Systems For Detection Of Pathogenesis 2 Flashcards
What is molecular gene targeting?
Aim to detect a gene or gene products that are pathogen specific
Nucleic acid amplification techniques (NAAT)
Polymerase chain reaction (PCR)
What is quantitative PCR?
Measures the speed at which a PCR amplicon product accumulates by the amount of fluorescence released
Which genes are suitable targets?
Constitutive
Virulence
Antibiotic resistance
Pathogenic phenotype Repetitive
What makes a good molecular gene test?
Specificity
Reliability - non essential? Transmissible?
Sensitivity - how many organisms does it take to suggest disease
Accuracy - do we need to detect liver organisms?
Rapidly
What are molecular signatures?
Aim to detect a gene or gene products that are pathogen specific
- single gene target
- Mass spectrometry
How can mass spectrometry be used for bio-signature profiling?
Isolate organism
Lyse with crystallising matrix
Ionise and detect time of flight for each
Calculate Mwt for each protein produced
Compare against archival databases
What are the advantages for MALDI TOF profiling?
Rapid
Specific identification
What are the disadvantages for MALDI TOF profiling?
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 are examples of cell wall antigens that are predictive of virulence?
Gram negative cell wall
Describe how shiva toxin is detected in E. coli O157
1) Enterohaemolysis
2) Agglutination with anti-toxin antibodies
3) 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 biomarkers
- ONLY in vivo testing of cultured pathogen
- infected into an animal model can prove virulence
What is rapid next generation sequencing?
Look at the whole product instead of the assembled one
What are the advantages of using rapid next generation sequencing?
Can show differences between single bases in strains or resistance mutations to antibiotics
What are the disadvantages of rapid next generation sequencing?
Not all of the possible mutations are involved in virulence
Silent mutations can be interagency (between genes) or synonymous (not altering coding)
What are the advantages of molecular detection methods?
Rapid - Faster detection of pathogens than traditional techniques
Allows appropriate, timely anti microbial therapy and infection control intervals
Increased sensitivity over culture and microscopy based techniques in posative samples
Can be automated and has potential for point of care testing
What are the disadvantages of molecular detection methods?
Expensive
Does not screen for unknowns
Requires expertise to interpret
Still requires intensive technical labour
Possibility of contamination
Require complex and efficient methods for extractions of nucleic acid
Negative samples STILL may need a gold standard culture
In culture-negative patients, PCR tests do not significantly decrease time to tuberculosis exclusion.
What does the future look like for bio signalling profiling?
Urine after malaria infection using nuclear magnetic resonance for excreted metabolites
Breath urine after tuberculosis infection using gas chromatography for volatile organic compounds
Rapid point of care testing is being developed
- microfluidics
- nanotechnology
- lab on chip
How can lab on chip become a reality?
Be able to take a sample and get results through phone
Still being developed
