Systems for Detecting Pathogens II Flashcards
- What is the aim of molecular gene testing?
Aim to detect a gene or gene products that are pathogen specific
- What are two examples of molecular gene testing that we can use?
Nucleic acid amplification techniques (NAAT)
Polymerase Chain Reaction (PCR)
- Give some examples of viruses that in St George’s we test for using molecular gene targeting?
Influenza/H1N1 Norovirus MRSA HIV Hepatitis B Hepatitis C Mycobacterium tuberculosis CMV EBV
- What are some ways we can visualise PCR?
- Can be seen in a gel electrophoresis as a band
- Spectroscopy
- Fluorescence
- Very briefly explain how PCR works?
Two DNA primers (18-20bp) specific for opposite
DNA strands, used to amplify
DNA region
Product is visualised by fluorescent tags or
staining in gels for an amplicon of an exact size
- Why is PCR amplicon important using qPCR important
PCR amplicon is important because we can detect what is it (specificity ) and it gives us a good approximation of how much is in there (quantification)
- How is qPCR different to regular PCR?
Measures the speed at which a PCR amplicon
product accumulates by the amount of
fluorescence released
- What SDA (Strand displacement amplification) similar to?
similar to PCR , you use primers and use primer to go along amplification on your target and producing fluorescent signal. But it is slightly different.
- What two diseases can we test for using SDA?
N. gonorrhoea and C. trachomatis
- How do we know which genes to target for what qualities do we look for?
Constitutive Virulence Antibiotic resistance Pathogenic phenotype Repetitive
- In Chlamydia trachomatis, which gene can we look for?
insertion seq IS711 is used for Chlamydia because multiple copies of this same gene is in every chlamydia organism – when we’re doing PCR or SDA because we can increase the sensitivity really well because of many copies of that single organism
- What are 5 qualities that a test needs, explain each one briefly?
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? for every sample type for every host type for every epidemiological niche
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
- What is multiple gene targeting (microarrays)?
Ordered short oligonucleotide probes (40-70mer) attached to slides in defined
spots.
Each spot represents a single gene
- What is one advantage and disadvantage of Multiple Gene Testing (microarrays)?
Advantage:
- Can test 1000s of genes to see if they are present
Disadvantage
-Doesn’t tell us how much of one gene is present
- What are microarrays - Tlled Arrays?
-Looks for genes as well as expression of genes and whether certain genes are being expressed eg are we expressing a certain toxin genes
- What are four disadvantages of Tiled Arrays?
Covers the whole genome
Strand dependant
Can be used for RNA and Transcriptomics
Can look for microRNA
- What are molecular signatures ?
Aim to detect a gene or gene products that are pathogen specific/ what the organism is producing
- What are some examples of what machinery /techniques you can use to finf molecular signatures?
Which one is fastest
—>Single gene target (PCR or qPCR)
–>Multiple gene target (Microarray)
—->Mass spectrometry (MALDI-TOF) = FASTEST
- What can MALDI-TOF tell us about an organism?
what it produces, what’s in on its cell surface, a metabolite of what the organism is producing that is unique to the organism
20. Briefly explain how MALDI-TOF works? Mentioning the three main steps: Isolate organism Lyse with crystalizing matrix Ionise and detect time of flight for each particle
Sample is broken up, laser de-absorption, ionised in flight , as its ionised it breaks it up, pushed against accelerator, as its pushed down it takes charge, deflects charge or decreases TOF when it reaches detector – peaks produces- specific to small fragments of peptides eg – makes a pattern – what we’re looking at systemic Taxonomy
- What are 2 advantages of MALDI-TOF?
Rapid
Specific Identification
- What are 3 disadvantages of MALDI-TOF?
Requires pure culture
Requires rigorous calibration and protocol standardisation
Will only identify known profiles
- What are Biomarkers of Virulence?
Looking for selected genes or gene products that drive the disease process
- What part of the cell can we look at biomarkers?
Cell walls
- What is the latex agglutination test?
Give an example of what sample we can take from the patient
uses particles coated with specific antibody to cell wall antigens if organism inside, will agglutinate . Due to antibody specificity. Takes about 30seconds to do
For eg CSF- cerebrospinal fluid
- What are two specific examples of biomarkers of virulence?
- Toxin Detection
- Serotyping
- Explain how serotyping can be a biomarker of virulence?
Specific cell wall antigens are predictive of invasiveness and virulence
- Give some examples of different serotypes and how they can be detected?
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:
eg. Paired sera for
Influenza Virus antibodies
- Give an example of a toxin we could detect and how we would detect it?
Shiga Toxin detection in E.coli O157
1) Enterohaemolysis
2) Agglutination with anti-toxin antibodies
3) PCR for the presence of the gene
- What are three advantages of using biomarkers of virulence?
Good Specificity
Good Sensitivity
Easily automated
- What are four disadvantages of using biomarkers of virulence?
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
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
- What is rapid sequencing?
used to determine the order of the four bases, ie all the genes
- Although rapid sequencing why can it be quite difficult once you have the data?
- Huge amount of Data- How do we analyse all of it, do we need THAT much data? Are we asking the right amount of questions, lots of different types of sequencing machines
- What is the difference between NGS and Direct Sequencing (eg Sanger)??
While the Sanger method only sequences a single DNA fragment at a time, NGS is massively parallel, sequencing millions of fragments simultaneously per run.
- What is the Oxford Nanopore USB memory stick platform and what type of rapid sequencing does it use?
Uses NGS
USB that codes billions of bases in a day
- What are some examples of machines that can be used for sequencing and their associated mechanism of sequencing?
454 = Pyrosequencing SOLiD = Rapid Ligation Illumina (Solexa)= Fixed slide amplification Ion Torrent = Semiconductor sequencing Heliscope = Single molecule sequencing Pacific Biosciences= Real Time SMS ONT = Nanopore electrical sensing
- What can sequencing tell us about an organism ?
- SNPs
- Resistance to bacteria (ie if they code for a certain protein)
- What are the two types of silent mutations?
Intragenic (between genes)
or
Synonymous (not altering coding)
- What are some advantages of molecular detection methods?
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
- What are some disadvantages of molecular detection methods?
Expensive
Does not screen for UNKNOWNS
Requires expertise
Labour intensive
Possibility of contamination
Require complex and efficient methods for extraction of nucleic acid
NEGATIVE samples may STILL need Gold Standard culture
Hospitalization costs accounted for 95% of health-system costs
among patients suspected of tuberculosis. In culture-negative
patients, PCR tests do not significantly decrease time to tuberculosis
exclusion
- What is the FUTURE for detecting pathogens?
- > Bio Signature Profiling (Following the host transcriptomic profile with microarrays)
- > Look at which genes are turned on during disease
- > Metabolic Profile (Smelling disease eg TB produces a smell, use gas chromatography or NMR to look for certain compounds)
- > Rapid Intrinsic Fluoresce
- > Point of care testing (lab on a chip, test yourself, data sent to lab -PCR and gene sequencing- results sent to your phone)
