Now that the testing season for the presence of the great crested newt (GCN) has begun it will be crucial over the next two and a half months for those using an eDNA (Environmental DNA) testing service to have confidence in the technique.

However, during lab proficiency testing in 2017 – provided by FAPAS – only four out of seven laboratories offering eDNA testing for GCNs during the 2017 season correctly identified all seven of the ‘blind’ samples sent out.

Read our investigation into the fate of the great crested newt and Brexit here.

In February 2018, ten ‘blind’ samples were sent out to nine laboratories wanting to offer eDNA testing for GCNs during the 2018 season – and only five of these nine laboratories correctly identified all ten samples.

Developing best practice

Testing for GCNs and other species using eDNA is still an evolving technology, and laboratories are in the process of developing best practice.

There are, however, some important standards for how labs offering this service can optimise performance in terms of proficiency testing, and ensure they operate to best practice standards when processing pond water samples for eDNA analysis to determine the presence or absence of GCNs.

The approved methodology, standards, and field and laboratory procedures are set out in appendix 5 of the 2014 edition of Analytical and methodological development for improved surveillance of the Great Crested Newt, by Biggs, Ewald et al. otherwise known as the Technical advice note.

All labs should be following these methodologies and protocols to the best of their ability and in good faith. However, the fact that four out of nine labs failed to correctly identify all of the ‘blind’ samples in the 2018 test means that some things are going wrong, with a knock on negative effect on the laboratories’ clients. 

Lab contamination issues

It t is important not to start pointing the finger at ‘bad labs’. Due to its stochastic nature, PCR (Polymerase chain reaction) detection can be variable when eDNA is present at very low concentrations. This means that different laboratories may return a result where 1, 2, or even 3 of the 12 required replicates are positive – as was the case in both years – whilst still being considered accurate.

This in turn underlines why it is important for laboratories to be able to demonstrate that they can detect GCN eDNA at the limit of detection or they will fail to detect GCN eDNA at very low concentrations meaning that they will be more likely to report false negative results.

The inability to detect low levels of GCN eDNA can be caused by something as simple as pipetting errors, incorrect quantification or storage of the DNA used to determine the limit of detection, incorrect storage or use of substandard/expired PCR reagents.

All laboratories should regularly check and calibrate their pipettes to ensure they are measuring the correct volumes and should ensure that DNA and PCR reagents are stored correctly. The occurrence of false positives is likely to be down to contamination issues within the laboratory.

Checks and balances

Ecologists also have an important role to play in the field when collecting samples, as erroneous readings can be the sometimes be as a result of errors made by the ecologist when taking the sample. Contamination can be a result of contamination of the sampling kit when it was prepared, cross-contamination of samples during DNA extraction, and contamination during PCR set up.

If not already doing so each of these aspects of the methodology should be carried out within separate laboratories to provide a physical separation of activities. Laboratories can also regularly run ‘blank’ plates (DNA free water rather than DNA) to monitor for contamination and run samples known to be positive/negative for GCN to increase confidence in the results and identify potential cross-contamination issues.

Laboratories cannot afford to ignore the results of proficiency testing and should be open and honest with their clients about their results.

Although Natural England are not ‘policing’ the scheme or accrediting laboratories who do correctly identify the ‘blind’ samples they will now only accept eDNA results from laboratories participating in this scheme to support GCN license applications and post-development presence or absence monitoring for mitigation licences.

This new technology is of great importance to the industries that need to test for newts, and laboratories offering this testing must continually review and improve their services is to increase confidence in the technique of eDNA testing and demonstrate its utility for monitoring of the presence of GCNs. Ecologists should be active participants in providing checks and balances on laboratory testing, as on occasion an eDNA result might contradict what they already known about a pond.

This Author

Helen Rees is a director at ADAS, which runs an eDNA testing service for GCNs.