Molecular diagnostics

Question 1

2 pros and one con to molecular assays

Answer 1

Pros: highly sensitive and specific
Cons: very expensive

Question 2

When should you use molecular methods?

Answer 2

When other methods fail or have difficulty:

1. Non-culturable agents
2. Non-viable organisms
3. Slow growing or difficult to grow
4. Culture confirmation - strange biochemical profiles
5. Agents present in low numbers
6. Sensitive detections is sought

Question 3

Incubation period

Answer 3

Period from infection until clinical symptoms

Question 4

Window period

Answer 4

Period from infection until laboratory detection

Question 5

4 Main types of molecular tests

Answer 5

1. Nucleic acid amplification techniques (NAATs)
2. Sequencing
3. Hybridization
4. Molecular epidemiology (outbreak investigations)

Question 6

Why do we purify nucleic acids

Answer 6

PCR inhibitors in clinical specimens can give false negative results

Question 7

PCR inhibitors in:

1. Urine
2. Feces
3. Blood
4. Tissue
5. Others

Answer 7

1. Urea
2. Bile salts, polysaccharides
3. Hemoglobin, anticoagulants (heparin, EDTA)
4. Collagen, melanin, myoglobin
5. Formalin, excess salts, detergents, alcohols (can be introduced during the process)

Question 8

PCR reaction components

5

Answer 8

DNA
Primers (forward and reverse)
dNTPs (G, T, A, C)
Heat stable DNA polymerase
Buffers and MgCl2 (cofactor)

Question 9

PCR temperatures

Answer 9

Denaturation (95degC)
Annealing (50-55 degC)
Extension (72 degC)

Question 10

Gel electrophoresis migration is based on…

Answer 10

Size and charge!
Smaller moves further
Moves towards positive electrode

Question 11

Ethidium bromide

Answer 11

Stain used to visualize DNA after gel electrophoresis

Binds dsDNA and fluoresces when exposed to UV

Question 12

Reverse-transcription PCR

Answer 12

Reverse transcriptase converts RNA to complementary DNA (cDNA), which can then be used as a template for PCR

Question 13

Multiplex PCR

Answer 13

Multiple targets can be detected in a single reaction

Needs primer pairs for each target

Question 14

Qualitative versus Quantitative NAATs

Answer 14

Qualitative: determine presence or absence of an organism
Quantitative: used to quantify an organism (often used to monitor therapy)

Question 15

Real time PCR

Answer 15

No electrophoresis (faster results)
Detection during amplification using fluorescent chemistries
Fluorescence is proportional to the quantity of DNA produced

Question 16

Ct value

Answer 16

PCR cycle where the fluorescence crosses the threshold (can detect)
Lower number of organisms = higher CT

Question 17

3 general ways to prevent amplicon contamination

Answer 17

1. Physical barriers (gloves, gowns, separate areas…)
2. Decontamination (chemicals ex: bleach)
3. Unilateral workflow

Question 18

2 ways to prevent amplicon contamination

Answer 18

1. dUTP and UNG

2. Psoralens

Question 19

UNG

Answer 19

Uracil DNA glycosylase
Degrades U-DNA (but not the primers or template DNA)
Heat labile

Question 20

Psoralens

Answer 20

Added before PCR amplication
Activated by UV following PCR
Psoralens will cross-link DNA = prevents denaturation
Amplicons can no longer be re-amplifed

Question 21

2 things you need for DNA sequencing (Sanger)

Answer 21

Reaction like PCR except use of:
Only one primer at a time
Dideoxynucleotides (ddNTPs) = fluorescently labeled chain terminator

Question 22

3 applications of DNA sequencing

Answer 22

1. Identification of organisms
2. Predict susceptibility to antimicrobials
3. Molecular epidemiology - outbreak investigations

Question 23

16S rRNA PCR and sequencing

Answer 23

PCR: targets highly conserved regions. If an amplicon present = positive for bacteria (detection)
Sequencing: identification - divergent regions are different between bacteria

Question 24

16S rDNA Sequencing advantages versus disadvantages

Answer 24

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

Question 25

Hybridization assays

Answer 25

Based on complementary base-pairing
Ex: line probe assay
Used for the identification of organisms and differentiation of closely related organisms “genotyping”

Question 26

2 ways to look at genetic fingerprints of strains of bacteria

Answer 26

1. Pulsed field gel electrophoresis

2. Whole genome sequencing

Question 27

Pulsed field gel electrophoresis

Answer 27

Chromosomal DNA (up to 12 Mb) - no amplification!
Digested with restriction endonucleases to generate fragments
Electrophoresis - system of alternating current angles