Methods Of Detection (Pathogenic Bacteria) Flashcards
Does universal surveillance work?
Yes
Requirements of bacterial surveillance [7]
Sensitive
Specific
Rapid
Cost effective
Reliable
Easy to perform
Doesn’t require specialised interpretation
Name PHENOTYPIC methods [3]
Biochemical
Chromogenic media
MALDI TOF
Name GENOTYPIC detection (molecular diagnosis) [3]
Real time polymerase chain reaction (PCR)
WHOLE genome sequencing
Multi omics approach
What is the colour reflected based on?
The wavelength at which the molecule absorbs light
What is a Chromophore
The part of a molecule (which contains the electrons)
Involved in an electronic transition!
What is an electronic transition (chromophore)
Electrons in molecule being excited from one energy level to another.
The difference in energy between highest occupied (HOMO) and lowest unoccupied (LUMO) = Decreases,
So the Max absorption increases
Requirements of a chromogen
1.FREE chromogen = has a strong colour
- Colour is MUTED = when attached to targeting molecule
- Target molecule is RECOGNISED by bacterial ENZYME
- LINK between targeting molecule and chromogen is broken by the SPECIFIC bacterial enzyme
Why are L-Alanyl and B-Alanyl containing substrates used as bacterial enzymes?
Gram -ve bacteria have L alanyl aminopeptidase and hydrolyse substrate to release colour =
Differentiates gram +ve (no colour) from gram -ve
also B-alanyl aminopeptidase is only expressed in PSEUDOMONAS AERUGINOSA (PURPLE COLOUR)
How does PCR technology work
- Denature DNA
- Annealing of upstream and downstream PRIMERS for DNA sequence of interest
- Thermus Aquaticus (Taq) = DNA polymerase then Catalyses DNA replication
In real time PCR of MRSA
which DNA primers are used?
Primers that are specific for the mecA and S.aureus specific orfX genes
To allow for variations in the staphylococcal cassette Chromosome (CSSmec)
What is the
Staphylococcal cassette Chromosome
SCCmec?
A mobile genetic element which carries the mecA gene
Which encodes the B-lactam-resistant Penicillin Protein (PBP2)
Drawbacks of quantitative PCR (REAL TIME PCR)
High level of AMPLICATION =
contamination or detection of nucleic acids which remained from previously cleared infections csn be present
In qPCR, how do you overcome discrimination of between ASYMPTOMATIC colonisation and clinically relevant infection?
From standardised quantitative cut off cycle threshold values (Ct)
What is a Ct value
Number if PCR cycles required for the flurorescene signal to cross the threshold (background level).
Lower values = higher pathogenic bacterial.loads
What is the difference between TRADITIONAL and REAL TIME
PCR
Traditional PCR = use immunoassays or agarose gels
REAL TIME = MOLECULAR BEACONS (single stranded probes which have a DNA recognition sequence with a fluorescent dye at one end and quencher at other.
What is the difference between TRADITIONAL and REAL TIME
PCR
Traditional PCR = use immunoassays or agarose gels
REAL TIME = MOLECULAR BEACONS (single stranded probes which have a DNA recognition sequence with a fluorescent dye at one end and quencher at other.
MALDI TOF MS,
How does it work
ENERGY transferred to matrix, from a laser beam
The matrix employed has a chromophore which absorbs at the wavelength of the laser
Matrix absorbs a pulse of energy and undergoes rapid heating
Heating leads to the vaporisation and ionisation of the analyte molecules
MALDI TOF MS
what is analysed
Molecular Weights of ions analysed by
Time taken to reach the detector
MALDI TOF MS
DETECTION OF MRSA vs. MSSA
what was found?
MRSA = MORE PEAKS
they are different bases in peptides they produce
What does WHOLE GENOME SEQUENCING PROVIDE US WITH…
- Identication of pathogens
- Exact profiling of resistance genes
- Recognition of outbreaks
- Immediate design of PCR probes (based on the generated genetic data jn the event of outbreaks)
Criteria for POINT OF CARE diagnosis [6]
- Affordable
- Specific
- User friendly
- Rapid and robust
- Equipment free
- Deliverable
