Systems for Detecting Pathogens II Flashcards
1
Q
Purpose of molecular gene targeting
A
Aim to detect a gene or gene products that are pathogen specific
2
Q
Techniques used for molecular gene targeting
A
- Nucleic acid amplification techniques (NAAT)
- Polymerase Chain Reaction (PCR)
3
Q
PCR
A
Amplifying fragments of DNA by using primers/oligonucleotides that decide where the PCR will fit onto the DNA and amplify it.
4
Q
Why is fluorescene used in molecular gene targeting?
A
Used to quantitate how many of the cells are in the samples.
5
Q
Viruses that are commonly looked for on PCR
A
- Influenza/H1N1
- Norovirus
- MRSA
- HIV
- Hepatitis B
- Hepatitis C
- Mycobacterium Tuberculosis
- CMV (looked for in pregnant ladies as causes abortion.
- EBV
6
Q
Summarise PCR
A
- Two DNA primers (18-20bp) specific for opposite DNA strands.
- Used to amplify DNA regions
- Product is visualised by fluorescent tags or staining in gels for an amplicon of an exact size.
7
Q
What is quantitative PCR (qPCR)?
A
Measures the speed at which a PCR amplicon product accumulates by the amount of fluorescene released
8
Q
Name an alternative to PCR
A
Strand Displacement Amplification (SDA)
9
Q
What is strand displacement amplification?
A
- Use primers along the amplicons and produce a fluorescent signal.
- It is slightly different from PCR however.
- This is used in the lab to look at N. gonorrhoeae and C. trachomatis
10
Q
Which genes are suitable targets for molecular gene targeting?
A
- Constitutive
- Virulence
- Antibotic resistance
- Pathogenic phenotype
- Repetitive
11
Q
How to tell if a molecular test for one gene is good enough?
A
- Specificity of the test
- Reliability of the test
- Sensitivity of the test
- Accuracy of the test
- Rapidity of the test
It depends on what we are looking for, and what we are going to do to decide how the test is designed.
12
Q
Why is multiple gene targeting used and how?
A
Microarrays are used and to look at more than one target.
- Ordered short oligonucleotide probes (40-70 mer) attached to slides in defined spots.
- Each spot represents a single gene. There can be thousands of genes on one slide.
- Then conduct comparative genomic hybridisation (CGH) used mostly for DNA.
13
Q
What are tiled arrays (microarrays) used for?
A
Used not just for genes but also for the expression of the gene
14
Q
Advantages of tiled arrays (microarrays)
A
- Covers the whole genome
- Strand dependent
- Can be used for RNA and transcriptomics
- Can look for microRNA
15
Q
What is expression analysis (microarrays) used for?
A
- To look for the cells to see over what the certain amount of time that the expressions will change.
- Important to use all of the above to see what type of organisms we have and when, where it will cause the disease.
16
Q
Why are molecular signatures important?
A
Used to aim to detect a gene or gene products that are pathogen specific
17
Q
How is a molecular signature found?
A
- Single gene target: PCR and qPCR
- Multiple gene target (microarrays)
- Mass spectrometry (MALDI-TOF) = Matrix Assisted Laser Desorption Ionisation - Time - Of - Flight
18
Q
How does a MALDI-TOF work?
A
- Take the sample
- Put into a laser which ionises it.
- When it is ionised, it breaks up and pushes up against the accelerator.
- When it goes to the detector, it takes charge and deflects it one way.
- The detector will produce a peak at the right time. These peaks will be specific for a certain small fragment or something else. This will make a pattern.
19
Q
What is the pattern made from the MALDI-TOF profiling used for?
A
- As the pattern produced can be for anything, it is unique for a pathogen. Then, it can be used against a database and identify the pathogen.
- Then compare the pattern against an archival database, 62500 unique spectral profiles. Identifying 1160 species and 233 genera to decide which pathogen it is.
20
Q
Advantages of MALDI-TOF profiling
A
- Rapid
- Specific identification
- Doesn’t necessarily need to look at DNA but rather what the organism is producing or a metabolite that is unique to that organism.
21
Q
Disadvantages of MALDI-TOI profiling
A
- Requires pure culture: cannot identify specific signals if they’re all on top
- Requires rigorous calibration and protocol standardisation
- Will only identify known profiles: 90% of things we already know, however, difficult if it is a new virus or a mutated virus
22
Q
What are biomarkers of virulence?
A
Selected genes or gene products that drive the disease process.
23
Q
How are biomarkers of virulence detected?
A
The body reacts completely differently to different pathogens but these differences are detected as biomarkers of virulence.
24
Q
What is an example of a biomarker?
A
An antibody that is produced through a protein system. It recognises the antibody and produces a biomarker.
25
What are biomarkers used for?
They are usually against things on the cell surface of the pathogen. These can be used to look for what we are trying to detect e.g. antibodies to look things on the cell wall of the pathogen.
26
What are agglutination tests? Give examples
Tests that are used to show the specific antibodies that are on the cell wall antigens.
Latex agglutination test
CSF direct aggultination tests
27
What is serology?
The scientifc study or diagnostic examination of blood serum.
28
How is serology conducted?
By ELISA e.g. paired sera for influenza virus antibodies to compare the different types of antibodies against different types of antigens.
29
Advantages of serology testing
- Good specificity
- Good sensitivity
- Easily automated
30
Disadvantages of serology testing
- Serological response is not rapid (rely on the body to make antibodies and this is not rapid) therefore, not useful in acute infections. Take 10-14 days
- Single sera results are meaningless due to possible previous exposure - may have already encountered the disease but does not mean you have the disease now.
- Some antibodies are cross-reactive
- Virulence is only inferred by the presence of a biomarker ONLY in vivo testing of culture
31
Rapid sequencing
- Rapid next generation sequencing gives a lot of data
| - There are many different ways of sequencing all done very quickly; however, it produces a lot of data to be analysed.
32
Direct sequencing
- Sequences one base after another
- Sequencing can show differences between single bases in strains or resistance mutations to antibiotics
- Can see easily the changes in bases as they are different colours etc.
33
What do direct sequences tell us?
- They tell us theoretically what is happening.
- Infer information from DNA for example, a specific sequence may be antibiotic resistance. and this can be recognised.
- It gives an idea of what is happening but not the phenotype - just the genotype.
34
How can the phenotype of an organism be identified?
- The only way of doing the phenotype is the traditional method of taking the organism and seeing if it kills the antibiotic.
35
Advantages of molecular detection
- Rapid
- Faster detection of pathogens than traditional techniques
- Allows appropriate, timely antimicrobial therapy and infection control interventions
- Increased sensitivity over culture and microscopy based techniques in POSITIVE samples
- Can be automated and has potential for Point of Care testing
36
Disadvantages of Molecular Detection
- Expensive = look for multiple bases when you may only be looking for one base, this is very expensive.
- Does not screen for UNKNOWNS = sequence blind - not sure what you're actually looking for
- Requires expertise
- Labour intensive
- Possibility of contamination = very common problem, important to have a pure sample
- Require complex and efficient methods of extraction of nucleic acid
- Negative samples may still need Gold standard culture
37
Bio-signature profiling
1. Take an active disease and see which genes are switched on when a patient has this disease.
2. Also look at what is being made, and biochemical problems that occur dependent on the disease.
3. A profile is then being made - this makes it easier to identify a disease based on what has already been observed or tested for.
4. It can also be used to predict which patients are at higher risk.
38
Metabolic Profiling
1. Looking at how the whole body has reacted to a specific disease e.g. smell of someone.
2. For example - bees were trained to know which patients had TB. This is because TBs patients have a specific genetic peak for a specific smell that was a fatty acid only made in TB patients.
39
What is a rapid point of care testing?
- Future of medicine: be able to buy tests to tell what disease you may be showing symptoms of.
- Others ways: Lab on a chip, Nanotechnology, Microfundics, Rapid sequencing of samples direct at the bedside; DNA extraction, PCR and sequencing on site.
40
Why may rapid point of care testing not be the way forward?
- It should still be in the hands of the trained professionals. Therefore, who should be able to do the testing.
41
Summarise what the new systems for detecting pathogens must be
- Reliable, sensitive, specific and preferably rapid
- Applied to the correct specimen
- Must derive from a large reference database
- Constantly updated with new species or variants
- Must be as good or better than the gold standard (direct culture)
