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Microbiology And Immunology > System For Detection Of Pathogens II > Flashcards

System For Detection Of Pathogens II Flashcards

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1
Q

What is molecular gene targeting used for?

A
  • Aim to detect a gene or gene products that are pathogen specific
  • Nucleic acid amplification techniques (NAAT)
  • Polymerase Chain Reaction (PCR)
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2
Q

Describe the types of PCR’s involved in molecular gene targeting?

A
  • Standard PCR: Two DNA primers (18-20bp) specific for opposite DNA strands, used to amplify DNA region -> Exponential amplification, Product is visualised by fluorescent tags or staining in gels for an amplicon of an exact size
  • Quantitive PCR (qPCR): Measures the speed at which a PC amplicon product accumulates by the amount of fluorescence released
  • Strand displacement amplification (SDA): Detects N. gonorrhoea and C. trachomatis
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3
Q

State what type of genes are suitable targets?

A
  • Constitutive -> Genes always expressed due to of high importance or function when expressed
  • Virulence -> Severity of a disease
  • Antibiotic resistance
  • Pathogenic phenotype
  • Repetitive
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4
Q

What type of PCR is used for gene targeting pathogens?

A
  • Multiplex PCR
  • Realtime qPCR
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5
Q

What factors are considered for the choice of a molecular test for one gene? (5)

A
  • Specificity: Is the test unique to the Genus? Species? Туре
  • 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 resistanc, Next Week? - Chronic persistent infections
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6
Q

What are molecular signatures and what methods are used for this?

A
  • Aim to detect a gene or gene products that are pathogen specific
  • Single gene target (PCR): PCR, qPCR
    2. Mass spectrometry (MALDI-TOF): MALDI-TOF = Matrix assisted laser desorption ionisation time of flight
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7
Q

Describe how bio-signature profiling occurs find MALDI-TOF?

A

VD

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8
Q

State the advantages of MALDI TOF profiling?

A
  • Rapid
  • Specific Identification
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9
Q

State the disadvantages of MALDI TOF profiling?

A
  • Requires pure culture
  • Requires rigorous calibration and protocol standardisation
  • Will only identify known profiles
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10
Q

What are biomarkers of virulence and how are they detected?

A
  • Looking for selected genes or gene products that drive the disease process
  • Specific cell wall antigens are predictive of invasiveness and virulence
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11
Q

State a key method that detects for biomarkers of virulence and state 2 examples?

A
  • Serotyping
  • Examples for use: E.coli Type 0157, Shigella flexneri OType 6
  • Also Serology by ELISA eg. Paired sera for Influenza Virus antibody titres
  • CSF Direct Agglutination test
  • Latex particles coated with specific antibodies to cell wall antigens
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12
Q

Describe the method behind shigella toxin detection in E.coli 0157?

A

1) Enterohaemolysis - Destruction of RBCs in intestine (effect of shiga toxin)
2) Agglutination with anti-toxin antibodies
3) PCR for the presence of the gene

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13
Q

Describe advantages for biomarkers of virulence (3)?

A
  • Good Specificity
  • Good Sensitivity
  • Easily automated
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14
Q

Describe disadvantages for biomarkers of virulence (4)?

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 (the reaction of an antibody with an antigen other than the one which gave rise to it.)
  • 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
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15
Q

State the use of the following rapid sequence techniques and what rapid sequencing is used for?

A
  • Rapid sequencing can also be used to identify pathogens. This is useful because it’s very quick sequencing however, the result is a large amount of data.
  • 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
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16
Q

What can direct sequencing show?

A

Sequencing can show differences between SINGLE bases in strains Or resistance mutations to antibiotics

17
Q

What is the key principle behind mutation and virulence?

A
  • Not all possible mutations are involved in virulence
  • Silent mutations can be Intragenic (between genes) or Synonymous (not altering coding)
  • Silent mutations are mutations in DNA that do not have an observable effect on the organism’s phenotype
18
Q

What are the advantages behind molecular detection methods? (5)

A
  • Rapid.
  • Faster detection of pathogens than traditional techniques.
  • Allows timely appropriate 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
19
Q

What are the disadvantages behind molecular detection methods? (8)

A
  • Expensive
  • Does not screen for UNKNOWNS
  • Requires expertise
  • Labour intensive
  • Possibility of contamination
  • Require complex and efficient methods for extraction of nuclei 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.
20
Q

What can bio-signature profiling be used for in future methods of detection of pathogens?

A

VD

21
Q

Describe the two methods metabolic profiling can be performed using urine?

A
  • Urine after malaria infection using nuclear magnetic resonance for excreted metabolites
  • Breath urine after tuberculosis infection using gas chromatography for volatile organic compounds
22
Q

Describe examples of rapid point of care testing as future methods of detecting pathogens?

A
  • Point of Care (POC) testing
  • Lab on a Chip
  • Nanotechnology
  • Microfuidics Rapid sequencing of samples direct at the bedside…..
  • DNA extraction
  • PCR
  • Sequencing on site
23
Q

What criteria should new systems for detecting pathogens include?

A
  • Reliable
  • Sensitive
  • Specific
  • 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)

Microbiology And Immunology (32 decks)

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