C5 Flashcards
Define molecular typing
Essential tool for the analysis of bacterial pathogen obtain from investigation, lab contamination & recurrent infection
Types of multi drug resistant pathogen
Gram +ve nosocomial
- Vancomycin resistant Enterococci
- MRSA
Gram -ve bacilli
- Betalactamase E.coli
- Fluoroquinolone resistant E.coli
Typing system characterisation
Typeability
- Ability of techniques to assign unambiguous result
Reproducibility
- Ability to yield same results upon repeat testing
Discriminatory
- Ability to differentiate among epidemiology unrelated isolate
Types of molecular typing
- Typing by RFLP
- Typing by PCR
- Ribotyping with Southern Blot Analysis
- Typing by Sequencing analysis
Principle of typing by RFLP
- Chromosomal DNA digested with restriction enzyme resulting in series of fragments w diff pattern
- Difference in pattern known as RFLP
- Analyse by PFGE to allow separation of DNA of 20-1000kbp
Procedure of RFLP-PAGE
- Bacteria cell embedded in gel block
- Cell lysis & release of intact chromosomal DNA by soaking the gel block in lysis solution (lysozyme)
- Restriction endonuclease digestion
- Separation of DNA fragments by PFGE at 14C for 22 hours
- Staining by ethidium bromide
- Analysis of DNA RFLP
Pros & Cons of typing by RFLP
Pros
- Fast & simple
- High reliability
- Co dominance - differentiate hetero & homozygotes
Cons
- Incomplete digestion
- Require large amount of sample
- Technically demanding
Application of typing by RFLP
- Detection of bacterial contamination in food
- Clustered patients with possible epidemiological links
- False positive culture investigation
Principle in typing by sequencing
- Reproduce typing profile that are highly amenable to standardisation & uniform interpretation due to simple data
- Use universal sequences
Types of sequence typing
- Single locus sequence typing
- Multilocus sequence typing
- Whole genome sequencing
Explain single locus sequence typing
- Target single gene or locus or sequencing
- Simple & cheap method
- Not provide same level of resolution as MLST & WGS
Explain multilocus sequence typing
- Sequencing several housekeeping gene & comparing sequences to assign unique allelic profile
- High resolution & result can be easily shared
- Time consuming & expensive
Explain whole genome sequencing
- Sequencing entire genome of microorganism
- Compare sequences to identify variations
- High resolution & detail information
- Expensive & complex data analysis
Procedure of typing by sequencing
- Sample preparation (Proteinase K extraction & Spin column DNA)
- DNA amplification (PCR)
- PCR purification (Spin column based PCR)
- Sequencing pre preparation (Denature, Label dNTPs)
- DNA sequencing
Pros & Cons typing by sequencing analysis
Pros
- Broad coverage
- High sensitivity
- High reproducibility
Cons
- Expensive
- Complex technique
- Limited throughput
Application of typing by sequencing analysis
- Microbial identification
- Study new bacterial species via metagenomics study
Define ribotyping
Molecular technique for bacterial identification & characterisation that use information from rRNA based phylogenetic analysis
Explain principle of ribotyping
- Use restriction enzyme to target & cut region of ribosomal RNA (16S, 23S & 5S)
- Generate DNA fingerprint that is unique to strain
- 16S, 23S & 5S is a polycistronic operon
Explain ribosome in ribotyping
- 16S most conserved rRNA & serve as gold standard for identification & taxonomic
- Eukaryotes: 28S, 5.8S, 5S, 18S
- Prokaryotes: 23S, 15S, 16S
Procedure in ribotyping
- Sample preparation
- Library construction
- Sequencing
- Data analysis
Pros & Cons of Ribotyping
Pros
- High sensitivity in differentiation diff taxa
- Less labor
- Abundance
Cons
- Expensive
- Limited information
- Unable differentiate closely related species
Application of ribotyping
- Species annotation
- Phylogeny
- Diversity analysis
Principle in DNA chip
- Use to measure expression level of large number of gene simultaneously
- DNA spot contain picomoles of specific DNA sequences (probes)
- Short DNA use to hybridise cDNA or cRNA
DNA chips are simply
- Glass surface
- Array of DNA fragments at discrete address
- Fragments for hybridisation
- DNA spot on chip are hybridised to complex sample of fluorescent label DNA or RNA
Types of DNA arrays
- Spotted array
- In situ, synthesised array
- Self assembled array
Explain spotted array
- Array made on pol-lysine coated glass microscope slide
- Provide binding high density DNA by slotted pin
- Allow fluorescent labelling of the sample
- When spotted array (pen) dip into solution contain DNA & physically deposited on 1x3 glass microscope slide
Explain In situ, synthesized array
- Array made by chemical synthesis on solid substrate
- Photolabile protecting group combined with photolithography to perform the action
Explain self assembled array
- Fibre optic array made by deposition of DNA synthesis on small polystyrene bead
- Bead are deposited on etched end of the array
- Different DNA can be synthesised on different beads & applying mixture of bead to fiber optic array
Procedure of DNA chip
- Gene extracted from specimen labelled fluorescent green
- Reference standard sample labelled fluorescent red
- Hybridise to DNA microarray
- DNA microarray washed
- Scanner detect signal
- Examinations of ratio of red to green signal
What happen to the normal & mutation DNA in DNA chip
Normal
- Red & green sample bind to sequence that represent normal sequence
Mutation
- DNA will not bind properly to DNA sequences that represent the normal sequence
- Bind to sequence that represent mutated DNA
Pros & Cons of DNA chip
Pros
- Fast
- Sensitivity
- Multiplexing
Cons
- Expensive
- Limited dynamic range
- Complex
Application for DNA chip
- To monitor expression in mRNA populations from living cell
- To detect DNA sequences polymorphism or mutation in genomic DNA
5 ideal sepsis diagnostic test
- Rapid detection
- Broad based detection
- High sensitivity &specificity
- Detect drug resistance
- Minimal invasiveness
Define Digital PCR
- Amplify DNA/RNA to generate million copies that partitioned into thousands of of individual reactions
Principle of digital PCR
- Each reaction independently
- Presence or absence of target sequence determine by measuring fluorescence signal
Procedure of digital PCR
- Sample preparation: extract & purify
- Partitioning: into thousand individuals well
- Amplification: PCR, results in binary signal
- Detection: fluorescent base detection
- Analysis
Pros & Cons of digital PCR
Pros
- High sensitivity
- High accuracy & reproducibility
- Fast
Cons
- Expensive
- Specialised equipment & expertise
- Limited detection (small sample size)
Explain LAMP technology
- Use 4 primer & 6 recognition sites per target to achieve high level of amplicon in 1 hour
- Inner primer: start target amplification
- Outer primer: start round of replication
- Generate ss template with denaturation
- Pyrophosphate ion precipitation by add of Mg
- Positive result: turbidity
Pros of LAMP
- Cheap (no need fluorescence probe)
- Fast
- High specificity
Explain HDA technology
- Isothermal amplification
- Use UvrD (DNA helicase) & MutL enzyme & ss binding protein & ss template
Procedure of HDA
- Denaturation
- Uses UvrD & MutL to catalyse temperature independent creation of ssDNA template
- UrvD/MutL unwind dsDNA
- SSB bind to denature strand
- Primer anneal
- DNA poly extend
Explain nanoparticle based diagnostic assay
- Size: 1-100nm
- Large surface area, self assemble, low toxicity
Types of nanoparticle
- Carbon based nanomaterial
- Organic based nanomaterial
- Composite based nanomaterial
- Inorganic based nanomaterial
Explain aptamer based diagnostic assay
- Replace antibody to capture molecule
- Short ssDNA/RNA that selectively bind to specific target
Pros & application of aptamer based diagnostic test
Pros
- Cheap
- Versatile
- High specificity & selectivity
Application
- Detect tuberculosis
- Cancer recognition
- Stem cell maker
