Which is a common cause of false positives in PCR testing?

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Contamination with target DNA is the most common cause of false positives in PCR. Because PCR amplifies whatever template is present, even trace amounts of DNA from a previous reaction, environmental DNA, or contaminated reagents can seed a new reaction and produce a detectable signal after many cycles, making it seem like the target DNA was in the sample when it wasn’t. This carryover can occur through aerosols during pipetting, shared work surfaces, or mixing in contaminated tubes or reagents. To prevent it, labs use negative controls, separate pre- and post-amplification work areas, dedicated equipment and pipettes, and sometimes strategies like uracil-D glycosylase to prevent carryover amplification.

Primer-dimers are non-specific byproducts formed by primers annealing to each other and can occasionally appear as a signal, but they are typically identified and minimized through careful primer design and analysis (for example, melting curves or product size checks). Inhibitors in the reaction block amplification, leading to false negatives rather than positives, and having too little template also tends to yield false negatives instead of false positives.