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MIC661:MOLECULAR DIAGNOSTIC > C5 > Flashcards

C5 Flashcards

1
Q

Define molecular typing

A

Essential tool for the analysis of bacterial pathogen obtain from investigation, lab contamination & recurrent infection

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2
Q

Types of multi drug resistant pathogen

A

Gram +ve nosocomial
- Vancomycin resistant Enterococci
- MRSA

Gram -ve bacilli
- Betalactamase E.coli
- Fluoroquinolone resistant E.coli

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3
Q

Typing system characterisation

A

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

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4
Q

Types of molecular typing

A
  • Typing by RFLP
  • Typing by PCR
  • Ribotyping with Southern Blot Analysis
  • Typing by Sequencing analysis
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5
Q

Principle of typing by RFLP

A
  • 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
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6
Q

Procedure of RFLP-PAGE

A
  • 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
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7
Q

Pros & Cons of typing by RFLP

A

Pros
- Fast & simple
- High reliability
- Co dominance - differentiate hetero & homozygotes

Cons
- Incomplete digestion
- Require large amount of sample
- Technically demanding

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8
Q

Application of typing by RFLP

A
  • Detection of bacterial contamination in food
  • Clustered patients with possible epidemiological links
  • False positive culture investigation
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9
Q

Principle in typing by sequencing

A
  • Reproduce typing profile that are highly amenable to standardisation & uniform interpretation due to simple data
  • Use universal sequences
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10
Q

Types of sequence typing

A
  • Single locus sequence typing
  • Multilocus sequence typing
  • Whole genome sequencing
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11
Q

Explain single locus sequence typing

A
  • Target single gene or locus or sequencing
  • Simple & cheap method
  • Not provide same level of resolution as MLST & WGS
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12
Q

Explain multilocus sequence typing

A
  • Sequencing several housekeeping gene & comparing sequences to assign unique allelic profile
  • High resolution & result can be easily shared
  • Time consuming & expensive
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13
Q

Explain whole genome sequencing

A
  • Sequencing entire genome of microorganism
  • Compare sequences to identify variations
  • High resolution & detail information
  • Expensive & complex data analysis
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14
Q

Procedure of typing by sequencing

A
  • 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
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15
Q

Pros & Cons typing by sequencing analysis

A

Pros
- Broad coverage
- High sensitivity
- High reproducibility

Cons
- Expensive
- Complex technique
- Limited throughput

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16
Q

Application of typing by sequencing analysis

A
  • Microbial identification
  • Study new bacterial species via metagenomics study
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17
Q

Define ribotyping

A

Molecular technique for bacterial identification & characterisation that use information from rRNA based phylogenetic analysis

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18
Q

Explain principle of ribotyping

A
  • 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
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19
Q

Explain ribosome in ribotyping

A
  • 16S most conserved rRNA & serve as gold standard for identification & taxonomic
  • Eukaryotes: 28S, 5.8S, 5S, 18S
  • Prokaryotes: 23S, 15S, 16S
20
Q

Procedure in ribotyping

A
  • Sample preparation
  • Library construction
  • Sequencing
  • Data analysis
21
Q

Pros & Cons of Ribotyping

A

Pros
- High sensitivity in differentiation diff taxa
- Less labor
- Abundance

Cons
- Expensive
- Limited information
- Unable differentiate closely related species

22
Q

Application of ribotyping

A
  • Species annotation
  • Phylogeny
  • Diversity analysis
23
Q

Principle in DNA chip

A
  • 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
24
Q

DNA chips are simply

A
  • 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
25
Types of DNA arrays
- Spotted array - In situ, synthesised array - Self assembled array
26
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
27
Explain In situ, synthesized array
- Array made by chemical synthesis on solid substrate - Photolabile protecting group combined with photolithography to perform the action
28
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
29
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
30
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
31
Pros & Cons of DNA chip
Pros - Fast - Sensitivity - Multiplexing Cons - Expensive - Limited dynamic range - Complex
32
Application for DNA chip
- To monitor expression in mRNA populations from living cell - To detect DNA sequences polymorphism or mutation in genomic DNA
33
5 ideal sepsis diagnostic test
- Rapid detection - Broad based detection - High sensitivity &specificity - Detect drug resistance - Minimal invasiveness
34
Define Digital PCR
- Amplify DNA/RNA to generate million copies that partitioned into thousands of of individual reactions
35
Principle of digital PCR
- Each reaction independently - Presence or absence of target sequence determine by measuring fluorescence signal
36
Procedure of digital PCR
- Sample preparation: extract & purify - Partitioning: into thousand individuals well - Amplification: PCR, results in binary signal - Detection: fluorescent base detection - Analysis
37
Pros & Cons of digital PCR
Pros - High sensitivity - High accuracy & reproducibility - Fast Cons - Expensive - Specialised equipment & expertise - Limited detection (small sample size)
38
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
39
Pros of LAMP
- Cheap (no need fluorescence probe) - Fast - High specificity
40
Explain HDA technology
- Isothermal amplification - Use UvrD (DNA helicase) & MutL enzyme & ss binding protein & ss template
41
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
42
Explain nanoparticle based diagnostic assay
- Size: 1-100nm - Large surface area, self assemble, low toxicity
43
Types of nanoparticle
- Carbon based nanomaterial - Organic based nanomaterial - Composite based nanomaterial - Inorganic based nanomaterial
44
Explain aptamer based diagnostic assay
- Replace antibody to capture molecule - Short ssDNA/RNA that selectively bind to specific target
45
Pros & application of aptamer based diagnostic test
Pros - Cheap - Versatile - High specificity & selectivity Application - Detect tuberculosis - Cancer recognition - Stem cell maker

MIC661:MOLECULAR DIAGNOSTIC (7 decks)

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