MICR5832 L20: PCR Diagnostic Methods 1/19/26

What can you use PCR to amplify?

-Viruses

-Bacteria

-Fungi

-Amoeba

-Protists

-Specific genes

Why is PCR constrained by design?

1) Sequence target must be in existing databases

2) Sequence length must be right size

3) Thermodynamically suitable

What is this?

-Sensitivity

Sequence target is conserved across pathogen

What is this?

-Specificity

Sequence target is different enough from non-target sequences

What are the principles of diagnostic PCR?

1) Extract

2) Amplify

3) Analyze

What happens during 1) PCR Extraction?

-Remove purified nucleic acid from cells

-Removes inhibitors

-Concentrate nucleic acid, convert -> liquid

What sample types would you expect to be ready to undergo nucleic acid extraction without being processed?

-Liquid samples

-Urine and blood

What is used to liberate nucleic acid from cells and virions?

-Chemical and physic process

-Solid phase extraction (Boom Method)

What is this extraction method?

-Simple, rapid, reliable, cost effective

-Convenient, ready to use

-Automated for high throughput

-Wide range

Solid phase extraction (Boom Method)

What happens during this step of extracting nucleic acids?

1) Lysing

-Nucleic acid liberated from cells/virions

-Detergents and chaotropic agents used

What is an example of a detergent used to lyse cells during extraction?

Triton-X100

What is an example of a chaotropic agent used to lyse cells during extraction?

Guanidinium thiocyanate

What happens during this step of extracting nucleic acids?

2) Binding

-Nucleic acid binds to silica solid phase

-Due to presence of chaotropic salt in alcohol

-Silica in filter columns, on magnetic beads

What agent enables nucleic acid to bind to silica solid phase?

Chaotropic salt in alcohol

Where is the Silica located?

-Filter columns

-Magnetic beads

How does Silica get the nucleic acid to bind to it?

-Silica attracts negatively-charged DNA molecules

-Chaotropic agents allow salt bridge to form

What happens during this step of extracting nucleic acids?

3) Washing

-Bound nucleic acid washed with polar buffers

-Multiple washes required to remove inhibitors

True or False: Wash buffers keep bonds intact whilst removing inhibitors and cellular debris

True

What happens during this step of extracting nucleic acids?

4) Elution

-Nucleic acid is eluted from silica

-Uses an aqueous solution (water buffered or unbuffered)

List the steps in Extraction of nucleic acids for PCR

1) Lyse

2) Bind

3) Wash

4) Elute

What is in the PCR sample being amplified?

-Purified nucleic acids

-DNA/RNA

What is in the PCR Mastermix for Amplification?

-DNA Polymerase enzyme

-Primers

-dNTPs

-MgCl2 in aqueous buffer

-Reverse transcriptase

-Probes for real-time PCR

What happens during 2) Amplification?

1) Denaturation at 95C

2) Annealing at 68C

3) Elongation at 72C

What happens during this step of Amplification?

1) Denaturation at 95-96C

-Make dsDNA -> ssDNA

-Reverse transcribe RNA to cccDNA

What happens during this step of Amplification?

2) Annealing at 68C

-Primers stick to complementary sequences

-Lowered temperature

What happens during this step of Amplification?

3) Elongation at 72C

-Raise temperature so DNA polymerase can work

-DNA Pol pulls in DNTPs to make complementary strand

What happens during 3) Analysis?

-Find signal if pathogen is present in sample.

-Presence of PCR amplified product

What are 5 examples of PCR analysis?

1) Conventional PCR

2) RT-PCR

3) Multiplex PCR

4) Quantitative PCR

5) Digital PCR

What are the benefits of Conventional PCR?

1) Not limited by primer and probe design constraints

2) DNA of PCR products can be sequenced

3) Gels provide a measure of fragment size and can be used to separate fragments of different sizes

What is the gold standard for PCR?

Real-Time PCR

What is this?

-Detection and amplification occur in the same machine

-Includes hybridization probes, intercalating dyes

-Fluorescence read at each cycle

Real Time PCR

What are the parts of a hydrolysis probe?

1) Fluorescent reporter

2) Quencher

3) Primer

What does this part of the Taqman hybridization probe do during RT-PCR?

-Fluorescent reporter

Causes fluorescence

What does this part of the Taqman hybridization probe do during RT-PCR?

-Quencher

Prevents fluorescence

What happens before the hydrolysis probe is bound to the template strand?

Forward primers bind in the 5' to 3' direction to bookend target DNA

What happens after the hydrolysis probe is bound to the DNA template strand?

-No more fluorescence

-DNA polymerase munches it, fluorescence probe is removed

What limits the amount of amplified DNA after it reaches the exponential phase?

Reagent (DNTPs)

What happens to intercalating dye when it finds target DNA?

-Attaches to dsDNA

-Fluoresces

What happens after the intercalating dye attaches to dsDNA?

-dsDNA denatures into separate strands

-Dye falls out, fluorophore returns to ground state

True or False: The higher the concentration of dsDNA the higher the fluorescence intensity signal

True

What are the benefits of RT-PCR compared to Conventional PCR?

1) Cheaper to multiplex

2) Less contamination, same machine

3) Increased specificity (probe binding)

4) Increased sensitivity (fluorescence detection)

5) Quantitate sample

6) Faster turnaround time

7) High-throughput, automated workflows with sample tracking

Describe the steps for Digital PCR

1) Sample in PCR mastermix

2) Split reaction into partitions containing target DNA molecules

3) Amplification occurs independently in each partition

4) Detection of target, apply Poisson statistics to ratio (positive : negative) to calculate absolute copy number

What are some benefits of Digital PCR?

1) Absolute quantification (doesn't need standard curve)

2) High sensitivity and precision

3) Tolerance to inhibitors

What are some limitations of Digital PCR?

1) Expensive

2) Difficult to automate

3) Complex preparation

What is this?

-Most widely used sequencing method in clinical labs

-After amplification, PCR products do another round of clean-up

-Loaded onto an automated sequencer

-96 samples can be run overnight

Sanger Sequencing

Describe the steps for Sanger Sequencing

1) Primer annealing and chain extension

3) ddNTPs bind and chain termination, DNApol leaves

3) Fluorescently labelled DNA samples with different lengths/colored nucleotides

4) Capillary gel electrophoresis, match nucleotide fluorescence to sequence samples

5) Sequence analysis and reconstruction, send to database for match

What is this?

1) Simultaneously sequences millions of DNA fragments for rapid, deep sequencing results

2) Limited application in clinical diagnostic fields

3) Identification of novel or uncommon pathogens

4) Identification of bacterial/fungal isolates

Next Gen Sequencing

What are some challenges to using Next Gen Sequencing (NGS)?

-Data management, huge bundles

-Long turnaround time

-Regulatory compliance

-Technical complexity

-Clinical interpretation is hard

How do we mitigate PCR contamination from samples and amplicons?

1) Negative controls

2) Lab "wipe tests"

3) Use uracil instead of thymine, chop up with uracilase to ensure no leftover

How do we prevent a false positive PCR result?

1) Positive control, non-infectious pathogen RNA or DNA that gets co-amplified with target of interest

2) Inhibitor control (internal control)

What is this?

-Inihibitor/internal control

-Known quantity of non-pathogenic nucleic acid -Always expected to amplify

-Ensures correct extraction and absence of PCR inhibitors

True or False: Mortuary samples easily contaminated during autopsy

True

Explain why we need to consider this:

-Not all samples have clinical value

-CMV in immunocompetent adults is not clinically valuable

Explain why we need to consider this:

-Send best sample type

URT Legionella is different from LRT Legionella

Explain why we need to consider this:

-Genotype may not result in phenotype

Not always applicable to antibiotic sensitivity testing

Explain why we need to consider this:

-Negative result may not rule out possibility of infection

Presence of GAS in blood may be below limit of detection or poor timing

Explain why we need to consider this:

-Timing of sample collection can be crucial

-Dengue is detected in blood within 10 days of symptom onset

-Viraemia can end before target is detectable

Explain why we need to consider this:

-Assay specificity

-Single gene target is vulnerable to genetic drift

-Regular review of assay required to keep up with mutation