PCR and Real-Time PCR

The polymerase chain reaction (PCR) amplifies a specific DNA sequence exponentially (about 10 to the power 9 copies after 30 cycles) through repeated cycles of denaturation (94 to 95 °C), primer annealing (50 to 65 °C) and extension (72 °C) by a thermostable DNA polymerase.

Real time (quantitative) PCR monitors amplification with fluorescent probes (hydrolysis probes, molecular beacons) or intercalating dyes, giving a cycle threshold (Ct) that is inversely related to starting target quantity, with detection of as little as 1 to 10 copies in each reaction and a dynamic range of 6 to 8 logs.

Reverse transcription PCR (RT-PCR) first copies RNA to complementary DNA for RNA viruses and gene expression. Multiplex PCR detects several targets in one tube, nested PCR increases sensitivity, and digital PCR gives absolute quantification without a calibration curve and detects rare variants (0.01 to 0.1%).

PCR is the foundation of molecular diagnostics: infectious diseases (SARS-CoV-2, HIV viral load, hepatitis B and C, cytomegalovirus, tuberculosis, malaria, sexually transmitted infections, meningitis panels, respiratory and gastrointestinal panels), oncology (BCR-ABL1, JAK2 V617F, FLT3, NPM1, minimal residual disease), genetic disease (cystic fibrosis, haemochromatosis, factor V Leiden, prothrombin G20210A, thalassaemia),

pharmacogenomics, transplant monitoring, identity testing and prenatal diagnosis.

Turnaround is 1 to 6 hours, and point-of-care cartridge systems give results in 15 to 90 minutes.

Indications. Detection of pathogens that are difficult to culture, slow growing, dangerous or present in low numbers. Rapid diagnosis in critical illness (meningitis, encephalitis, sepsis panels). Viral load monitoring and resistance testing. Diagnosis and monitoring of leukaemia and other malignancies with defined molecular markers.

Confirmation of inherited mutations and carrier status for known variants. Detection of drug resistance genes (mecA, rpoB, carbapenemases). Donor and recipient testing in transplantation. Not as a screening test in people without symptoms or risk, and not when culture or antigen testing is sufficient.

Specimen. Depends on target: whole blood (EDTA, never heparin, which inhibits polymerase), plasma, serum, CSF, swabs in viral transport medium, sputum, urine, stool, tissue (fresh, frozen or formalin fixed), bone marrow, amniotic fluid, dried blood spots.

Collect in sterile, nuclease free containers, avoid cross-contamination, store at 2 to 8 °C for up to 72 hours or freeze at minus 20 to minus 80 °C for longer (RNA targets need prompt processing or stabilising medium). Extraction removes inhibitors (haem, heparin, bile salts, humic acids, polysaccharides), and an internal control monitors extraction and amplification.

Reference values. Target not detected, with a valid internal control. For quantitative assays, results are reported in copies/mL or international units/mL, with the assay's lower limit of detection (for example HIV RNA under 20 to 50 copies/mL, hepatitis B DNA under 10 to 20 IU/mL) and lower limit of quantification.

Interpretation.

• Detected: nucleic acid of the target is present. This shows presence of the organism or mutation, but not necessarily viability, active infection or disease, and results are read with the clinical picture.
• Cycle threshold (Ct): lower values mean more target (Ct under 25 high, 25 to 35 moderate, over 35 low). Ct values are assay specific and not directly comparable between platforms; they are used qualitatively for infectiousness only with validated assays.
• Not detected: target absent or under the limit of detection; false negatives occur from low load, poor sampling, inhibition, degradation, primer mismatch from variants, and timing (too early or too late in infection).
• Invalid or inhibited: internal control fails; repeat with diluted or re-extracted sample.
• Quantitative monitoring: a change of more than 0.5 log (3-fold) in viral load, or 1 log for HIV, HBV and HCV, is clinically significant; assay variability is about 0.2 to 0.3 log.
• Residual disease: BCR-ABL1 transcript levels on the international scale (major molecular response is 0.1% or less) and 3-log reduction direct leukaemia therapy.
• Genotyping: heterozygous or homozygous mutation status by melt curve, allele specific probes or sequencing confirmation.
• Positive control, negative control and no template control must perform to validate each run.
• Contamination is suspected when no template controls are positive or when positive results cluster in time.
• A positive PCR with negative culture is common in treated infection, low organism load, or non-viable organisms (tuberculosis, chlamydia, SARS-CoV-2 after recovery).

Critical values and findings needing immediate action.

• Positive PCR for a life threatening pathogen (Neisseria meningitidis, herpes simplex virus in encephalitis, Mycobacterium tuberculosis with rifampicin resistance, Plasmodium falciparum, Ebola virus, Bacillus anthracis): immediate clinician contact, isolation or precautions as relevant, specific treatment and public health notification.
• High viral load (HIV viral load rising on therapy, cytomegalovirus in transplant recipients): review of adherence, resistance testing and change of therapy or pre-emptive treatment.
• PML-RARA, BCR-ABL1, or other leukaemia defining transcripts: urgent haematology action.
• Resistance gene detected (mecA, vanA, carbapenemase, rpoB): adjust therapy and activate infection control.
• Rising BCR-ABL1 levels (loss of major molecular response): mutation analysis and therapy change.
• Positive result in blood donor or organ donor: discard or restrict use and counselling.

Interferences and pitfalls.

• Contamination by amplicons is the main cause of false positives: physically separated areas (reagent preparation, sample extraction, amplification), dedicated equipment, uracil-N-glycosylase and closed systems prevent it.
• Inhibitors in samples (heparin, haemoglobin, urea, bile salts, anticoagulants, antiseptics) cause false negatives, so internal controls are essential.
• Primer or probe mismatches due to mutations in the target (for example SARS-CoV-2 variants, HIV-2, hepatitis B genotypes, Chlamydia trachomatis Swedish variant) reduce sensitivity; multiple targets lower this risk.
• Detection of nucleic acid does not distinguish live from dead organism, so test of cure by PCR may be positive for weeks after successful treatment.
• Specimen quality (inadequate swab, wrong transport medium, delay, freeze thaw cycles) determines sensitivity more than the assay.
• Quantitative results vary between platforms and laboratories unless calibrated to international standards, and patients are followed on one assay.
• Ct values depend on sample type, extraction, and swab technique and should not be used as a surrogate for viral load without validation.
• Clinical interpretation requires attention to pre-test probability: a low positive Ct in a low prevalence setting may be a false positive.
• Panels detect organisms of uncertain significance (colonisers, latent viruses), and over interpretation leads to unnecessary treatment.
• Regulatory validation, quality control, external quality assessment and trained staff are needed, and costs and infrastructure limit access in low resource regions (point-of-care platforms help).

Special groups.

• Neonates and infants: PCR for congenital infections (cytomegalovirus, HIV, herpes simplex virus) on small samples, with dried blood spot testing.
• Immunocompromised patients: serology is unreliable and PCR monitoring (cytomegalovirus, Epstein-Barr virus, BK virus, adenovirus) guides pre-emptive therapy.
• Pregnancy: amniotic fluid PCR for toxoplasma, cytomegalovirus and parvovirus; HIV and hepatitis B viral load for prevention of transmission.
• Patients with cancer: minimal residual disease monitoring and circulating tumour DNA.
• Outbreaks and surveillance: pooled testing, wastewater and sequencing of positives.
• Regions: GeneXpert and similar cartridge platforms have made PCR accessible in district hospitals for tuberculosis, HIV viral load and early infant diagnosis.

Follow up tests. Culture and susceptibility for positive PCR as appropriate, sequencing to identify resistance or genotype, repeat PCR with fresh sample after inhibition or invalid result, quantitative viral load trend, serology, antigen tests, whole genome sequencing for outbreaks, minimal residual disease monitoring at defined intervals, and clinical review.