Internal Control in Real-Time PCR Testing

In this article, we explain how internal controls work in Real-Time PCR.
We also discuss which errors they detect and why they are essential in modern molecular diagnostics.

What Is Internal Control?

built-in control is a defined nucleic acid sequence.
It is amplified together with the target genes during a Real-Time PCR reaction.
This process confirms that extraction and amplification steps were successful.

Typically, the control is detected in a separate fluorescence channel.
Therefore, it does not interfere with target detection.

Why Is Internal Control Essential?

Without built-in control, negative PCR results may be misleading.
In some cases, a negative result occurs due to technical failure rather than true absence of the target.
For this reason, internal controls are critical.

• They confirm DNA or RNA extraction
• They identify PCR inhibitors
• They monitor amplification efficiency
• They reduce false-negative results

Which Errors Can Be Detected?

built-in control helps laboratories detect technical problems early.
This prevents the release of unreliable results.

Extraction Failure

If extraction fails, built-in control will not amplify.
In this case, the test must be repeated.

PCR Inhibition

Some substances inhibit PCR reactions.
Examples include hemoglobin or residual paraffin in FFPE samples.
built-in control makes these issues visible.

Technical Errors

Pipetting mistakes or reagent errors may occur.
When this happens, Internal Control signals the problem immediately.

nternal process control vs Other Controls

nternal process control is different from positive and negative controls.
Each control type has a unique role.

• Internal control: validates each individual sample
• Positive control: confirms reagent performance
• Negative control: detects contamination

built-in control in Multiplex PCR

In multiplex assays, multiple targets are tested at once.
Because of this complexity, nternal process control becomes even more important.
It confirms that negative results are technically valid.

Clinical Example: Easy® THYROID

The Easy® THYROID kit is a Multiplex Real-Time PCR assay.
It detects KRAS, NRAS, HRAS, and BRAF mutations in thyroid cancer.

In this assay,nternal process control  is co-amplified with target mutations.
This approach verifies extraction quality and amplification performance.

Internal Control in HPV Testing

HPV samples often contain PCR inhibitors.
Therefore, nternal process control is essential for reliable HPV testing.
It ensures that negative results are trustworthy.

Internal Control in Viral PCR Kits

Viral assays for HSV, EBV, and CMV require high sensitivity.
Here, nternal process control confirms that low viral loads are not missed due to technical failure.

Internal Control in Fungal Multiplex PCR

Multiplex fungal assays detect several species at once.
nternal process control validates negative results in complex clinical samples.

Reducing False-Negative Results

One major advantage of nternal process control is error prevention.
False-negative results may delay diagnosis and treatment.
Internal controls significantly reduce this risk.

Internal Control and CE-IVD Standards

Many CE-IVD certified kits require nternal process control.
This requirement highlights its importance in quality assurance.

Real-Time PCR Kits from Fardad Azma Rad

Fardad Azma Rad supplies advanced Real-Time PCR kits.
These kits include robust internal control systems.
They support accurate diagnostics in oncology and infectious diseases.

Conclusion

Internal Control is a core element of reliable Real-Time PCR testing.
It detects technical errors and ensures result accuracy.

By using validated kits with internal control systems,
laboratories can improve diagnostic confidence and clinical decision-making.

Scientific Disclaimer

This content is for educational purposes only.
It does not replace official laboratory guidelines or professional medical advice.