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
- 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).
- Aliquot reagents for single time use, especially nuclease-free water.
- 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.
- 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