysed at once
Inconclusive results
Labour-intensive
What are the advantages of DNA probes and PCR?
Less time-consuming than culture methods
Very sensitive
Can directly detect bacterial DNA within clinical samples
Do not require viable cells, samples do not have to be analysed immediately
Can detect uncultivable species
What are the disadvantages of DNA probes and PCR?
May detect dead cells
Detect only pre-selected species
What is PCR-RFLP?
Identification of bacterial isolates
Digest PCR-amplified bacterial rNA gene with restriction enzymes
Yields specific patterns for individual species
Rapid, cheaper, more specific alternative to culture and biochemical tests for identifying oral bacteria
What is subtyping?
Identification of different strains among isolates of the same species
Why is it important to subtype bacteria?
Track routes of transmission during disease outbreaks
Study pathogenicity of specific strains
How do you test for one or more phenotypic marker?
Serotyping
Biotyping
What is restriction enzyme analysis (REA)?
Problem?
Digest the whole genomic DNA with restriction enzymes
Too many DNA fragments obtained, makes interpretation difficult
What is gene probe typing?
Can reduce number of DNA fragments generated by REA using a suitable gene probe
What are the molecular (genetic) typing methods?
Restriction Enzyme Analysis
Gene Probe Typing
Ribotyping
16S-23S intergenic spacer region
DNA sequencing
What is ribotyping?
Use E.coli rRNA operon as a DNA probe following REA
rRNA operon present in multiple copies in bacterial genomes, well conserved in overall structure and sequence due to their evolutionary role in all bacteria
Variation in number and size of fragments in bacterial DNA digests which are complementary with rRNA
What is 16S-23S intergenic spacer region (IGS)?
Very variable sequence
Amplify this region by PCR using consensus primers
Digest PCR product with restriction enzymes to obtain strain-specific fingerprints
What is DNA sequencing?
The ultimate typing method
Can detect single base differences between strains
