A multiplex real-time RT-PCR for respiratory viruses is run on an extraction batch of 22 samples. Each batch includes a negative extraction control (viral transport medium taken through extraction) and a positive control, and each plate has a no-template control of water added at master-mix set-up. Results: sample 7, influenza A Ct 14.2; samples 6 and 8, from different wards, influenza A Ct 35.9 and 36.4; negative extraction control, in position 9, influenza A Ct 37.1; no-template control, no amplification; internal control normal in every well. The most likely explanation for the weak positives is:

Carry-over from sample 7 during extraction

Low viral loads in two true infections

PCR inhibition in samples 6, 8 and 9

Amplicon contamination of the PCR master mix

Some PCR master mixes contain dUTP in place of dTTP, together with uracil-N-glycosylase (UNG). This combination is used to:

Destroy amplicons carried over from earlier runs

Raise the specificity of probe hybridization

Show whether the specimen contains inhibitors

Prevent primers from annealing to each other

Nested PCR is more sensitive than single-round PCR, but it carries a greater risk of false positives. The main reason is that:

Tubes are opened to move first-round product

Twice as much specimen goes into the reaction

The second primer pair binds outside the first

The second round uses a lower annealing temperature

In a SYBR Green real-time PCR run, the no-template control amplifies at Ct 37 with a single melt peak at 74 °C. All positive controls and positive patient samples melt at 84 °C. The no-template control signal is most likely due to:

Primer-dimers, not contamination

Carryover of amplicon from earlier runs

Breakdown of a hydrolysis probe

Target DNA contaminating the reagents

To prevent contamination with amplified product, staff and materials in a PCR laboratory should move:

One way, from pre-amplification to post-amplification areas

Both ways, if gloves are changed at each door

From the amplification room back to reagent preparation

Freely, if all benches are cleaned with bleach daily

Molecular diagnostics principles

7 DOH Abu Dhabi (HAAD) lab licensing practice questions on Molecular diagnostics principles, part of General Laboratory, Quality and Safety. The first 7 are below; the first 5 with answers and explanations (tap Show answer after you have chosen), the rest in Learn with a free account.

Question 1 · Hard
A multiplex real-time RT-PCR for respiratory viruses is run on an extraction batch of 22 samples. Each batch includes a negative extraction control (viral transport medium taken through extraction) and a positive control, and each plate has a no-template control of water added at master-mix set-up. Results: sample 7, influenza A Ct 14.2; samples 6 and 8, from different wards, influenza A Ct 35.9 and 36.4; negative extraction control, in position 9, influenza A Ct 37.1; no-template control, no amplification; internal control normal in every well. The most likely explanation for the weak positives is:
1. ALow viral loads in two true infections
2. BPCR inhibition in samples 6, 8 and 9
3. CCarry-over from sample 7 during extraction
4. DAmplicon contamination of the PCR master mix
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Answer: C · Carry-over from sample 7 during extraction
The negative extraction control turned positive while the no-template control, added only at the PCR step, stayed negative, so the contamination entered before the master mix, during extraction. The weak positives sit right next to a very strong sample (Ct 14), the typical pattern of splash or aerosol carry-over between neighboring tubes or wells. Contaminated master mix or amplicon would also make the no-template control positive. Samples 6 and 8 are not reported as positive; they are re-extracted from the original specimens away from strong positives, and the extraction technique and plate layout are reviewed.
- A: The negative extraction control is positive too, so the signal is contamination.
- B: Internal controls are normal, and inhibition causes false negatives, not false positives.
- D: Contaminated master mix would also make the no-template control positive.
Question 2 · Medium
Some PCR master mixes contain dUTP in place of dTTP, together with uracil-N-glycosylase (UNG). This combination is used to:
1. ARaise the specificity of probe hybridization
2. BShow whether the specimen contains inhibitors
3. CPrevent primers from annealing to each other
4. DDestroy amplicons carried over from earlier runs
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Answer: D · Destroy amplicons carried over from earlier runs
Every product made with this mix contains uracil instead of thymine. Before cycling begins, UNG in a new reaction removes uracil from any contaminating product of earlier runs, and these strands break at the first high temperature, so they cannot be amplified. The patient's own DNA contains thymine and is not affected, and UNG is inactivated by heat before amplification. This protects against amplicon carryover but not against target DNA carried over from other specimens.
- A: UNG does not change how probes bind to their target.
- B: Inhibition is detected with an internal control.
- C: Primer-dimers are reduced by hot-start enzymes and primer design, not by UNG.
Question 3 · Medium
Nested PCR is more sensitive than single-round PCR, but it carries a greater risk of false positives. The main reason is that:
1. ATwice as much specimen goes into the reaction
2. BThe second primer pair binds outside the first
3. CTubes are opened to move first-round product
4. DThe second round uses a lower annealing temperature
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Answer: C · Tubes are opened to move first-round product
Nested PCR runs a first round with an outer primer pair, then transfers some of that product to a second reaction with an inner pair that binds within the first product. Opening tubes full of amplified product releases aerosols that can contaminate the work area and later specimens. Closed-tube real-time PCR avoids this step and has largely replaced nested methods in diagnostic laboratories. Where nested PCR is still used, the transfer is done in a separate area under strict controls.
- A: The specimen is added only to the first round.
- B: The inner primers bind within the first product, not outside it.
- D: Annealing temperature is not what makes nested PCR prone to contamination.
Question 4 · Hard
In a SYBR Green real-time PCR run, the no-template control amplifies at Ct 37 with a single melt peak at 74 °C. All positive controls and positive patient samples melt at 84 °C. The no-template control signal is most likely due to:
1. APrimer-dimers, not contamination
2. BCarryover of amplicon from earlier runs
3. CBreakdown of a hydrolysis probe
4. DTarget DNA contaminating the reagents
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Answer: A · Primer-dimers, not contamination
SYBR Green binds any double-stranded DNA, so the melt curve shows which product made the signal. Contamination with target or amplicon would give the same melt peak as the true product, 84 °C here. A distinct, lower peak points to a short nonspecific product, usually primer-dimers, which form late in reactions with no template. Hot-start enzymes, changes to primer concentration or annealing temperature, or redesigned primers reduce them, and a patient curve with only the low peak should not be read as positive.
- B: Amplicon carryover would melt at the target's temperature, 84 °C.
- C: SYBR Green assays use no hydrolysis probe.
- D: Target DNA in the reagents would also give the 84 °C peak.
Question 5 · Easy
To prevent contamination with amplified product, staff and materials in a PCR laboratory should move:
1. AOne way, from pre-amplification to post-amplification areas
2. BBoth ways, if gloves are changed at each door
3. CFrom the amplification room back to reagent preparation
4. DFreely, if all benches are cleaned with bleach daily
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Answer: A · One way, from pre-amplification to post-amplification areas
A single PCR can make billions of copies of its target, and a tiny aerosol of product can contaminate later reactions and cause false positives. Work is therefore split into separate areas for reagent preparation, specimen preparation and extraction, and amplification and detection. People, specimens, coats and equipment move only forward, from clean to amplified areas, and not back on the same day. Each area has its own pipettes, aerosol-barrier tips and coats.
- B: Changing gloves does not stop amplicons carried on coats, hair or equipment.
- C: Moving from the amplification room to reagent preparation carries amplicons into the cleanest area.
- D: Bleach cleaning helps but does not replace a one-way workflow.
Question 6 · Hard
A real-time PCR standard curve from tenfold dilutions has a slope of -3.58 (Ct against log10 concentration). Efficiency = 10^(-1/slope) - 1, and the laboratory accepts 90-110%. What is the efficiency?
1. AAbout 190%, far above the range
2. BAbout 110%, at the upper limit
3. CAbout 90%, at the lower limit
4. DAbout 100%, the ideal value
Answer and explanation in LearnFree account
Question 7 · Easy
Why is a heat-stable DNA polymerase, such as Taq, used in PCR?
1. AIt proofreads, so it makes no copying errors
2. BIt survives the repeated 94-95 °C denaturation steps
3. CIt starts new strands without any primers
4. DIt copies RNA templates directly into DNA
Answer and explanation in LearnFree account

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Molecular diagnostics principles

Some PCR master mixes contain dUTP in place of dTTP, together with uracil-N-glycosylase (UNG). This combination is used to:

Nested PCR is more sensitive than single-round PCR, but it carries a greater risk of false positives. The main reason is that:

In a SYBR Green real-time PCR run, the no-template control amplifies at Ct 37 with a single melt peak at 74 °C. All positive controls and positive patient samples melt at 84 °C. The no-template control signal is most likely due to:

To prevent contamination with amplified product, staff and materials in a PCR laboratory should move:

A real-time PCR standard curve from tenfold dilutions has a slope of -3.58 (Ct against log10 concentration). Efficiency = 10^(-1/slope) - 1, and the laboratory accepts 90-110%. What is the efficiency?

Why is a heat-stable DNA polymerase, such as Taq, used in PCR?