Medical Microbiology: Systems for Detection of Pathogens II Flashcards
What is the aim of molecular gene targeting?
- Aim is to detect a gene or gene products that are pathogen specific
What are some techniques that use molecular gene targeting?
- Nucleic acid amplification techniques (NAAT)
- Polymerase Chain Reaction (PCR)
What are some examples of viruses that we use molecular gene targeting techniques for?
- Influenza/H1N1
- Norovirus
- MRSA
- HIV
- Hepatitis B
- Hepatitis C
- Mycobacterium tuberculosis
- CMV (Cytomegalovirus)
- EBV (Epstein-barr virus)
What does qPCR measure?
- Measures the speed at which a PCR amplicon product accumulates by the amount of fluorescence released
What are some of the advantages of using qPCR?
- Good specificity
- Good quantitative measurements
- Not highly sensitive - can start with just one cell
What is strand displacement amplification (SDA)?
- Technique very similar to PCR - also uses primers and can also produce fluoresence BUT it is differerent
- Used for Neisseria gonorrhoea and Chlamydia trachomatis
What genes are suitable targets for molecular gene targets?
- Specific, sensitive and reliable genes are suitable examples include:
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Constitutive genes - many copies found in one organsism so increases sensitivity
- e.g. IS711 for chlamydia
- Virulence genes
- Antibiotic resistance genes
- Pathogenic phenotype genes
- Repetitive genes
What are the different factors that make a good molecular test?
- Specificity - Is test unique to particular genus or species?
- Reliability - Gene being tested has to be in every single organism
- Sensitivity - How many organisms does it take to suggest disease?
- Accuracy - Is the detection system susceptible to genomic shifts/mutations?
- Rapidity - How quickly does the result need to be found out?
Give an example of a technique that can be sued for multiple gene targeting
- Microarrays
- They’re ordered short oligonucleotide probes attached to slides in defined spots
- Each spot represents a single gene - entire microarray could therefore represent all pathogen genes associated with particlar disease
- Comparative Genomic Hybridisation (CGH) used mostly for DNA
What are the advantages of using microarrys
- Covers the whole genome
- Strand dependant
- Can be used for RNA and Transcriptomics
- Can look for microRNA
- Expression analysis can look at how expression of genes changes over time
- Tiled arrays can look at level of expression of genes
What are molecular signatures?
- Biological features, e.g. protein or DNA sequene, used to detect a gene or gene products that are pathogen specific
What can mass spectrometry (MALDI-TOF) be used to detect when used for molecular signature profiling?
- Not always looking for what’s inside the organism, sometimes looking for:
- Something on the cell surface
- Something the organism is producing
- A metabolite that is unique to that organism (can then be used to produce a biochemical pattern)
How does mass spectrometry work?
- Isolate organism
- Organism Lysed/broken down with crystalizing matrix
- Particles placed into machine and Ionised “in flight”
- Particles detected and time of flight for each particle calculated
- Detector then produces a specific peak for each of the different small peptides
- Each of these small peptides will make a specific biochemical pattern
How do you analyse the specific patterns that are produced from mass spectrometry?
- Calculate Mwt (Daltons) for each protein produced
- Compare pattern against an archival database - 62,500 unique spectral profiles Identifying 1,160 species
What are the advantages and disadvantages of mass spectrometry (MALDI-TOF) profiling?
-
Advantages
- Rapid
- Automated
- Specific identification
-
Disadvantages
- Requires pure culture
- Requires rigorous calibration and protocol standardisation
- Will only identify known profiles
What are biomarkers of virulence?
- Biological substances that are used to look for selected genes or gene products that drive the disease process
Name some tests that use antibodies as biomarkers of virulence
- Latex agglutination test - uses particles coated with specific antibody to cell wall antigens
- CSF Direct Agglutination test - Antibodies present on spots; CSF placed on those spots and if pathogen present then aggultination occurs. No pathogen = no agglutination
What other tests use biomarkers of virulence?
- Serology by ELISA e.g. Paired sera for Influenza Virus antibodies
- Toxin detection e.g. Shiga Toxin of E.coli O157
What tests could you use to detect Shiga Toxin in E.coli O157?
- Enterohaemolysis
- Agglutination with anti-toxin antibodies
- PCR for the presence of the gene
What are the advantages of biomarkers of virulence?
- Good Specificity
- Good Sensitivity
- Easily automated
What are the disadvantages of 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 can rapid next generation sequencing show about the genome of an organism?
- Can sequence entire genome of an organsim -
- Can show differences between single bases in strains or resistance mutations to antibiotics
- Problem with this is that not all possible mutations are involved in virulence
- These are called silent mutations
- Silent mutations can be Intragenic (between genes) or Synonymous (not altering coding)
What are the advantages of molecular detection methods (NGS)?
- 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 the disadvantages of molecular detection methods (NGS)?
- Expensive
- Does not screen for Unknowns - Can’t screen for new pathogens as genome sequence isn’t known
- Requires expertise
- Labour intensive
- Possibility of contamination
- Produces massive amounts of data
- Require complex and efficient methods for extraction of nucleic acid
What techniques are considered the future for detecting pathogens?
- Bio-signature profiling
- Metabolic profiling
- Rapid point of care testing
What is Bio-signature profiling?
- Can identify which human genes are turned on/off during active disease
- Can be used to make a profile of all the different genes that whose expression is altered for different diseases
What is metabolic profiling?
- Can look at how genes as well as whole body reacts to active disease
- E.g. People with tuberculosis infection breathe out voltile fatty acid which has very distinct smell
What is rapid point of care testing?
- Use blood/urine to see if you have a disease
- At home kit - uses DNA extraction, PCR and sequencing
