Environmental DNA tests and kits can only be trusted if quality can be assured. Different parameters and controls must be taken in account during development and performing of the test to trust the results. All kits offered contain a validation report with these parameters. Only if all parameters were approved, the kit is offered in the shop.
Parameters for development and validation of the test
“Influence of temperature variations and inhibiting factors present in environmental samples on the performance and specificity of the primer set”
“Lowest quantitative and qualitative limits in which the analysis can be reliably applied”
“The comparison of what is actually produced with what can be achieved with the same consumption of resources”
“The degree of similarity between the results of measurements of the same measured quantity that were performed under different measurement conditions”
“The ability of the method to do what it ‘says’ to do”
Controls during analysis
The qualitative analyses of the samples are performed minimally in eightfold. A sample is considered positive if at least one of these analyses gives a positive signal. Checks during analysis:
PCR positive control (PPC)
The PCR positive control provided in this kit contains cloned DNA of the target organism. This is a control for checking the reactions during analyses. This control should give a positive signal. Each time the kit is used, at least two positive control reactions must be included in the run. A positive result indicates that the primers and probes for detecting the target worked properly in that particular experimental scenario. If a negative result is obtained, the test results are invalid and must be repeated.
PCR negative control (PNC)
To validate any positive findings a negative control reaction should be included every time the kit is used. DNA contaminations will be checked with this control. A negative signal should be obtained with this control. A positive signal indicates a DNA contamination somewhere during the procedure. In that case the qPCR results are not reliable and the analysis should be repeated.
Internal positive control (IPC)
The Internal positive control is an efficiency control of the DNA isolation procedure and a quality and purity control of the isolated DNA. The internal positive control is a small piece of synthetic DNA present in the conservation buffer of the eDNA isolation kit. The chosen DNA sequence of the positive control is unknown to the aquatic environment and will not interfere in any detection of target organisms. A positive signal should be obtained from this control in all cases (reactions). A negative signal indicates inhibiting substances in the eDNA isolate or a failure during isolation. In that case the sampling and isolation procedure should be repeated.
During DNA isolation an additional negative control will be used. This control contains only preservation solution will be analysed as an normal sample. If a positive signal is detected with this control, a DNA contamination was obtained during isolation. Results are not reliable anymore. The sampling and isolation procedure should be repeated after cleaning the lab and equipment.
Limit of detection (LDqPCR)
Lowest analysis limit of the qPCR which corresponds to the minimum number of DNA copies in the qPCR which gives a positive result. This will determent with dilution series of the positive control.
Limit of quantification (LQqPCR)
Lowest limit of the qPCR where with a reliability of 95% the number of DNA copies can be quantified. This will determent with dilution series of the positive control.