The DNA extract is tested for PCR inhibition using a real-time PCR assay. If inhibition is found the sample is diluted prior to testing for the presence of great crested newts
The sample is tested for degradation using a real-time PCR assay
The sample is tested for presence of GCN DNA using a real-time PCR assay
According to the Natural England technical advice note once collected, the samples can be stored for 1 month in the refrigerator prior to analysis. If possible, samples should be stored refrigerated before they are returned to us.
A positive result means that great crested newts are present in the water or have been present in the water in the recently.
A negative result indicates that DNA from the great crested newt has not been detected in the sample.
An inconclusive result can be issued for a couple of reasons.
DNA from the great crested newt has not been detected but the controls have indicated that the sample has been degraded.
DNA from the great crested newt has not been detected but the controls have indicated that the PCR has been inhibited.
An inconclusive result may be due to the water chemistry, sediment or other debris in the sample. The pond could be retested (a new sample would be required) but if the water chemistry was the cause of the inconclusive result - the re-test would also be inconclusive.
If degradation (the results from the degradation control assay were outside of accepted limits) or inhibition (something in the DNA extract prevented PCR amplification) were observed, negative results will be reported as inconclusive.
