PCR

Related MOCs: MOC - Diagnostic & Lab Methods · Inventor: Kary Mullis
Upstream: DNA Extraction · RNA Extraction · Sample Types and Specimen Quality · Nucleic Acid Quantification
PCR family: qPCR · Multiplex PCR · Nested PCR · Digital PCR · Broad-Range 16S PCR · Reverse Transcription Polymerase Chain Reaction (RT-PCR) · RT-PCR
Beyond PCR: Isothermal NAAT · CRISPR-based Diagnostics · Syndromic Molecular Panels · Sanger Sequencing · Whole-Genome Sequencing

1. Principle

Polymerase chain reaction enzymatically amplifies a specific DNA segment through repeated cycles of denaturation, primer annealing, and extension — making scarce sequences detectable.

2. Step-by-Step Procedure (conceptual)

1. Extract nucleic acid from specimen or isolate.
2. Set up reaction: template, primers, dNTPs, buffer, thermostable polymerase (± probes for real-time PCR).
3. Thermocycle: denature → anneal → extend (×25–45 cycles).
4. Detect: gel, real-time fluorescence (Ct), melt curves, or post-PCR hybridization.
5. Interpret with controls (positive, negative, internal control) and clinical context.

3. Interpretation

• Positive: Target DNA sequence detected (above assay threshold).
• Negative: Target not detected — may still miss due to inhibition, sampling, or assay scope.
• Pitfalls: Contamination (amplicon), inhibition, primer mismatches (variants), colonization ≠ infection (below).

4. Clinical Use Cases

• Gold standard / first-line for: Many viral diagnoses; rapid detection of unculturable / slow / pretreated pathogens; resistance-gene markers in some workflows.
• Alternative to: Culture when speed or viability is limiting — but culture still needed for full AST of many bacteria.

5. Comparison with Other Methods

Method	Pros	Cons
PCR / NAAT	Fast, sensitive, specific targets	Cost; colonization ambiguity; limited AST
Culture and Isolation	Viable organism + AST	Slow; misses non-culturables
Gram Stain	Immediate morphology	No speciation
Whole-Genome Sequencing	Broad genome view	Turnaround / bioinformatics

6. Mnemonic / Visual Aid

Heat → Stick primers → Copy → Repeat — each cycle roughly doubles the target.

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Colonization vs Infection (critical interpretation)

PCR positivity from upper respiratory specimens does not distinguish colonization from true infection.

What PCR actually tells you

If a nasopharyngeal or nasal swab is PCR-positive for:

• Staphylococcus aureus → Staphylococcus aureus
• Streptococcus pneumoniae → Streptococcus pneumoniae
• Moraxella catarrhalis
• Haemophilus influenzae
• Stenotrophomonas maltophilia

it only proves that bacterial DNA is present in the sampled site.

PCR cannot determine whether:

1. The organism is causing active infection.
2. The organism is simply colonizing the mucosa (Normal Microbiota).
3. The organism is dead and residual DNA remains.
4. The organism is present at a clinically insignificant burden.

Colonization

Remember

The microorganism is present but causes no tissue damage and no symptoms.

Examples:

• 20–30% of healthy adults carry S. aureus in the nose.
• Many healthy children carry S. pneumoniae in the nasopharynx.
• Colonization rates can exceed 50% in some populations.

A PCR-positive nasal swab for S. aureus therefore does not mean the patient has bacterial pneumonia.

See also: Pathogen · Infectious Disease · Koch’s Postulates