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Implementing External Quality Control in PCR Workflows: A Step-by-Step Laboratory Guide

Polymerase Chain Reaction (PCR) has revolutionized molecular biology and microbial detection. Yet the high sensitivity of PCR also makes it vulnerable to technical errors, false positives, and reagent degradation. To ensure accurate, reproducible, and high-confidence results, External Quality Control (EQC) must be embedded into every PCR workflow.

This in-depth guide explains how laboratories can implement robust EQC protocols, supported by validated data sources from government and academic institutions. This blog avoids any sensitive health-related content, focusing purely on lab workflow optimization and technical validation strategies for high-sensitivity applications.

What Is External Quality Control in PCR?

External Quality Control refers to the use of known, standardized reference samples that are included in each PCR run to assess the full assay process—from extraction to amplification. These controls are critical for detecting:

• PCR efficiency loss

• Instrument drift

• Contamination

• Lot-to-lot reagent variability

Relevant resources:

• CDC Molecular Testing Standards

• NIH Reproducibility Resources

• FDA Molecular Diagnostic Evaluation Guidelines

Types of External PCR Quality Controls

Different control materials are used depending on the application. Common types include:

• Synthetic RNA or DNA fragments

• Plasmid-based SARS-CoV-2 PCR External Quality Control reagents

• Inactivated viral particles

• Full-process spike-ins like AffiCHECK SARS-CoV-2 Control

These are designed to mimic sample behavior and are used to assess PCR accuracy from nucleic acid extraction to signal detection.

Explore:

• NIST Certified Reference Materials

• WHO COVID-19 Molecular Reference Panels

• NIH COVID-19 Research Tools

 Step-by-Step Implementation of External Quality Control

Step 1: Select a Validated EQC Material

Choose a control matched to your assay target, such as:

• GenMark ePlex PCR Control for automated syndromic panels

• AffiCHECK SARS-CoV-2 Control for viral gene targets (e.g., N, E, RdRp)

Make sure your selected EQC is traceable, consistent across lots, and stable under your workflow conditions.

For example: FDA EUA Reference Panel

Step 2: Validate Control Range and Acceptance Limits

For each EQC material, establish:

• Mean Ct values across 10+ runs

• Acceptable variation (e.g., ±1 Ct)

• Amplification curves and melt peak integrity

• Use both PCR Quality Control Low Level and high-level controls

Refer to:

• NIH PCR Validation Guidelines

• CDC Molecular Proficiency Testing

Step 3: Include Controls in Every Run

In every qPCR plate or run, include at least:

• One External Quality Control for COVID-19 PCR

• A no-template control (NTC)

• An internal positive control (if applicable)

All controls should be extracted and amplified using the same workflow as the clinical or research samples.

• FDA EUA Extraction Protocols

Step 4: Record and Analyze Data Continuously

Use a spreadsheet or a LIMS system to record:

• Ct values from EQC wells

• Instrument ID

• Date, operator, lot number

• Deviations from accepted ranges

Monitor using Levey-Jennings plots or Z-score tracking. This ensures PCR Accuracy Control for SARS-CoV-2 performance across time.

Reference: CDC QC Monitoring Techniques

Step 5: Interpret Deviations and Respond Rapidly

If an EQC sample fails (e.g., no amplification or out-of-range Ct), consider:

• Degraded enzyme or primer mix

• Pipetting error

• Contamination or inhibition

Do not release or interpret any associated sample data. Re-run with fresh reagents and document the error.

• FDA Corrective Action Guide

Step 6: Participate in Proficiency Testing

Enroll in an external program such as:

• WHO EQAP

• CAP SARS-CoV-2 PCR External Control Testing

• CDC International Quality Assurance Program

These programs help benchmark your lab’s performance against thousands of peers.

 Integration with Specific SARS-CoV-2 Platforms

For laboratories using the GenMark ePlex SARS-CoV-2 platform, certified EQC materials such as the ePlex SARS-CoV-2 Quality Control are available commercially. These mimic clinical sample behavior and help validate instrument accuracy daily.

Also recommended:

• FDA Cleared Control Kits for ePlex

• NIH Instrument Calibration Standards

Best Practices for EQC in COVID-19 and Beyond

Final Takeaways

Implementing a robust External Quality Control system in PCR workflows helps labs ensure consistent performance, reduce batch-to-batch variability, and increase confidence in reported results. Whether working with SARS-CoV-2 PCR External Quality Control materials or general DNA/RNA assays, every lab can benefit from:

• Routine integration of validated EQC samples

• Continuous performance logging

• Participation in international quality assurance initiatives

• Strict contamination prevention protocols

By following this guide and incorporating proven tools such as ePlex SARS-CoV-2 Quality Control, AffiCHECK PCR Control, and PCR Quality Control Low Level reagents, laboratories can greatly enhance their reliability and reproducibility.