Contamination

In the molecular laboratory, contamination is the introduction of unwanted nucleic acids into a reaction, leading to false positive results. Because amplification techniques (like PCR) are designed to detect trace amounts of DNA/RNA and multiply them billions of times, even a microscopic amount of contaminant can ruin a patient result. Contamination generally falls into three categories: Amplicon/Carryover (previous PCR products), Biological (cross-contamination between patient samples), and Environmental (genomic DNA from staff or surroundings)

Prevention

Prevention is the primary defense against contamination. It relies on a combination of strict physical barriers, unidirectional workflow, and enzymatic chemistry

• Unidirectional Workflow (One-Way Traffic)
  • The most critical physical control is the flow of personnel and specimens from “Clean” to “Dirty.”
  • Pre-PCR (Clean): Reagent preparation. No DNA or patient samples allowed
  • Sample Prep (Gray Zone): Nucleic acid extraction
  • Post-PCR (Dirty): Amplification and analysis. This area contains billions of amplicons
  • Rule: Equipment (pipettes, racks) and PPE (lab coats) must be dedicated to their specific rooms. Nothing moves from Post-PCR back to Pre-PCR
• Engineering & Consumable Controls
  • Aerosol Barrier Tips: All pipetting requires filter tips to prevent liquid aerosols from contaminating the interior barrel of the pipette
  • Aliquot Reagents: Reagents should be separated into single-use aliquots. This ensures that if one tube is contaminated, the entire stock lot is not lost
  • Positive Pressure: Clean rooms should be kept under positive air pressure to push dust/spores out when doors open
• Enzymatic Control: The dUTP-UNG System
  • To prevent carryover of amplicons from previous runs, dTTP (Thymine) is replaced with dUTP (Uracil) in the PCR Master Mix
  • The enzyme Uracil-N-Glycosylase (UNG/UDG): is added to the mix
  • Mechanism: Before the PCR starts, the mix is heated (approx. 50°C). UNG degrades any DNA containing Uracil (contaminants from previous runs). The mixture is then heated to 95°C, killing the UNG, and the true patient sample (containing Thymine) is amplified

Monitoring & Detection

Since physical barriers are not infallible, the laboratory must actively monitor for breaches using Quality Control (QC) and surveillance

• Experimental Controls
  • No Template Control (NTC) / Reagent Blank: Contains water instead of DNA. If this amplifies, the reagents or the Pre-PCR area are contaminated
  • Negative Extraction Control: A negative sample (e.g., water or negative plasma) run through the entire extraction process. A positive result here indicates cross-contamination between samples during extraction
• Data Analysis Trends
  • Late Ct Values: In Real-Time PCR, contamination often presents as weak amplification with high Crossing Threshold (Ct) values (e.g., >35 cycles)
  • Melt Curve Analysis: Contamination (especially primer-dimers or off-target environmental bacteria) often produces a different melt temperature (Tm) peak than the specific target
  • Positivity Rate: A sudden, unexplained spike in positive results for a specific pathogen suggests a systemic contamination event (e.g., a contaminated reagent lot)
• Environmental Wipe Tests
  • Routine surveillance involves swabbing laboratory surfaces (bench tops, pipette handles, door knobs) and running PCR on the swabs
  • The “Clean” room must be completely free of amplifiable DNA. The presence of DNA in the clean room indicates a breakdown in the unidirectional workflow

Elimination

Once contamination is confirmed, patient testing must stop. The laboratory must implement a “Deep Clean” protocol to destroy the nucleic acids before resuming operations

• Chemical Decontamination
  • 10% Sodium Hypochlorite (Bleach): The most effective agent for destroying DNA. It chemically breaks the sugar-phosphate backbone
  • The Rinse Step: Because bleach is corrosive to stainless steel and pipettes, it must be followed by a rinse with 70% Ethanol or DI water to remove residue
  • Note: Alcohol alone is a disinfectant, not a DNA decontaminant. It precipitates DNA but does not destroy it.
• Physical Decontamination
  • UV Irradiation: UV light (254 nm) causes the formation of thymine dimers in DNA. These dimers create kinks in the strand that prevent DNA Polymerase from replicating the template
• Remediation Protocol
  • Discard Reagents: Any open reagents in the affected area must be thrown away
  • Clean-to-Dirty: Decontamination cleaning must start in the Clean room and move toward the Dirty room to prevent back-flow of contaminants
  • Verification: Before resuming patient testing, a “Dummy Run” consisting entirely of NTCs and negative controls must be performed to prove the laboratory is clean