MALDI-TOF and Molecular Methods in Microbiology Flashcards
Objective 1: Differentiate genotypic and phenotypic characteristics for organism identification.
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
Objective 2: Compare turnaround time for identification using conventional (biochemical)
approaches to that of MALDI-TOF MS.
Conventional Methods
- TAT: 1-3 DAYS after specimen rec’d in lab
MALDI-TOF MS
- TAT: ID obtained within MINUTES
Objective 3: Recognize the full name for the MALDI-TOF MS system.
Matrix Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry
Objective 4: State the identification approach used by the MALDI-TOF system.
Proteomic approach: analysis of protein profiles
Objective 5: Describe the principle of each stage of MALDI-TOF MS
- Mass Spectrometry
Mass Spectrometry
- Chemical compounds ionized into charged molecules
- Ratio of mass to charge is measured
- Compare mass/charge profile to database to obtain
ID
Objective 5: Describe the principle of each stage of MALDI-TOF MS
- Matrix-assisted
Matrix-assisted
- Matrix components include: Solvent (and water) extracts intracellular proteins and then vaporizes allowing it to crystalize with an organic compound
Objective 5: Describe the principle of each stage of MALDI-TOF MS
- Laser Desorption Ionization
Laser Desorption Ionization
- Laser light is converted into heat, causing desorption of sample, creating charged ions
Objective 5: Describe the principle of each stage of MALDI-TOF MS
- Time of Flight
Time of Flight
- Separation (by mass/charge ratio) and detection of charge ions
Objective 6: Identify the function of the matrix added to the organism isolate during sample preparation
Matrix converts laser light into heat
Objective 7: Outline the 3 sample preparation methods discussed for MALDI-TOF MS analysis
- Direct Transfer
- On target plate: bacterial colony – matrix
- Extended Direct Transfer (“On-target extraction”)
- On target plate: bacterial colony – formic acid –
matrix
- On target plate: bacterial colony – formic acid –
- In-Tube Extraction
- Washing and chemical extraction before sample
is loaded on target plate
- Washing and chemical extraction before sample
Objective 8: Evaluate which sample preparation method would be best suited for a given isolate.
Extended Direct Transfer (“On-target extraction”)
- Gram-positives, mucoid colonies, yeast
In-Tube Extraction
- ambiguous results, Nocardia, Mycobacteria
Objective 10: Summarize how ions are separated and measured by TOF analyzers using MALDI-TOF MS.
- Separated by mass/charge ratio
- TOF analyzers measure mass/charge ratio by time required to travel flight tube
Objective 12: Identify factors that affect specimen integrity when using molecular techniques for
direct detection from clinical specimens.
- Specimen type
- Collection device
- Time of collection
- Transport and storage conditions
Objective 13 & 14: Describe the basic principle of nucleic acid hybridization/Define duplex or hybrid as it relates to nucleic acid hybridization
Ability of two (2) nucleic acid strands with complementary base sequences to bond with each other forming a duplex/hybrid
Objective 15: Classify which nucleic acid strand is considered the probe and which is the target during
hybridization
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
Objective 16: Outline the four steps in nucleic acid hybridization
- Select probe
- Purify specimen
- Hybridization
- Signal detection
Objective 17: Discuss how a nucleic acid probe is designed for hybridization
Designed to have a complimentary sequence to the targeted gene
Objective 18: Identify the purpose of attaching a reporter molecule (label) to the probe strand during
hybridization.
To signal detection of hybridization by instrumentation
Objective 19: Define stringency
Conditions set for optimal target-probe binding
Objective 20: Evaluate the degree of base-pairing required between target and probe in low stringency and high stringency conditions.
Low stringency: forgiving – less base-pairing required
High stringency: strict – high degree of base-pairing
Objective 21: Outline the 3 sequential reactions that occur during PCR
- Denaturation of target nucleic acid
- Primer annealing to single strand target
- Elongation of primer-target duplex
Objective 22: State the purpose of an automated thermal cycler instrument.
Enables temperature changes between cycle repeats
Objective 23: Recognize the temperature at which each stage of the PCR process occurs
Denaturing: 94 deg. C
Annealing: 55-62 deg. C (primer-dependent)
Elongation: 72 deg. C
Objective 24: Discuss the purpose of nucleic acid extraction and purification during the pre-PCR
process.
- Releases DNA/RNA from organism/clinical sample
- Removes protein/contaminants preventing degradation of target
Objective 25: Identify the ingredients included in the PCR reaction mix.
- Buffer
- Nucleotides
- Primer pairs
- Polymerase
Objective 26: State the method of DNA denaturation during PCR.
Heat
Objective 27: Describe the general design of a primer used in PCR.
Short, single-strand sequences complimentary to the target gene — genus-specific/species-specific/antimicrobial resistance-specific
Objective 28: Identify the most common target used for PCR amplification of bacterial nucleic acid.
16s rRNA
Objective 30: Discuss why Taq DNA polymerase is the enzyme utilized in most PCR reactions.
Stable at higher temperatures through several amplification cycles
Objective 31: Calculate how many copies of a target nucleic acid sequence (amplicons) are made given
the number of PCR cycles.
2^n
Objective 32: Define amplicon
Amplified PCR product
Objective 33: Differentiate real-time analysis and end-point analysis of PCR products
Real-time analysis: detecting product in real-time
End-point analysis: detecting product after all cycles of PCR are completed
Objective 34: Recognize what phase of the PCR reaction is monitored during real-time PCR versus
end-point PCR analysis
Real-time PCR: Exponential Phase
End-point PCR: Plataea Phase
Objective 35: Define threshold cycle (Ct value) detected during real-time PCR
Cycle number at which the fluorescence crosses background threshold
Objective 36: Analyze the relationship between a given Ct value and the amount of initial target sequence present in the sample
Inversely proportional
Objective 38: Define melting temperature (Tm)
Temperature at which 50% of DNA denatures into two (2) strands
Objective 39: Given a set of nucleic acid sequences, predict which sequence would have the highest
melting temperature (Tm)
- More G-C base pairs & length of nucleic acid product:
higher melting temp.
Objective 40: Explain the principle of multiplex PCR
Allows detection of multiple targets in a single reaction utilizing more than one primer pair
Objective 41: Recognize common sources of PCR contamination
- DNA contaminating RNA samples
- Cross-contamination among different patient
samples processed simultaneously - Lab contamination of cloned target sequences
- Carryover of PCR products from other PCR
reactions
Objective 42: State how risk of contamination can be minimized
- 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
Objective 43: List 3 applications of molecular testing in microbiology
- Detection of organisms directly from patient specimens
- ID of organisms grown in culture
- Detection of virulence factors/antimicrobial drug resistance markers (target genes)
Objective 45: Evaluate situations in which molecular identification of organism grown in culture would
be favored over conventional identification methods
- Helpful for slow growing organisms
- MALDI-TOF is not working
