Infectious Syndromic Testing (IST) relies heavily on the reliability and precision of multiplex Polymerase Chain Reaction (PCR) systems. Quality control (QC) is a cornerstone of IST workflows, directly affecting the accuracy of molecular detection for multiple pathogens in a single panel. This article presents a deep technical breakdown of IST PCR Quality Control, written for laboratories, researchers, and developers. It integrates protocols, public resources, and non-sophisticated but comprehensive technical detail, with over 20 hyperlinks to .edu and .gov websites for reference.
What Is IST PCR?
IST PCR is a type of multiplexed nucleic acid amplification testing that allows researchers to identify multiple infectious agents from a single biological specimen. These assays are used in studies of respiratory pathogens, enteric diseases, wound microbiota, and more. Popular IST PCR systems include QIAstat-Dx, BioFire FilmArray, ePlex, and Luminex.
Because IST panels contain dozens of primer-probe sets in one assay, rigorous quality control is essential to ensure performance and minimize false positives or false negatives.
Internal Controls (ICs)
Internal controls are synthetic or non-pathogenic sequences that are co-amplified in each PCR reaction. They:
- Confirm successful amplification
- Detect inhibition
- Provide baseline Ct values
Most IST platforms use bacteriophage MS2 or armored RNA/DNA as internal controls. ICs must not interfere with target amplification.
Reference: FDA IVD Quality Control
External Controls (ECs)
External controls are processed alongside test samples and are used to validate the run. They include:
- Positive Controls (contains target DNA/RNA)
- Negative Controls (nuclease-free water or irrelevant DNA)
They confirm that:
- Amplification reagents are working
- Contamination is absent
- Cross-reactivity is controlled
Reference: CDC Clinical Laboratory Improvement
Extraction Controls
To verify nucleic acid extraction efficiency, external or spiked internal extraction controls are used. These are essential for:
- Samples with inhibitors (feces, blood, sputum)
- Surveillance samples from the environment
Reference: NIH Research Tools
Proficiency Testing and Regulatory Compliance
CLIA Certification
Laboratories conducting IST must comply with CLIA requirements:
- Validate all methods
- Run control materials
- Document calibration and maintenance
Reference: CMS CLIA Overview
Proficiency Testing (PT)
PT ensures that labs can detect the correct pathogen in blind samples. PT should be conducted:
- Biannually
- Through organizations like CAP or CDC PT Programs
Analytical Validation Metrics
1. Ct Value Reproducibility
- Repeats of same sample should show <1 Ct variation
2. Amplification Efficiency
- Calculated using standard curves
- Optimal range: 90–110%
3. Limit of Detection (LOD)
- Lowest copy number detectable with >95% confidence
4. Precision and Accuracy
- Measured via intra- and inter-assay coefficient of variation (CV)
5. Specificity
- Evaluate cross-reactivity using non-target organisms
Reference: NIH NCBI Guide to qPCR
Calibration and Instrument QC
Instruments used in IST PCR workflows include:
- Thermal cyclers
- Optical detection systems
Calibration should include:
- Thermal block uniformity (±0.3°C)
- Fluorescence detection alignment
- Software version control
Reference: FDA Thermocycler Guidelines
Common Failure Modes
- Inhibitory samples (blood, sputum)
- Evaporation or poor sealing
- Incorrect pipetting
- Degraded reagents
Reference: NIH Sample Prep QC
Reference Materials
NIST SRMs
The National Institute of Standards and Technology provides:
- RNA/DNA concentration references
- Multiplex assay standards
Academic Controls
Many universities offer in-house validation data:
Automation and Digital QC
Modern labs use software for:
- Run tracking
- Barcode verification
- Ct trend analysis
- Control template tracking
HL7-based LIMS integration allows for real-time data tracking. Learn more at:
Training and Competency
Training resources are vital for sustaining high-quality operations. The CDC Laboratory Training Program provides e-learning modules on:
- IST platforms
- Internal/external controls
- Data review
NSF and NIH are funding new AI-driven systems that monitor QC parameters in real time. These systems:
- Learn from Ct patterns
- Flag inhibition early
- Predict reagent degradation
Reference: NSF Grant Portal
Best Practices Summary
- Run ICs in every sample
- Verify ECs for each assay run
- Log all deviations and reruns
- Use NIST standards for calibration
- Join PT programs from CDC or CAP
- Integrate LIMS or spreadsheet QC tracking
- Train personnel and document competency