Systems for Detection of Pathogens II Flashcards
What are the three ways in which we can look for pathogen molecular signatures?
We aim to detect a gene or gene products that are pathogen specific.
The ways to do this are:
- single gene targeting (PCR)
- multiple gene targeting (microarray)
- mass spectroscopy (MALDI-TOF)
Describe molecular gene targeting using PCR for detecting pathogens.
The aim is to detect a gene or gene products that are pathogen specific.
PCR is amplifying DNA specifically, while using primers of oligonucleotides that decides where the PCR is going to fix onto the DNA, so it is very specific.
So, if you’ve got a gene that in the DNA that is only in one particular organism, you can design PCR to amplify the signal. That can be seen in a gel as either a band of a particular size (and you can use a size chart to identify it) or you can use different types of spectroscopy or fluorescence or different types of dyes to decide how much signal it is. The fluorescence it gives off is a good quantitation of how many of those cells were in that sample when you did it.
What does quantitative PCR do?
Quantitative PCR measures the speed at which a PCR amplicon product accumulates by the amount of fluorescence released.
Describe strand displacement amplification (SDA).
This is very similar to PCR. You’re using primers to go along the DNA strand, and it’s producing a fluorescent signal. However, there are slight differences.
SDA relies on a strand-displacing DNA polymerase, typically Bst DNA Polymerase, Large Fragment or Klenow Fragment (3’-5’ exo–), to initiate at nicks created by a strand-limited restriction endonuclease or nicking enzyme at a site contained in a primer. The nicking site is regenerated with each polymerase displacement step, resulting in exponential amplification.
It is used for N. gonorrhoea and C. trachomatis routinely in the lab.
Make sure not to confuse SDA with PCR.
How do we decide which gene to test for using molecular genetic tests?
We want to use a gene that is only found in the pathogen. Maybe it produces a toxin, so we could look for the gene for the toxin.
The test we are looking for has to be designed depending on what we want the answer to be.
We want it to be sensitive, specific, and reliable. To ensure those are kept up, we have to do lots of tests with thousands of samples.
What are some different questions we need to ask ourselves when designing these genetic tests?
SPECIFICITY:
- Is the test unique to the genus/species/type?
RELIABILITY:
- Is the target non-essential/transmissible?
SENSITIVITY:
How many organisms does it take to suggest disease?
ACCURACY:
- Do we need to detect live organisms?
- Is the detection system susceptible to genomic shifts/mutations?
RAPIDITY:
- Is the result generated going to be beneficial to the patient?:
Instant Bedside? - Diagnosis of paediatric meningitis
Same Day? - Transmission/Quarantine
Next Day? - Antibiotic resistance
Next Week? - Chronic persistent infections
Describe multiple gene targeting (microarrays) for detecting pathogens.
This system uses hybridisation of DNA onto little spots, and each spot represents a gene, and we can have hundreds of thousands of these on one slide.
Since we can tell exactly which gene is present, we have our specificity down.
Here, we don’t just look for the genes, but we also look for the expression of those genes. It may be present, but not being expressed.
Using expression analysis, we can determine whether a gene is turned on, and whether an organism is likely to cause disease.
Describe how we can use mass spectroscopy to detect pathogens.
Mass spectrometry is another way of looking at organisms.
Here, we don’t look for DNA; we look for what the organism consists of. Maybe, it could be something on the cell surface or something that the cell is producing. It could also be a metabolite (that is unique to that organism).
The sample is broken up, then there is laser desorption, then it’s ionised in flight. As it is ionised, it breaks it and pushes it against this accelerator, and it goes down to the detector. As it goes down, the sample bits take charge and get deflected, which increases the time of flight that it takes to get to the detector. The detector, when stimulated, will produce a peak depending on how much of the sample was there at the time.
The peaks will be specific for a certain small fragment of either peptides or other materials. These fragments will make a pattern, which is put together to present a mass spectroscopy graph.
When we obtain the pattern, we can check it against the database, and identify the substance in the sample we put in. Each organism has a unique profile as it behaves in a certain way.
List some advantages and disadvantages of MALDI-TOF profiling.
ADVANTAGES:
- rapid
- specific identification
DISADVANTAGES:
- requires a pure culture
- requires rigorous calibration and protocol standardisation
- will only identify known profiles
What can we use to detect pathogens besides the pathogens themselves?
The virulence factors produced by the pathogen will induce certain symptoms that we can use to work backwards and figure out the type of disease.
So, sometimes instead of looking for foreign organisms, we can look at what’s changed in the body. The simplest way to look for a biomarker would be using an antibody.
What are some ways in which we can identify cell wall antigens?
SEROTYPING:
- E.coli Type O157
- Shigella flexneri OType 6
CSF Direct Agglutination test:
- Neisseria meningitidis Group B
- Haemophilus influenzae type B
- Streptoccoccus pneumoniae
Serology by ELISA:
- paired sera for
Influenza Virus antibodies
Toxin detection:
- Shiga Toxin of E.coli O157
Very briefly, how would you detect the Shiga toxin in E.Coli O157?
1) Enterohaemolysis
2) Agglutination with anti-toxin antibodies
3) PCR for the presence of the gene
What are some advantages and disadvantages of biomarkers of virulence?
ADVANTAGES:
- good specificity
- good sensitivity
- easily automated
DISADVANTAGES:
- serological response is not rapid therefore not useful in acute infections
- single sera results are meaningless due to possible previous exposure
- some antibodies are cross-reactive (they may not be specific enough)
- virulence is only INFERRED by the presence of a biomarker ONLY in vivo testing of cultured pathogen
infected into an animal model can prove virulence
List some advantages and disadvantages of molecular detection methods.
ADVANTAGES
- 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
DISADVANTAGES:
- expensive
- does not screen for UNKNOWNS
- requires expertise
- labour intensive
- possibility of contamination
- requires complex and efficient methods for extraction of nucleic acid
- NEGATIVE samples may STILL need Gold Standard culture
Describe how bio-signature profiling would take place.
Here, we can take a human with an active disease and check to see which one of the genes are turned on when they have the disease. We can also check if they are making antibodies, different types of cytokines, etc.
Then, we can generate a profile that will tell us which genes are on and off that we can use to compare to patients that will help us predict their course of disease. It can help us identify which patients will only be getting worse, and so to focus on them.
