PCR and Molecular Diagnostic Variants
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Direct answer
PCR, or polymerase chain reaction, amplifies a specific DNA sequence exponentially, so a few copies can be detected directly from clinical material. Real-time PCR measures amplification as it happens using fluorescence, while RT-PCR converts RNA to DNA first. Variants such as nested, multiplex, LAMP and line probe assays trade off speed, sensitivity and the number of targets detected.
What you must remember
- Principle: denaturation, primer annealing and extension repeated for about 30 to 40 cycles, roughly doubling the product each cycle.
- Real-time PCR: fluorescence from a TaqMan probe or SYBR Green is read every cycle, and the Ct value allows quantification.
- Nested PCR: two rounds with an inner primer pair, giving higher sensitivity and specificity but a high carry-over risk.
- Multiplex PCR: several targets amplified in one reaction, as in meningitis and respiratory panels.
- RT-PCR: reverse transcriptase makes cDNA from RNA before amplification, used for HIV, hepatitis C and influenza RNA.
- LAMP: loop-mediated isothermal amplification at about 60 to 65 °C, needing no thermal cycler and read by turbidity or colour.
- Line probe assay: amplified DNA hybridised to strips to detect resistance mutations, as in rifampicin and isoniazid resistance.
- GeneXpert: Xpert MTB/RIF detects Mycobacterium tuberculosis and rifampicin resistance within hours under the national programme.
Choosing the right variant
Each variant changes one part of the PCR workflow, and that change determines what it is good for. The table compares the variants most often asked in examinations.
| Variant | Key feature | Typical use |
|---|---|---|
| Conventional PCR | Product read on a gel after the run | Confirmatory identification |
| Real-time PCR | Fluorescence and Ct value | Quantification, viral load |
| Nested PCR | Two rounds with inner primers | Very low copy numbers |
| Multiplex PCR | Several targets in one tube | Syndromic panels |
| RT-PCR | RNA converted to cDNA first | RNA viruses |
| LAMP | Isothermal, no cycler | Point-of-care settings |
| Line probe assay | Strip hybridisation | Drug resistance mutations |
Real-time PCR is preferred for viral load and quantification because the signal is read during amplification. Nested PCR is powerful but risky, since carry-over of a previous product can produce false positives. Multiplex panels save time and sample, but each target has its own sensitivity.
The choice of variant follows the question being asked: quantity, speed, number of targets or resistance pattern. A laboratory selects the test that gives the needed answer with the least risk of error.
Where the exam tries to catch you
Molecular diagnostics questions test the trade-off each variant makes and the source of its errors.
- Trap: nested PCR is always the best test Correct: nested PCR is more sensitive, but its open-tube carry-over causes false positives, so strict controls are needed.
- Trap: real-time PCR cannot quantify Correct: real-time PCR quantifies by comparing Ct values against a standard curve, which is how viral loads are reported.
- Trap: LAMP needs a thermal cycler Correct: LAMP is isothermal, running at about 60 to 65 °C in a water bath or heat block.
Frequently asked questions
What is the difference between real-time PCR and conventional PCR?
Real-time PCR detects the amplified product during cycling through fluorescence, while conventional PCR is read after the run on a gel. Real-time PCR also gives quantitative results through Ct values, which conventional PCR cannot provide.
Why is RT-PCR used for detecting HIV or hepatitis C?
HIV and hepatitis C are RNA viruses, so their RNA is first reverse transcribed into complementary DNA before amplification. Quantitative RT-PCR is therefore the standard method for viral load testing of these viruses.
What does a line probe assay detect in tuberculosis diagnosis?
A line probe assay detects specific mutations in rifampicin and isoniazid resistance genes, such as rpoB, katG and inhA, using a strip-based hybridisation format. It is used to identify drug-resistant tuberculosis from amplified DNA.
Why is LAMP useful in low-resource laboratories?
LAMP amplifies DNA at a single constant temperature of about 60 to 65 °C, so it needs no thermal cycler. Results can be read by turbidity or a colour change, which keeps the equipment simple and inexpensive.