PCR controls: how to know whether a negative is really negative

The problem of the silent negative

A positive PCR result leaves a visible trace: a fluorescence curve that rises. A negative, in contrast, is the absence of that curve. And the absence of a signal has more than one possible explanation: that the microorganism is not in the sample, or that the reaction did not work. A polymerase inhibited by food components, a pipetting error, a failed extraction or a malfunctioning instrument produce exactly the same picture as a clean sample.

That is why a PCR without controls cannot distinguish a true negative from a false negative. In food microbiology that difference matters a great deal: a false negative for Salmonella or Listeria monocytogenes means releasing a contaminated product with a report saying the opposite.

The reference framework: ISO 22174 and MIQE

ISO 22174 establishes the general requirements for nucleic acid amplification (DNA or RNA) in the detection of food chain microorganisms and viruses. Its second edition, from 2024, replaced the 2005 version and three other PCR standards (ISO 20837, ISO 20838 and ISO 22119), incorporated requirements for digital PCR and for monitoring laboratory workflows, and expanded the section on control reactions with a description of the different controls.

In parallel, the MIQE guidelines (Minimum Information for Publication of Quantitative Real-Time PCR Experiments), revised as MIQE 2.0 in 2025, set what information on controls, assay design and data analysis must be documented for a qPCR result to be reproducible. Although they were created for scientific publications, they are a good checklist for any laboratory.

The controls that make a run interpretable

Internal amplification control (IAC)

It is a DNA sequence that does not belong to the target microorganism and that is added to the same tube as the sample, to be amplified at the same time. If the IAC amplifies, the reaction worked in that tube; if it does not, something prevented it. Hoorfar and colleagues (2004) sum it up like this: the IAC makes it possible to distinguish a true negative from a PCR failure caused by inhibitors, by the instrument or by poor polymerase activity. The same authors point out that the EN ISO 22174 standard requires its presence in the PCR detection of foodborne pathogens.

There are two design strategies:

• Competitive IAC: shares the primer binding sites with the target. It avoids primer interference problems, but competes for reagents and may reduce sensitivity.
• Non-competitive IAC: uses its own primer pair. It requires careful optimization so that competition for reagents and polymerase does not affect detection of the target, something similar to the design challenge of multiplex PCR.

The IAC concentration is critical. The authors recommend keeping it as low as possible, as long as it still generates a signal: an excess IAC can tolerate partial inhibition that does affect a target present at a low copy number, and also take resources away from the reaction.

Positive control

A reaction with the genetic material of the target microorganism that demonstrates that the primers, probes, reagents and temperature program detect what they should detect in that run.

Negative control and no-template control (NTC)

The no-template control contains all the reagents but no nucleic acid. If it amplifies, there is reagent contamination or carryover of amplicons from earlier runs, and the positives of that run lose credibility. A negative extraction control, processed alongside the samples from the start, extends that vigilance to the whole preparation stage.

Process controls

Some protocols add a control that accompanies the sample from enrichment or extraction, to verify that the whole process (not just amplification) worked. It is especially useful in new or difficult matrices.

How the combination of signals is read

With an IAC in each tube, the interpretation of a sample falls into three cases, following the scheme of Hoorfar and colleagues:

• IAC amplifies and the target does not: valid negative. The reaction was working and the target was not there.
• Target amplifies and the IAC does not: valid positive. When the target is present in large amounts, it can displace the IAC in the competition for reagents.
• Neither amplifies: invalid result. The most likely cause is inhibition or a failure of the reaction; it cannot be reported as negative.

All of this holds only if the run controls behaved as expected: the positive control amplified and the no-template control stayed clean. If either fails, the whole run is reviewed before interpreting any sample.

Inhibition: where it comes from and what to do

The review by Schrader and colleagues (2012) describes PCR inhibitors as a diverse group of substances with different properties and mechanisms, some typical of certain sample types, which can reduce assay sensitivity or produce false negatives. In food, matrices high in fat, protein, polysaccharides or polyphenols, such as chocolate or spices, are the ones that most often require specific preparation protocols. The authors recommend evaluating the influence of inhibitors in each particular assay, not assuming that one protocol suits all matrices.

When the IAC indicates inhibition, the usual responses are:

• Dilute the extract and repeat: the FDA's BAM indicates that, for screening results suspected of inhibition, the analysis must be repeated with diluted template DNA. Dilution reduces the inhibitor more than it reduces sensitivity, as long as the target is not near the limit of detection.
• Improve the extraction: use a protocol designed for matrices with a high load of inhibitors.
• Switch to the culture method: the BAM establishes that, if molecular methods give indeterminate results, the culture method is completed, and that if a spiked sample (matrix spike) is negative on screening, the analysis must continue by culture.

Contamination: the inverse risk

The other classic error is the false positive from contamination. A PCR generates millions of copies of the amplified fragment, and an invisible amount of that product on a pipette, a glove or a surface can contaminate the next run. That is why laboratories physically separate the reagent preparation, extraction and amplification areas, with one-way flow. The 2024 edition of ISO 22174 incorporated precisely requirements for monitoring those flows, including environmental monitoring of the PCR laboratory itself. The no-template control is the first-line alarm: if it amplifies, the run is not reliable.

Controls and automated interpretation

In a laboratory that processes hundreds of reactions a day, checking each IAC, each positive control and each NTC by hand is slow and opens room for errors. That is why interpretation software, such as TxA in TAAG's kits, applies the validity rules systematically before assigning a result. Automation does not replace control design, but ensures the rules are applied the same way across all runs and shifts.

And even with all the controls in order, a screening positive remains presumptive in most regulatory schemes. What happens next is explained in presumptive vs. confirmed result.