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Updated: Dec 18, 2025

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Pitfalls in SARS-CoV-2 PCR diagnostics.

Kerstin Wernike1, Markus Keller1, Franz J Conraths1

  • 1Friedrich-Loeffler-Institut, Greifswald-Insel Riems, Germany.

Transboundary and Emerging Diseases
|June 15, 2020
PubMed
Summary

Contaminated SARS-CoV-2 PCR reagents can cause false positives, impacting COVID-19 test accuracy. Rigorous validation of all reagent batches is crucial to ensure reliable diagnostic specificity and positive predictive value.

Keywords:
COVID-19contaminationcoronavirusdiagnosticspoolingreal-time PCRswab

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Area of Science:

  • Molecular Biology
  • Infectious Disease Diagnostics

Background:

  • Millions of reverse transcription quantitative polymerase chain reaction (RT-qPCR) tests are vital for COVID-19 pandemic control.
  • Deviations in diagnostic sensitivity and specificity compromise the predictive values of PCR tests.

Purpose of the Study:

  • To report contaminations in commercial SARS-CoV-2 primers/probe sets as a cause of false-positive RT-qPCR results.
  • To highlight the impact of reagent contamination on diagnostic specificity.

Main Methods:

  • Commercial primers/probe sets for SARS-CoV-2 RT-qPCR were evaluated.
  • Negative control samples were tested to detect contaminations.
  • Internal validation processes were employed to identify low-level contaminations.

Main Results:

  • Contamination of commercial primers/probe sets with SARS-CoV-2 sequences was detected.
  • Quantification cycle (Cq) values as low as 17 were observed in negative controls with contaminated reagents.
  • Some reagent batches showed very low-level contamination detectable only through thorough internal validation.

Conclusions:

  • Extensive pre-testing of each reagent batch is imperative before routine use in COVID-19 diagnostics.
  • Reagent contamination can lead to false-positive results, particularly affecting positive predictive value in low-prevalence settings.
  • Retrospective re-assessment of COVID-19 diagnostic results may be necessary to validate epidemiological data and control measures.