Molecular diagnostics Flashcards
2 pros and one con to molecular assays
Pros: highly sensitive and specific
Cons: very expensive
When should you use molecular methods?
When other methods fail or have difficulty:
- Non-culturable agents
- Non-viable organisms
- Slow growing or difficult to grow
- Culture confirmation - strange biochemical profiles
- Agents present in low numbers
- Sensitive detections is sought
Incubation period
Period from infection until clinical symptoms
Window period
Period from infection until laboratory detection
4 Main types of molecular tests
- Nucleic acid amplification techniques (NAATs)
- Sequencing
- Hybridization
- Molecular epidemiology (outbreak investigations)
Why do we purify nucleic acids
PCR inhibitors in clinical specimens can give false negative results
PCR inhibitors in:
- Urine
- Feces
- Blood
- Tissue
- Others
- Urea
- Bile salts, polysaccharides
- Hemoglobin, anticoagulants (heparin, EDTA)
- Collagen, melanin, myoglobin
- Formalin, excess salts, detergents, alcohols (can be introduced during the process)
PCR reaction components
5
DNA Primers (forward and reverse) dNTPs (G, T, A, C) Heat stable DNA polymerase Buffers and MgCl2 (cofactor)
PCR temperatures
Denaturation (95degC)
Annealing (50-55 degC)
Extension (72 degC)
Gel electrophoresis migration is based on…
Size and charge!
Smaller moves further
Moves towards positive electrode
Ethidium bromide
Stain used to visualize DNA after gel electrophoresis
Binds dsDNA and fluoresces when exposed to UV
Reverse-transcription PCR
Reverse transcriptase converts RNA to complementary DNA (cDNA), which can then be used as a template for PCR
Multiplex PCR
Multiple targets can be detected in a single reaction
Needs primer pairs for each target
Qualitative versus Quantitative NAATs
Qualitative: determine presence or absence of an organism
Quantitative: used to quantify an organism (often used to monitor therapy)
Real time PCR
No electrophoresis (faster results)
Detection during amplification using fluorescent chemistries
Fluorescence is proportional to the quantity of DNA produced
Ct value
PCR cycle where the fluorescence crosses the threshold (can detect)
Lower number of organisms = higher CT
3 general ways to prevent amplicon contamination
- Physical barriers (gloves, gowns, separate areas…)
- Decontamination (chemicals ex: bleach)
- Unilateral workflow
2 ways to prevent amplicon contamination
- dUTP and UNG
2. Psoralens
UNG
Uracil DNA glycosylase
Degrades U-DNA (but not the primers or template DNA)
Heat labile
Psoralens
Added before PCR amplication
Activated by UV following PCR
Psoralens will cross-link DNA = prevents denaturation
Amplicons can no longer be re-amplifed
2 things you need for DNA sequencing (Sanger)
Reaction like PCR except use of:
Only one primer at a time
Dideoxynucleotides (ddNTPs) = fluorescently labeled chain terminator
3 applications of DNA sequencing
- Identification of organisms
- Predict susceptibility to antimicrobials
- Molecular epidemiology - outbreak investigations
16S rRNA PCR and sequencing
PCR: targets highly conserved regions. If an amplicon present = positive for bacteria (detection)
Sequencing: identification - divergent regions are different between bacteria
16S rDNA Sequencing advantages versus disadvantages
Pro: identification of most bacteria whether viable or not
Con: Only good for monomicrobial infections (or pure cultures), if polymicrobial PCR still positive but sequencing is ruined, so only applied to normally sterile tissues or body fluids
Hybridization assays
Based on complementary base-pairing
Ex: line probe assay
Used for the identification of organisms and differentiation of closely related organisms “genotyping”
2 ways to look at genetic fingerprints of strains of bacteria
- Pulsed field gel electrophoresis
2. Whole genome sequencing
Pulsed field gel electrophoresis
Chromosomal DNA (up to 12 Mb) - no amplification! Digested with restriction endonucleases to generate fragments Electrophoresis - system of alternating current angles
