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IUMICRO > MALDI-TOF and Molecular Methods in Microbiology > Flashcards

MALDI-TOF and Molecular Methods in Microbiology Flashcards

1
Q

Objective 1: Differentiate genotypic and phenotypic characteristics for organism identification.

A

Phenotypic
- Observable characteristics and features – analysis of gene products
- Micro I: Conventional Methods (growth requirements, staining, biochemicals)

Genotypic
- ID based on organism’s nucleic acid makeup

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2
Q

Objective 2: Compare turnaround time for identification using conventional (biochemical)
approaches to that of MALDI-TOF MS.

A

Conventional Methods
- TAT: 1-3 DAYS after specimen rec’d in lab

MALDI-TOF MS
- TAT: ID obtained within MINUTES

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3
Q

Objective 3: Recognize the full name for the MALDI-TOF MS system.

A

Matrix Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry

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4
Q

Objective 4: State the identification approach used by the MALDI-TOF system.

A

Proteomic approach: analysis of protein profiles

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5
Q

Objective 5: Describe the principle of each stage of MALDI-TOF MS
- Mass Spectrometry

A

Mass Spectrometry
- Chemical compounds ionized into charged molecules
- Ratio of mass to charge is measured
- Compare mass/charge profile to database to obtain
ID

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6
Q

Objective 5: Describe the principle of each stage of MALDI-TOF MS
- Matrix-assisted

A

Matrix-assisted
- Matrix components include: Solvent (and water) extracts intracellular proteins and then vaporizes allowing it to crystalize with an organic compound

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7
Q

Objective 5: Describe the principle of each stage of MALDI-TOF MS
- Laser Desorption Ionization

A

Laser Desorption Ionization
- Laser light is converted into heat, causing desorption of sample, creating charged ions

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8
Q

Objective 5: Describe the principle of each stage of MALDI-TOF MS
- Time of Flight

A

Time of Flight
- Separation (by mass/charge ratio) and detection of charge ions

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9
Q

Objective 6: Identify the function of the matrix added to the organism isolate during sample preparation

A

Matrix converts laser light into heat

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10
Q

Objective 7: Outline the 3 sample preparation methods discussed for MALDI-TOF MS analysis

A
  1. Direct Transfer
    • On target plate: bacterial colony – matrix
  2. Extended Direct Transfer (“On-target extraction”)
    • On target plate: bacterial colony – formic acid –
      matrix
  3. In-Tube Extraction
    • Washing and chemical extraction before sample
      is loaded on target plate
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11
Q

Objective 8: Evaluate which sample preparation method would be best suited for a given isolate.

A

Extended Direct Transfer (“On-target extraction”)
- Gram-positives, mucoid colonies, yeast

In-Tube Extraction
- ambiguous results, Nocardia, Mycobacteria

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12
Q

Objective 10: Summarize how ions are separated and measured by TOF analyzers using MALDI-TOF MS.

A
  • Separated by mass/charge ratio
  • TOF analyzers measure mass/charge ratio by time required to travel flight tube
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13
Q

Objective 12: Identify factors that affect specimen integrity when using molecular techniques for
direct detection from clinical specimens.

A
  • Specimen type
  • Collection device
  • Time of collection
  • Transport and storage conditions
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14
Q

Objective 13 & 14: Describe the basic principle of nucleic acid hybridization/Define duplex or hybrid as it relates to nucleic acid hybridization

A

Ability of two (2) nucleic acid strands with complementary base sequences to bond with each other forming a duplex/hybrid

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15
Q

Objective 15: Classify which nucleic acid strand is considered the probe and which is the target during
hybridization

A

Probe strand: the reporter-labeled sequence that is complimentary to the sequence of the pathogen we are attempting to detect

Target strand: purified nucleic acids from specimen

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16
Q

Objective 16: Outline the four steps in nucleic acid hybridization

A
  1. Select probe
  2. Purify specimen
  3. Hybridization
  4. Signal detection
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17
Q

Objective 17: Discuss how a nucleic acid probe is designed for hybridization

A

Designed to have a complimentary sequence to the targeted gene

18
Q

Objective 18: Identify the purpose of attaching a reporter molecule (label) to the probe strand during
hybridization.

A

To signal detection of hybridization by instrumentation

19
Q

Objective 19: Define stringency

A

Conditions set for optimal target-probe binding

20
Q

Objective 20: Evaluate the degree of base-pairing required between target and probe in low stringency and high stringency conditions.

A

Low stringency: forgiving – less base-pairing required
High stringency: strict – high degree of base-pairing

21
Q

Objective 21: Outline the 3 sequential reactions that occur during PCR

A
  1. Denaturation of target nucleic acid
  2. Primer annealing to single strand target
  3. Elongation of primer-target duplex
22
Q

Objective 22: State the purpose of an automated thermal cycler instrument.

A

Enables temperature changes between cycle repeats

23
Q

Objective 23: Recognize the temperature at which each stage of the PCR process occurs

A

Denaturing: 94 deg. C
Annealing: 55-62 deg. C (primer-dependent)
Elongation: 72 deg. C

24
Q

Objective 24: Discuss the purpose of nucleic acid extraction and purification during the pre-PCR
process.

A
  • Releases DNA/RNA from organism/clinical sample
  • Removes protein/contaminants preventing degradation of target
25
Q

Objective 25: Identify the ingredients included in the PCR reaction mix.

A
  • Buffer
  • Nucleotides
  • Primer pairs
  • Polymerase
26
Q

Objective 26: State the method of DNA denaturation during PCR.

A

Heat

27
Q

Objective 27: Describe the general design of a primer used in PCR.

A

Short, single-strand sequences complimentary to the target gene — genus-specific/species-specific/antimicrobial resistance-specific

28
Q

Objective 28: Identify the most common target used for PCR amplification of bacterial nucleic acid.

A

16s rRNA

29
Q

Objective 30: Discuss why Taq DNA polymerase is the enzyme utilized in most PCR reactions.

A

Stable at higher temperatures through several amplification cycles

30
Q

Objective 31: Calculate how many copies of a target nucleic acid sequence (amplicons) are made given
the number of PCR cycles.

A

2^n

31
Q

Objective 32: Define amplicon

A

Amplified PCR product

32
Q

Objective 33: Differentiate real-time analysis and end-point analysis of PCR products

A

Real-time analysis: detecting product in real-time

End-point analysis: detecting product after all cycles of PCR are completed

33
Q

Objective 34: Recognize what phase of the PCR reaction is monitored during real-time PCR versus
end-point PCR analysis

A

Real-time PCR: Exponential Phase
End-point PCR: Plataea Phase

34
Q

Objective 35: Define threshold cycle (Ct value) detected during real-time PCR

A

Cycle number at which the fluorescence crosses background threshold

35
Q

Objective 36: Analyze the relationship between a given Ct value and the amount of initial target sequence present in the sample

A

Inversely proportional

36
Q

Objective 38: Define melting temperature (Tm)

A

Temperature at which 50% of DNA denatures into two (2) strands

37
Q

Objective 39: Given a set of nucleic acid sequences, predict which sequence would have the highest
melting temperature (Tm)

A
  • More G-C base pairs & length of nucleic acid product:
    higher melting temp.
38
Q

Objective 40: Explain the principle of multiplex PCR

A

Allows detection of multiple targets in a single reaction utilizing more than one primer pair

39
Q

Objective 41: Recognize common sources of PCR contamination

A
  1. DNA contaminating RNA samples
  2. Cross-contamination among different patient
    samples processed simultaneously
  3. Lab contamination of cloned target sequences
  4. Carryover of PCR products from other PCR
    reactions
40
Q

Objective 42: State how risk of contamination can be minimized

A
  • Separate lab work areas for pre-PCR and post-PCR
    set-up and analysis
  • All PCR procedures should include the use of
    negative and positive controls
41
Q

Objective 43: List 3 applications of molecular testing in microbiology

A
  1. Detection of organisms directly from patient specimens
  2. ID of organisms grown in culture
  3. Detection of virulence factors/antimicrobial drug resistance markers (target genes)
42
Q

Objective 45: Evaluate situations in which molecular identification of organism grown in culture would
be favored over conventional identification methods

A
  • Helpful for slow growing organisms
  • MALDI-TOF is not working

IUMICRO (25 decks)

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