Topic: PCR, ELISA, and Autoradiography — Real-World Applications
PCR Clinical Applications
HIV Early Diagnosis:
- Window period (2-12 weeks): antibodies absent → ELISA negative
- PCR detects HIV RNA/DNA when viral load is as low as a single copy
- Critical for blood bank screening (HIV NAT — Nucleic Acid Testing)
Prenatal Genetic Diagnosis:
- Source: Chorionic Villus Sampling (CVS, 10-12 weeks) or amniocentesis (15-18 weeks)
- PCR amplifies fetal DNA from sample
- Detects sickle cell anemia (MstII RFLP), cystic fibrosis (CFTR mutations), DMD (deletions)
- PCR-based diagnosis is faster and more specific than karyotyping for single-gene disorders
Forensic DNA Profiling:
- STR (Short Tandem Repeat) analysis via PCR
- 13-20 loci analyzed for paternity testing, crime scene evidence
- PCR amplifies trace DNA from blood, hair, saliva, skin cells
ELISA Clinical Applications
| Disease | ELISA Format | What Is Detected |
|---|---|---|
| HIV screening | 4th-gen combo | HIV-1/2 antibodies + p24 antigen |
| Hepatitis B | Sandwich ELISA | HBsAg (surface antigen) |
| Pregnancy test | Lateral flow ELISA | hCG (human chorionic gonadotropin) |
| Thyroid disease | Sandwich ELISA | TSH (thyroid stimulating hormone) |
| Allergy testing | Indirect ELISA | IgE antibodies to specific allergens |
ELISA Pitfalls
- False positive: Cross-reactive antibodies (autoimmune conditions, other infections, pregnancy)
- False negative: Window period (no antibodies yet); HIV strain variation in antigen; reagent degradation
- Remedy: 4th-gen ELISA (detects p24 antigen + antibodies); confirmatory Western blot
Autoradiography Applications
- Combined with Southern blot (DNA) or Northern blot (RNA) + membrane hybridization
- Radioactive probe (^{32}P-labeled) → hybridize → X-ray film exposure
- Applications: gene mutation detection, RFLP analysis, chromosome mapping, mRNA expression studies
- Largely replaced by fluorescent probes in clinical diagnostics but remains in research