Environmental DNA tests and kits are only reliable when their quality is assured. During both assay development and routine testing, several performance parameters and quality controls must be evaluated to ensure trustworthy results. Every kit we offer includes a validation report covering these parameters. A kit is only made available in our webshop once all validation criteria have been successfully met.
Parameters for assay development and validation
Robustness
“Influence of temperature variations and inhibiting factors present in environmental samples on the performance and specificity of the primer set”
Detection limits
“Lowest quantitative and qualitative limits in which the analysis can be reliably applied”
Efficiency
“The comparison of what is actually produced with what can be achieved with the same consumption of resources”
Repeatability
“The degree of similarity between the results of measurements of the same measured quantity that were performed under different measurement conditions”
Correctness
“The ability of the method to do what it ‘says’ to do”
Quality controls during analysis
Number of replicate reactions
Qualitative analyses are performed in at least eight replicate reactions per sample. A sample is considered positive when at least one of these reactions produces a valid positive signal.
The following quality controls are included during analysis:
PCR positive control (PPC)
The PCR positive controls provided in our kits contain cloned DNA of the target organism. It is used to verify that the qPCR reagents, primers and probe function correctly during the analysis.
At least two positive-control reactions must be included each time the kit is used. These reactions should produce a positive signal. A valid positive result confirms that the assay performed correctly under the conditions of that particular run.
When the positive control does not produce the expected signal, the results of the analysis are invalid and the qPCR run must be repeated.
PCR negative control (PNC)
A PCR negative control must be included each time the kit is used to check for DNA contamination in the qPCR reagents or during reaction preparation.
This control should not produce a positive signal. A positive signal indicates that DNA contamination may have occurred during the procedure. In that case, the qPCR results are considered unreliable and the analysis must be repeated.
Internal positive control (IPC)
The internal positive control is used to assess the efficiency of the DNA-isolation procedure and the quality and purity of the isolated DNA.
The IPC consists of a small fragment of synthetic DNA that is added to the preservation solution supplied with the eDNA isolation kit. The selected DNA sequence does not occur naturally in aquatic environments and does not interfere with the detection of the target organism.
A positive IPC signal should be obtained from every sample. A missing or substantially delayed signal may indicate the presence of PCR-inhibiting substances in the eDNA isolate or a problem during DNA isolation.
In that case, the sample should be investigated further and the isolation or sampling procedure may need to be repeated.
Blank procedure
An additional negative control is included during DNA isolation. This procedure blank contains preservation solution but no environmental sample and is processed in the same way as a regular sample.
The procedure blank should not produce a positive target signal. A positive signal indicates that DNA contamination may have occurred during the isolation procedure.
In that case, the results are considered unreliable. The laboratory and equipment should be cleaned thoroughly, and the DNA-isolation procedure should be repeated.
Limit of detection (LDqPCR)
The limit of detection is the lowest number of target-DNA copies per qPCR reaction that can be detected reliably.
The LOD is determined using a dilution series of the positive control.
Limit of quantification (LQqPCR)
The limit of quantification is the lowest number of target-DNA copies per qPCR reaction that can be quantified with an acceptable level of accuracy and precision, typically at a confidence level of 95%.
The LOQ is determined using a dilution series of the positive control.