diagnostic and typing methods Flashcards
(47 cards)
1
Q
what bacteria are associated with periodontal disease
A
- Porphyromonas gingivalis
- Actinobacillus actinomycetemcomitans
- Prevotella intermedia
- Bacteroides forsythus
2
Q
what bacteria are associated with dental caries
A
- streptococco mutans
3
Q
what bacteria are associated with root canal infections
A
- Porphyromonas endodontalis
* Fusobacterium nucleatum
4
Q
what are bacterial detection methods
A
- microbiological culture
- molecular biological
5
Q
what is microbiological culture
A
- This is the traditional way to identify bacteria within clinical specimens
- Culture on suitable agar medium
- Isolate bacteria
- Identify by characterisation of enzyme activities, sugar fermentation tests
6
Q
what is molecular biological
A
- DNA probes
* Polymerase chain reaction (PCR)
7
Q
what is the microbiological culture methods
A
- Vortex mix sample for 30 seconds
- Serial dilutions (to 10-6) in FAB
- Spiral plate to agar media:
- a) Fastidious Anaerobe Agar (FAA) + 7.5% v/v defibrinated horse blood
- b) As a) but supplemented with vancomycin
- (selective agent for Gram-negative anaerobes)
- Incubate anaerobically for 10 days
- Obtain total bacterial counts
8
Q
what is biochemical identification
A
- Anaerobes noted by their sensitivity to metronidazole disc (5μg/disc)
- Gram stain
- Rapid API 32 A: enzymatic activities, sugar fermentation
9
Q
how can you tell the difference between gram positive and negative
A
-We can do Gram staining, this will stain Gram-positive bacteria a violet colour because they have a very thick peptidoglycan layer in their cell wall, they will retain the Gram stain; whereas Gram-negative bacteria only have a very thin peptidoglycan layer in their bacterial cell wall, they will not retain the Gram stain and will not stain
10
Q
what are the advantages of culture methods
A
- yields bacterial isolates for future testing and study
- antibiotic sensitivities
11
Q
what are the disadvantages of culture methods
A
- requires viable cells
- insensitive = won’t detect bacteria unless present within a clinical specimen at levels between 10^5 or 10^6
- only small number of samples analysed at once
- inconclusive results
- labour-intensive
12
Q
what are DNA probes
A
• Segments of DNA that have been labelled with chemoluminescent, fluorescent or radioactive agents
13
Q
what are types of DNA probes
A
- Whole genomic – entire genome
- Cloned gene
- Oligonucleotide – 20-50 bases
14
Q
how do DNA probes work
A
- prepare probe and sample
- heat each to denature it and pull strands apart to expose bases
- label one strand in the probe with label (chemiluminescent, radioactive or fluorescent)
- mix probe with sample in process called hybridisation
- probe binds to complementary sequence of DNA in sample
- remove any non-binding DNA and we then have the labelled DNA probe identified within the sample
15
Q
when were genomic probes used
A
- in the 80’s when we didn’t have any genetic information on different bacterial species
16
Q
how large is the genome of bacterial species
A
- around 4 million bases
17
Q
what is the major pbolemw with whole genomic probe
A
- extremely non-specific
- lots of cross-reactivity between whole genomic probes and DNA from other bacterial species
- fairly unreliable
18
Q
how do cloned gene probes work
A
- these cloned gene probes would be prepared targeting a particular gene that might be unique to that particular bacterial species that you wish to identify within the clinical specimen
- So the gene of interest to be used as a probe would be cloned into E. coli, the cloned fragment isolated, purified and LABEL attached.
- Now cloned gene probes are much more specific than whole genomic probes = deal with a single gene rather than thousands
19
Q
what are oligonucleotide probes
A
- most specific due to small size
- target 16S ribosomal RNA gene
- The synthesised single-stranded oligonucleotide is then labelled and used as a probe as previously described.
- Oligonucleotides are the DNA probes of choice due to their high specificity
20
Q
how long is the 16S rRNA gene
A
- around 1500 base pairs in length
21
Q
why is 16S gene ideal for preparing species-spefic probes
A
possesses nine hypervariable regions (V1 to V9) that contain unique DNA sequences that provide a specific signature for each bacterial species.
22
Q
what is the 16S rRNA gene
A
- Found in all bacteria
- Essential for survival
- Gene sequence for all known bacteria
- Highly variable regions provide unique signatures to any bacterium = species-specific probes or primers
- Conserved regions = ‘broad range’ (consensus) probes or PCR primers
23
Q
what is PCR
A
- polymerised chain reaction
- highly specific
- sensitive
- used to directly detect bacteria in clinical specimens
- PCR is an exponential amplification procedure whereby a single molecule of DNA can be amplified many billions of times to produce large amounts of DNA. Hence, it is of huge value in forensics, but can also be used to detect bacterial within clinical specimens.
24
Q
what does PCR need
A
• PCR requires a double stranded DNA template (from the sample), primers (specific for a particular gene, for example the 16S ribosomal RNA gene of the bacterial species one wishes to detect in a clinical sample), deoxynucleotide triphosphates (dNTPs; the building blocks of DNA) and the enzyme Taq DNA polymerase which catalyses the synthesis of new DNA strands.
25
how does PCR work
* Firstly, the double stranded DNA from the sample is heat denatured at 94C to pull the two DNA strands apart. This is known as the denaturation step.
* The PCR primers then hybridise to their target sequences on each DNA strand. This known as the primer annealing step.
* The Taq DNA polymerase then synthesises the opposite strand, incorporating the dNTPs. This is known as the primer extension step.
* Thus, after each cycle of PCR amplification, the amount of DNA is doubled.
* The cycle is then repeated up to 35 times, so after 35 cycles of PCR amplification there has been a 2 to the power of 35-fold amplification of the original DNA.
26
what do PCR primers usually target
16S rRNA gene
27
what type of PCR primers are there
* General bacterial primers
* Group-specific primers
* Species-specific primers = single or more than one pair (multiplex)
28
what do general bacterial primers do
, which target the conserved (consensus) region of the 16S ribosomal RNA gene
29
what do group-specific primers do
• Or group-specific primers can be developed, for example those that will detect all members of a particular bacterial genus, for example Prophyromonas
30
why are multiplex primers goof
- look at more than one species at a time
| - but no more than 3 primer pairs in a single reaction as can have different requirements for reaction
31
what is a DNA size marker used for in PCR
- allows size of PCR products to be estimated
32
what are the advantages of DNA probes and PCR
- less time consuming = get results in same day
- ver sensitive
- detect bacterial DNA
- don't require viable cells
- detect uncultivated species
33
what are the disadvantages of DNA probes and PCR
- detect dead cells
| - detect only pre-selected species
34
what is PCR-RFLP
* Identification of bacterial isolates
* Digest PCR-amplified bacterial 16S rRNA gene with restriction enzymes
* Yields specific patterns (‘fingerprints’) for individual species
* Rapid, cheaper, more specific alternative to biochemical tests for identifying bacterial isolates from clinical samples
* It can be used for identifying bacterial isolates that have been obtained by culture methods and is far more reliable than biochemical identification methods
35
how is it possible to predict size of DNA fragments when digesting gene with restriction enzymes in PCR-RFLP
- since sequence of 16S rRNA gene has been determined for all known bacterial species
36
why is it important to subtype bacteria
- track routes of transmission during disease outbreaks
| - study pathogenicity of specific strains
37
what are traditional methods for typing of bacterial isolates
- testing for one r more phenotypic marker = serotyping or bio typing
38
what are the problems with typing of bacterial isolates
- limited discriminatory capacity
- organism specific methods
- specialised reagents required
39
what are molecular (genetic) tying methods
* An alternative to traditional subtyping methods are molecular (genetic) typing methods which are DNA-based
* Restriction enzyme analysis (REA)
* Gene probe typing
* Ribotyping
40
what is REA
* Digest whole genomic DNA with restriction enzymes
* The DNA fragments tend to merge into each other, producing a smear effect.
* Because of this, it is almost impossible to discriminate between different strains.
41
what is the problem with REA
* Too many DNA fragments obtained, makes interpretation difficult
* Since many thousands of DNA fragments are generated, they cannot be resolved on an agarose gel, which makes interpretation extremely difficult
42
what is gene probe typing
• Can reduce number of DNA fragments generated by REA using a suitable gene probe
43
what is rubotyping
* Use E. coli rRNA operon (16S-23S-5S) as a DNA probe following REA
* rRNA operon present in multiple copies in bacterial genomes, well conserved in overall structure and sequence due to its evolutionary role in all bacteria
* Variation in number of size of fragments in bacterial DNA digests which are complementary with rRNA
44
how is ribotyping done
* Genomic DNA is extracted from the bacterial isolates or clinical sample and digested with one or more restriction enzymes to give many DNA fragments.
* The DNA fragments are separated into distinct bands by agarose gel electrophoresis – the smaller more mobile DNA fragments migrate more quickly than larger DNA fragments.
* DNA bands in the gel are transferred to a nylon membrane in preparation for hybridisation to the labelled DNA probe (for ribotyping, the probe is the E. coli ribosomal RNA operon).
* The DNA probe binds to specific DNA fragments on the membrane that all or part of the probe sequence.
* The probe is then visualised, and the DNA fragments compared
45
what is the 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 DNA fingerprints
* A very rapid and technically simpler DNA-based typing method is PCR-RFLP analysis of the 16S-23S intergenic spacer region.
46
what is DNA sequencing
* The ultimate typing method
* Can detect single base differences between strains
* Sequences all the bases in a stretch of DNA and detects even single base changes between different bacterial strains of the same species
47
how is molecular identification of uncultivable and novel bacteria done
* Genomic DNA is extracted from the clinical sample
* General bacteria PCR primers are then used to amplify the 16S ribosomal RNA gene from all the bacteria in the clinical sample
* If 200 bacteria are present in the sample, then 200 different bacterial 16S ribosomal RNA genes will be amplified
* Since the 16S ribosomal RNA genes are of a very similar size (approximately 1500 base pairs in length), they must be separated by cloning into a suitable plasmid vector and then introduced into E. coli.
* From the E. coli library generated, 50 clones are randomly chosen and their 16S ribosomal RNA genes sequenced using the Sanger method
* A gene sequence match of 98% or higher with a known bacterial 16S ribosomal RNA gene sequence in the database identifies the clone sequence as having originated from that particular bacterial species, which was therefore present in the sample.
* A gene sequence match of less than 97% with known gene sequences in the public database indicates that a potentially novel bacterial species has been identified in the clinical sample.
