Causes of PCR Inhibition in Urine Samples
Urea is the primary inhibitor of PCR in urine samples, causing inhibition at concentrations above 50 mM, which is particularly problematic in samples from older children and adults compared to neonates due to higher urea concentrations. 1
Major Inhibitory Components in Urine
Chemical Inhibitors
- Urea: The major inhibitory component that affects PCR at concentrations >50 mM 1
- Organic acids: Present in urine and can interfere with PCR reactions 2
- Betaines: While glycine betaine helps bacteria adapt to high osmolarity, other dietary betaines like trigonelline may enhance urea toxicity and affect PCR 2
Organic Substances
- Humic acid-like substances: These can cause significant RT-PCR inhibition resulting in >3-log10-unit underestimation of viral loads 3
- Protein-like fractions: Can be concentrated during sample processing 3
- Organic fractions in 10-100 kDa size range: Particularly suspected to be PCR inhibitors 3
Sample Collection and Processing Factors
Collection Issues
- Contamination: Improper specimen collection can introduce contaminants that inhibit PCR 4
- Storage conditions: Urine samples should be processed within 2 hours or refrigerated to prevent degradation and false results 4
Processing Challenges
- Concentration procedures: Virus concentration methods can co-concentrate organic inhibitory substances 3
- Recovery rates: Over 60% of inhibitory organics can be recovered in concentrates during processing 3
Prevalence of PCR Inhibition by Sample Type
- Urine samples: Relatively low inhibition rate of approximately 1.1% 5
- Cervical swabs: Moderate inhibition rate of about 7% 5
- Urethral swabs: High inhibition rate of approximately 45% 5
Methods to Eliminate PCR Inhibition
Physical Methods
- Storage at 4°C: Eliminates inhibition in 86% of specimens 5
- Freezing at -70°C: Eliminates inhibition in 72% of specimens 5
- Dialysis or ultrafiltration: Effectively removes inhibitory effects of urine 1
Chemical Methods
- Dilution: 1:10 dilution (89% effective) or 1:100 dilution (86% effective) after storage at 4°C 5
- Phenol-chloroform extraction: Most effective method (99% success rate) 5
- Addition of bovine serum albumin (BSA): Eliminates inhibitory effects in stool samples and may be applicable to urine 6
PCR Optimization
- Nested primers: Improves sensitivity and specificity 1
- Modified PCR protocol: Using reduced reactants in the first cycles of a two-stage PCR 1
Clinical Implications
- PCR inhibition can lead to false negative results, potentially missing important pathogens
- Understanding the source of inhibition helps in selecting appropriate sample processing methods
- Proper specimen collection and handling are critical to minimize inhibition
- For diagnostic purposes, incorporating internal controls is essential to detect inhibition 7
When processing urine samples for PCR testing, it's important to consider these inhibitory factors and implement appropriate mitigation strategies to ensure accurate results.