Diagnostic And Typing Methods Flashcards
List the bacteria associated with periodontal disease (4)
Porphyromonas gingivalis (P gingivalis)
Actinobacillus actinomycetenconitans
Prevotella intermedia
Bacteroides forsythus
What is the main bacteria associated with dental caries?
Streptococcus mutans
What are the 2 main bacteria associated with root canal infection?
Porphyromonas endodontalis
Fusobacterium nucleatum
What are the types of bacterial detection methods?
Microbiological culture
Molecular biological
Why are serial dilutions carried out?
Otherwise, there would be so much bacteria on the agar, would be impossible to detect colonies of bacteria
What is Vancomycin?
Supplement on agar - selective agent for gram-negative anaerobes
How long are agar plates incubated anaerobically for?
10 days
Which bacteria do black, pigmented colonies generally belong to?
Prevotalla and Porphromonous
(Associated with oral disease eg perio)
How do you obtain a pure colony?
Remove an isolated individual colony from agar and grown in pure culture
How are bacteria identified?
Metronidazole disc (5ug/ disc) - anaerobes sensitive to this, will die out when in vicinity
Gram staining (positive= purple, thick peptidoglycan layer in cell wall, retains stain, negative= thin layer, will not stain, pink).
Enzymatic and sugar fermentation tests - Rapid API 32 A (identify bacteria by creating activity and fermentation profile and compare to database)
What are some advantages and disadvantages of culture methods?
Advantages:
- yields bacteria isolates for future testing and study
Disadvantages:
- requires viable cells (often bacteria which have caused infection will die off)
- insensitive (need 10^5-6 cells)
- small no samples can be analysed at once
- inconclusive
- labour-intensive/ expensive
What are DNA probes
Segments of DNA that have been labelled with chemoluminescent/ fluorescent/ radioactive agents
Can be:
- whole genomic (entire gene)
- cloned gene (particular gene)
- oligonucleotide (20-50 bases- short)
What is the main advantage of DNA probe
Much more sensitive than culture (only need 10^3 cells to detect bacteria)
How is the DNA probe prepared?
Heat denatured to make single stranded
Label one of the strands (chemoluminescent etc..)
How is the sample prepared
Describe the process of DNA probe
The probe is prepared- heat denatured to make single stranded, labelled at one end (chemoluminescent etc…).
The DNA is extracted from sample and heat denatured to make single stranded.
Probe and sample mixed in hybridisation reaction- probe binds to complementary sequence in sample.
Non-binding DNA is removed.
What was the first type of DNA probe used, and describe.
Genomic:
DNA extracted and purified from bacterial species and cut into fragments and labelled to create a whole genomic probe (non-specific, lots of cross reactivity between species).
Then,
Cloned gene probe:
Gene of interest cloned into E. coli, fragment isolated and purified and label attached. Specific gene is specific to the bacterial species.
Describe Oligonucleotide probes
Most specific as so small
Target 16S ribosomal RNA gene (all bacteria possess, 1500 base pairs in length) - this gene is good for preparing species specific probes as posses 9 hyper specific regions (V1-V9) that contain unique DNA sequencing for each species.
Synthesise a species specific probes to target one or more hyper specific regions- this is labelled and used as a probe.
What is 16S ribosomal RNA gene?
Found in all bacteria
Highly variable rations (V1-V9) providing a unique signature to any bacterium - species specific probes/ primers
Conserved regions are the same across all species allowing creation of a broad range of probes/ PCR primers - used when not essential to know the species, but just the presence of bacteria
What is PCR?
Polymerase chain reaction
Highly specific and sensitive
Can be used to directly detect bacteria in clinical specimens
Describe the process of PCR
Exponential amplification procedure
Need:
Double stranded DNA template
Primer is specific to gene
DNPTs -building blocks of DNA
DNA polymerase- catalyses synthesis of new DNA strands
Double stranded DNA is heat denatured
PCR primer hybridise to target sequences (annealing)
DNA polymerase catalyses the synthesis of opposite strand (incorporating dNTPs) (primer extension)
Doubles the amount of DNA, cycle can be repeated up to 35 times.
What gene do the PCR primers target?
16S RNA gene
What are the 3 different types of primers?
General bacterial primers (target consensus region of gene)
Group specific primers (to identify a genus of bacteria)
Species-specific primers (can be a single primer pair or more than one (multiplex- cheaper and quicker however, can only use up to 3 primer pairs as all have different optimal PCR conditions eg, temp for annealing).
Give some advantages of DNA probes and PCR
Less time-consuming than culture methods (24-48 hours)
Very sensitive (DNA probes, 103 cells; PCR, 10 cells, culture methods can only detect upwards of 100,000 cells)
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
Give some disadvantages of DNA probes and PCR
May detect dead cells (may not have been actively involved in infection)
Detect only pre-selected species
What is PCR RFLP
Identification of bacterial isolates (that have been obtained by culture methods)
16S RNA gene is amplified from bacterial isolate and digested by restriction enzymes
Can predict sizes of DNA fragments when restricting the gene- yields specific fingerprints for each species
Can identify species.
Rapid/ cheaper/ more specific than biological tests to identify a species from clinical samples.
Why is it important to subtype bacteria?
To track routes of transmission during disease outbreaks
To study pathogenicity of specific strains
What are 5 methods of DNA typing?
- Restriction Enzyme Analysis (REA)
- Gene probe typing
- Ribotyping
- 16S-23S intervenir spacer region (IGS)
- DNA sequencing
What is restriction enzyme analysis?
Digest whole genomic DNA with restriction enzymes
However- there are too many DNA fragments obtained (produces a strain effect), making interpretation difficult
What is gene probe typing
Use a suitable gene probe to reduce the number of DNA fragments generated by REA
Gene probe is hybridised to DNA fragments, only fragments with all/ part of gene will hybridise to the probe and create a signal
Makes interpretation much easier and can identify different strains.
What is ribotyping?
Use E. coli rRNA operon as a DNA probe following REA
rRNA operon is present in multiple copies in bacterial genomes and is well conserved in overall structure and sequence due to its evolutionary role in all bacteria
Variation in number and size of fragments in bacterial DNA digests which are complementary with rRNA
Describe the gene typing method
Genomic DNA extracted from bacterial isolate/ clinical smaple
Digested by one or more restrictive enzymes to give fragments which are separated into bands by agar electrophoresis (small, mobile fragments migrate towards positive electrode)
DNA bands in gel are transferred to nylon membrane in preparation for hybridisation to labelled DNA probe
DNA probe bonds to specific DNA fragments on membrane that are all/ part of probe sequence- this is visualised and the DNA fragments are compared.
What is 16s-23s intergenic spacer region (IGS)
Rapid and simple method
This region is very variable in sequence (even different strains of same species will have different sequence)
Amplify the spacer region by PCR using consensus primers, digest PCR products with restriction enzymes to obtain strain-specific DNA fingerprints
What is DNA sequencing?
The ultimate typing method
Can detect single base differences between strains of same species
How are uncultivable and novel bacteria identified?
Genomic DNA extracted from sample
General PCR amplification of 16s rRNA gene from bacterial DNA
These genes are separated by cloning into suitable plasma vector and introduced into Ecoli
50 clones are randomly chosen and sequenced using Sanger method.
16s rRNA genes of unknown bacterial species in the sample are analysed using BLAST (compares this of non-bacterial species in database)
- gene sequence match of <97% match, potential novel bacterial species is in the sample.
