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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
Q

Types of DNA arrays

A
  • Spotted array
  • In situ, synthesised array
  • Self assembled array
26
Q

Explain spotted array

A
  • 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
Q

Explain In situ, synthesized array

A
  • Array made by chemical synthesis on solid substrate
  • Photolabile protecting group combined with photolithography to perform the action
28
Q

Explain self assembled array

A
  • 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
Q

Procedure of DNA chip

A
  • 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
Q

What happen to the normal & mutation DNA in DNA chip

A

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
Q

Pros & Cons of DNA chip

A

Pros
- Fast
- Sensitivity
- Multiplexing

Cons
- Expensive
- Limited dynamic range
- Complex

32
Q

Application for DNA chip

A
  • To monitor expression in mRNA populations from living cell
  • To detect DNA sequences polymorphism or mutation in genomic DNA
33
Q

5 ideal sepsis diagnostic test

A
  • Rapid detection
  • Broad based detection
  • High sensitivity &specificity
  • Detect drug resistance
  • Minimal invasiveness
34
Q

Define Digital PCR

A
  • Amplify DNA/RNA to generate million copies that partitioned into thousands of of individual reactions
35
Q

Principle of digital PCR

A
  • Each reaction independently
  • Presence or absence of target sequence determine by measuring fluorescence signal
36
Q

Procedure of digital PCR

A
  • Sample preparation: extract & purify
  • Partitioning: into thousand individuals well
  • Amplification: PCR, results in binary signal
  • Detection: fluorescent base detection
  • Analysis
37
Q

Pros & Cons of digital PCR

A

Pros
- High sensitivity
- High accuracy & reproducibility
- Fast

Cons
- Expensive
- Specialised equipment & expertise
- Limited detection (small sample size)

38
Q

Explain LAMP technology

A
  • 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
Q

Pros of LAMP

A
  • Cheap (no need fluorescence probe)
  • Fast
  • High specificity
40
Q

Explain HDA technology

A
  • Isothermal amplification
  • Use UvrD (DNA helicase) & MutL enzyme & ss binding protein & ss template
41
Q

Procedure of HDA

A
  • 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
Q

Explain nanoparticle based diagnostic assay

A
  • Size: 1-100nm
  • Large surface area, self assemble, low toxicity
43
Q

Types of nanoparticle

A
  • Carbon based nanomaterial
  • Organic based nanomaterial
  • Composite based nanomaterial
  • Inorganic based nanomaterial
44
Q

Explain aptamer based diagnostic assay

A
  • Replace antibody to capture molecule
  • Short ssDNA/RNA that selectively bind to specific target
45
Q

Pros & application of aptamer based diagnostic test

A

Pros
- Cheap
- Versatile
- High specificity & selectivity

Application
- Detect tuberculosis
- Cancer recognition
- Stem cell maker

MIC661:MOLECULAR DIAGNOSTIC (7 decks)

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