Contamination of reagents used for molecular detection of SARS-CoV-2 risks compromising diagnostic assays

Hi All

Very important information highlighted by the Coronavirus Standards Working Group on the extent of contamination in reagents (especially oligonucleotides) used for SARS-CoV-2 detection.

Please see this slide deck for more information, which is available on the working group website.

I quote directly from the presentation’s suggestion below! These are extremely useful and important both for testing labs but also anyone making test kits and reagents.

Test for it

  1. Assume reagents may contain contamination (Figure 1A). Quality control reagents prior to their use (primers, probes, PCR mastermix, water) using at least 10 negative control replicates (alongside a positive control).
  2. Aliquot reagents for single time use, especially nuclease-free water.
  3. Implement control procedures that include extraction blanks that contain carrier RNA; the latter (present in negative patient extracts) is important for measuring low level contamination. Consider using multiple extraction blanks to detect low level contamination.
  4. Further information on the source of contamination can be provided by including reverse transcription negative reactions; this will confirm DNA and not viral RNA as the source

Apply caution when results are close to assay the limit of detection

  • Beware of large numbers results with high Cq values.
  • Consider the pattern of results. If low signal positives are not randomly distributed (e.g. if they occur adjacent to a high titre sample) this could point at cross-contamination. Consider repeating such low positive samples.
  • Consider influences of pre-analysis and sample cross-contamination.
  • If possible test for more than one SARS-CoV-2 target gene.

Take preventive measures

  • Physically separate PCR setup and sample handling steps (and equipment) from those used
  • for PCR analysis. Ideally use pre- and post-PCR rooms.
  • Consider steps during preparation that may lead to contamination through aerosol production: pipetting (high throughput), centrifuges, etc. may lead to small amounts of aerosol that can result in cross-contamination.

Get rid of it

  • Discard all reagents linked to contaminated reactions. While systematic evaluation may
  • determine which reaction component is the culprit, it is often more resource efficient to start
  • from scratch.
  • Deep clean the laboratory using proven solutions that destroy nucleic acids (e.g. bleach and UV)
  • If contamination persists, users may need to redesign assay to different part of the pathogen’s
  • genome