SP2 Flashcards
What are the two molecular genetic gene targeting for pathogens?
→Nucleic acid amplification techniques (NAAT)
→Polymerase Chain Reaction (PCR)
How long are DNA primers?
→18-20bp
What is qPCR?
→Measures the speed at which a PCR amplicon product accumulates by the amount of fluorescence released
What is strand displacement amplification?
→Relies on astrand displacement DNA polymeraseand a DNA nicking event targeted via primer design and anicking endonuclease
Which genes are suitable targets for for molecular testing for pathogens?
→Antibiotic resistance →Pathogenic phenotype Repetitive
What pathogens can be detected using strand displacement amplification?
→Neisseria meningitidis- genital, IS711
→Trophyrema whipplei
Examples of pathogens detected using PCR
→Respiratory Syncytial →Virus Influenza A / B
→Hepatitis B & C →Coronavirus →Adenovirus →Cytomegalovirus →Epstein Barr Virus
What factors need to be considered to decide whether molecular test for one gene is good eneough?
→specificity →reliability →sensitivity →accuracy →rapidity
What is MALDI-TOF?
→Matrix Assisted Laser Desorption Ionization-Time-of-Flight
Describe the test for MALDI-TOF
→Isolate organism
→Lyse with crystalizing matrix
→Ionise and detect time of flight for each particle
→Calculate Mwt (Daltons) for each protein produced
→Compare against an archival database
What are the advantages of MALDI-TOF?
→Rapid
→Specific Identification
What are the disadvantages of MALDI-TOF?
→Requires pure culture
→Requires rigorous calibration and protocol standardisation
→Will only identify known profiles
What type of test can be done for biomarkers of virulence?
→serotyping
What are biomarkers of virulence for E.coli?
→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 for virulence by serotyping?
→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
Why look at the assembled product when you can look at ALL the pieces?
→Rapid Next Generation Sequencing
What are the 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 the 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
What is the future of molecular testing?
→Following the host transcriptomic profile with microarrays
→metabolic profiling
Give two examples of metabolic profiling
→urine after malaria infection using nuclear magnetic resonance
for excreted metabolites
→Breath urine after tuberculosis infection using gas chromatography
for volatile organic compounds
Examples of point of care testing
→Lab on a Chip →Nanotechnology
→Microfuidics
