A Contaminated PCR Screen for Invasive Mussels
A watershed council uses PCR to detect environmental DNA (eDNA) from zebra mussels. A 450 bp cytochrome oxidase I fragment is amplified from 1 L water samples after filtration. Six sites are sampled: three from a lake with known mussels, three from a river thought to be uninvaded. The gel shows the positive control at 450 bp, the negative control (sterile water) at 450 bp, all three lake samples at 450 bp, one river sample faint at 450 bp, and two river samples with a 300 bp band. The lab concludes the river is negative except for one 'false positive.' The council must decide whether to close a boat ramp. No replicate extractions were run. The PCR reagent batch was shared with another lab. The council has one week before the holiday boating season.
1. Identify the most likely source of error and explain why the negative control changes the interpretation of every lane.
Read the worked answer
The negative control band at 450 bp indicates contamination of the PCR run. Sterile water should produce no band; a band at the target size means template or amplicon contamination entered the reaction - likely from shared reagents, pipetting, or laboratory aerosols. Because the negative control failed, the entire gel is invalid: the lake positives cannot be used to validate the assay, the faint river band cannot be called a true positive, and the 'false positive' label is premature. The 300 bp bands are a separate issue, but they also cannot be interpreted on a contaminated plate. The council should not close or open the ramp based on this run. Repeat with fresh reagents, separate pre- and post-PCR work areas, aliquoted reagents, and multiple negative controls.
2. What follow-up would you require before closing the boat ramp, and how would you interpret a faint band at the expected size?
Read the worked answer
Require a clean repeat with replication and confirmation. Collect 3–5 field replicates per site, include extraction blanks and no-template PCR controls, and add an internal positive control to check for PCR inhibition. Re-extract with new reagents and run a second primer set targeting a different zebra mussel gene. Sequence any band at 450 bp to confirm species identity. A faint band at the expected size could be low-level eDNA from a few mussels, but it could also be contamination or nonspecific amplification. It is not sufficient for a management closure by itself; it is a trigger for targeted resampling and, if possible, physical inspection of the ramp and water. Given the one-week deadline, the council should use the precautionary principle at the ramp - signage and inspection - without declaring the river invaded.
3. Explain why the 300 bp bands are not evidence for zebra mussels and what they might represent.
Read the worked answer
The assay targets a 450 bp fragment; a 300 bp product is the wrong size. Gel electrophoresis separates by size, so a 300 bp band does not match the expected amplicon and cannot be scored as a zebra mussel positive. It could represent nonspecific amplification from another organism's DNA in the environmental sample, a secondary product from primers binding elsewhere, or a degraded or aberrant product. It is not a primer dimer, which would be much smaller. Without sequencing, the band's identity is unknown. The correct response is to sequence the product and improve PCR stringency, not to count it as evidence for or against mussels.