Inconclusive results may arise because DNA is degraded or samples are inhibited. DNA degradation is a process by which DNA has been damaged to the point that it cannot be amplified and subsequently detected. The use of preservatives in our kits minimises the risk of DNA damage, but inappropriate storage conditions may still lead to DNA degradation. PCR inhibition occurs when compounds present in the sample (e.g. acids) interfere with the qPCR reaction at a molecular level, preventing or reducing target DNA amplification and subsequent detection. Inhibitors may interact directly with target DNA, or interfere with the enzyme that drives the PCR reaction.
A synthetic control is included in kits to test for degradation, and a synthetic control added to samples after DNA extraction to test for inhibition. A qPCR assay specific to each control is used to test the original DNA extract. If the signal from either synthetic control is lower than expected or absent, then degradation and/or inhibition are present. If degradation is identified, there is no way to resolve this and the sample will be reported as inconclusive. If inhibition is identified, the sample is diluted twice prior to testing for GCN presence in accordance with the Natural England protocol. If dilution fails to resolve inhibition, then the sample will be reported as inconclusive.
Ponds with an inconclusive result should be resampled, adhering strictly to the Natural England protocol, and new samples returned to the lab for analysis as soon as possible.