Multiplex PCR
Related: PCR · qPCR · RT-PCR · Syndromic Molecular Panels
1. Principle
Multiplex PCR amplifies multiple targets in one reaction using primer (/probe) sets distinguished by amplicon size, melt temperature, or fluorophore channel — enabling syndromic testing from a single extract.
2. Step-by-Step Procedure (conceptual)
- Extract total nucleic acid (often DNA+RNA kits for respiratory/GI panels).
- Run multiplex mix on validated thermocycler / cartridge system.
- Detect via multi-channel fluorescence, arrays, or microfluidic endpoints.
- Interpret each analyte with panel-specific rules and cross-reactivity notes.
3. Interpretation
- Multi-positives common (co-detection ≠ co-pathogenicity).
- Pitfalls: Primer competition; preferential amplification; unexpected channel crosstalk; colonization (PCR).
4. Clinical Use Cases
- Meningitis/encephalitis CSF panels; respiratory virus/bacteria panels; GI pathogen panels; pneumonia panels; blood culture identification panels.
- Resistance markers bundled with ID (mecA, carbapenemase genes) on some platforms.
5. Comparison with Other Methods
| Method | Pros | Cons |
|---|---|---|
| Multiplex PCR | Breadth + speed | Cost; fixed target list |
| Singleplex qPCR | Optimized per target | More reactions |
| Metagenomic NGS | Untargeted | Slower, complex, cost |
6. Mnemonic / Visual Aid
One tube, many questions — still only answers the questions printed on the menu.
Active Recall
- Why can three positives appear in one CSF multiplex run?
- What is primer competition?
- When is culture still required after a positive multiplex?