Medical Microbiology: Systems for Detection of Pathogens II Flashcards

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1
Q

What is the aim of molecular gene targeting?

A
  • Aim is to detect a gene or gene products that are pathogen specific
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2
Q

What are some techniques that use molecular gene targeting?

A
  • Nucleic acid amplification techniques (NAAT)
  • Polymerase Chain Reaction (PCR)
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3
Q

What are some examples of viruses that we use molecular gene targeting techniques for?

A
  • Influenza/H1N1
  • Norovirus
  • MRSA
  • HIV
  • Hepatitis B
  • Hepatitis C
  • Mycobacterium tuberculosis
  • CMV (Cytomegalovirus)
  • EBV (Epstein-barr virus)
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4
Q

What does qPCR measure?

A
  • Measures the speed at which a PCR amplicon product accumulates by the amount of fluorescence released
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5
Q

What are some of the advantages of using qPCR?

A
  • Good specificity
  • Good quantitative measurements
  • Not highly sensitive - can start with just one cell
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6
Q

What is strand displacement amplification (SDA)?

A
  • Technique very similar to PCR - also uses primers and can also produce fluoresence BUT it is differerent
  • Used for Neisseria gonorrhoea and Chlamydia trachomatis
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7
Q

What genes are suitable targets for molecular gene targets?

A
  • Specific, sensitive and reliable genes are suitable examples include:
  • 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
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8
Q

What are the different factors that make a good molecular test?

A
  • 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?
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9
Q

Give an example of a technique that can be sued for multiple gene targeting

A
  • 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
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10
Q

What are the advantages of using microarrys

A
  • 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
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11
Q

What are molecular signatures?

A
  • Biological features, e.g. protein or DNA sequene, used to detect a gene or gene products that are pathogen specific
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12
Q

What can mass spectrometry (MALDI-TOF) be used to detect when used for molecular signature profiling?

A
  • 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)
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13
Q

How does mass spectrometry work?

A
  • 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
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14
Q

How do you analyse the specific patterns that are produced from mass spectrometry?

A
  • Calculate Mwt (Daltons) for each protein produced
  • Compare pattern against an archival database - 62,500 unique spectral profiles Identifying 1,160 species
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15
Q

What are the advantages and disadvantages of mass spectrometry (MALDI-TOF) profiling?

A
  • Advantages
    • Rapid
    • Automated
    • Specific identification
  • Disadvantages
    • Requires pure culture
    • Requires rigorous calibration and protocol standardisation
    • Will only identify known profiles
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16
Q

What are biomarkers of virulence?

A
  • Biological substances that are used to look for selected genes or gene products that drive the disease process
17
Q

Name some tests that use antibodies as biomarkers of virulence

A
  • 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
18
Q

What other tests use biomarkers of virulence?

A
  • Serology by ELISA e.g. Paired sera for Influenza Virus antibodies
  • Toxin detection e.g. Shiga Toxin of E.coli O157
19
Q

What tests could you use to detect Shiga Toxin in E.coli O157?

A
  • Enterohaemolysis
  • Agglutination with anti-toxin antibodies
  • PCR for the presence of the gene
20
Q

What are the advantages of biomarkers of virulence?

A
  • Good Specificity
  • Good Sensitivity
  • Easily automated
21
Q

What are the disadvantages of biomarkers of virulence?

A
  • 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
22
Q

What can rapid next generation sequencing show about the genome of an organism?

A
  • 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)
23
Q

What are the advantages of molecular detection methods (NGS)?

A
  • 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
24
Q

What are the disadvantages of molecular detection methods (NGS)?

A
  • 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
25
Q

What techniques are considered the future for detecting pathogens?

A
  • Bio-signature profiling
  • Metabolic profiling
  • Rapid point of care testing
26
Q

What is Bio-signature profiling?

A
  • 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
27
Q

What is metabolic profiling?

A
  • 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
28
Q

What is rapid point of care testing?

A
  • Use blood/urine to see if you have a disease
  • At home kit - uses DNA extraction, PCR and sequencing