The Development of a dPCR assay for American mink (Neovison vison)

The development of a dPCR assay for American mink (Neovison vison). 1. qPCR validation a. The successful contractor will evaluate four existing assays for American mink that target either the COI gene (Pugh 2022), cyt-b gene (Di Girolamo 2022) or ND2 gene (Takaba and others 2024) using the validation scale for single species eDNA assays (Thalinger and others 2021). b. Areas for assay improvement should be identified, and further in silico and in vitro testing conducted using qPCR to ensure at least one assay reaches Level 3 minimum on the validation scale (Thalinger and others 2021). The assay(s) should be able to achieve a R-squared value ≥0.990 and efficiency of 90-110%. c. If these validation criteria are not met, the successful contractor must design a new assay that does meet the validation criteria. d. A confidence assessment should be undertaken for the best-performing assay following the requirements set out in NECR359 "A framework for assessing confidence in environmental DNA qPCR assays and results" (K.J. Harper and others 2021). This framework (also known as COASTER) requires a standardised data input, as well as user-defined settings, and can be accessed through common web browsers. The tool operates in R under the Shiny framework. A link to the tool will be made available to the successful contractor (it is planned for this to be open access in the near future).

2. ddPCR or dPCR validation a. The successful contractor will transition the best-performing assay with qPCR technology to ddPCR or dPCR technology. b. In vitro testing should be repeated to confirm assay specificity as well as redetermine the Limit of Detection (LOD) and Limit of Quantification (LOQ) due to the higher detection sensitivity of ddPCR and dPCR technology. c. Costs of running ddPCR or dPCR vs. qPCR should be compared.

3. Field validation a. The successful contractor will design a sampling strategy to test the assay in areas of known American mink presence and absence determined via trapping in consultation with the Natural England Project Officer. At least three sites with known mink presence and three sites with known mink absence should be sampled, and field negative controls should be included. b. Water sampling equipment, protocols and collection forms will be provided for the agreed sampling strategy by the contractor. Consideration should be given to in-field applicability (e.g. single-use filters) by Natural England staff and the need to reduce or eliminate cross-contamination in the field. Freezing samples is not generally possible so alternative methods should be considered for sample preservation prior to shipping to the laboratory. Shipping should be arranged by the contractor and sample storage and packaging instructions provided.

DESCRIPTION CONTINUED IN ADDITIONAL TEXT FIELD

Additional information: c. The contractor will extract DNA from samples (including extraction negative controls) and analyse resulting DNA extracts with the best-performing assay using qPCR and ddPCR or dPCR technology to compare detection sensitivity. At least three technical replicates per sample should be performed with both technologies for DNA amplification, and PCR positive and negative controls should be included. d. Samples that fail to amplify should be tested for inhibition. If inhibition is identified, affected samples should treated for inhibition and retested using qPCR and ddPCR or dPCR technology. e. Positive samples should be sequenced to confirm species identity.

The results should be compiled into a final detailed report.

To apply for this opportunity you must submit your quotation meeting the requirements detailed in the Request for Quotation (RFQ) attached. Your response must be sent to [email protected] by 12 midday on 5th September 2024.

If you have any clarification questions linked to this opportunity or the procurement process please submit these via email to [email protected]. Please note that, unless commercially sensitive, both the question and the response will be circulated to all tenderers.