Biosecurity molecular screening using eDNA technology

Summary

This project is focusing on how we can quickly and easily detect biosecurity threats through the international ornamental fish trade.

The ornamental fish trade in Australia has an estimated value of about $350 million, with 12 to 14 million fish imported each year.  Of the 37 established alien fish species, 25 are though to have arrived via the ornamental fish trade and species used in this industry are now the main source of new introductions. Alien fish species impact on native fishes through predation, competition, habitat change, disease spread, and potential for hybridisation and introgression. The best approach to prevention of further alien fish introductions is to have a border detection system that can immediately and accurately identify pests and diseases present in shipments.

The ornamental fish trade is an ideal system for multi-species detection technology using eDNA, as the DNA from both the fish present and any potential pathogens are contained within a relatively small amount of water. It is likely that DNA will be very concentrated in these samples as the fish experience stress during transportation. Also, this is a great example of a commodity importation that could harbour a range of unidentifiable risks.

Status

Commenced

Objectives

The aims of this project are to:

  • Develop standardised protocols for sample collection and preparation of eDNA;
  • Construct reference databases for potential target species and for various taxonomic levels for identification of novel species.
  • Trial various real-time DNA sequencing platforms, specifically the Oxford Nanopore Minion device
  • Provide a bioinformatic pipeline to support the analysis and interpretation of the results as they occur in ‘real-time’

Project Leader



Prof. Dianne Gleeson
Project Team

Dr Alejandro Trujillo-González, University of Canberra

Project Partners
  • University of Canberra
  • University of Sydney
  • Cawthron Institute

 

 

Outputs

February 2020:

  • Environmental DNA-based collection, extraction and amplification methods for the use of metabarcoding in Australian biosecurity have been developed and tested overseas by collecting water samples in an international aquarium trade facility in Bangkok, Thailand.
  • Two collection methods using the Biomeme M1 sample preparation kit and Smith-Root Single Use eDNA Filter Packs, proved to be suitable for operational use in Australian Biosecurity.
  • Similarly, DNA has been collected and extracted from four non-permitted fish species of importance to biosecurity officers at border control and DNA for each species has been amplified for four mitochondrial gene regions. Genetic sequences have been curated for accession to the National Centre for Biotechnology Information (NCBI) and used to create custom barcode reference databases.
  • Standard operating procedures have been prepared for eDNA-based detection methods suitable for Australian biosecurity officers. Such standard operating procedures include the collection, extraction and amplification of eDNA using portable thermocyclers, preparing metabarcoding libraries for sequencing using Illumina and Nanopore technologies, and the analysis of sequenced data using a custom user-friendly bioinformatics pipeline.
  • These procedures are currently being used to test multiple sequencing methodologies for their suitability in detecting non permitted fish species in water samples at border control. Sequencing of water samples using the Illumina MySeq and Oxford Nanopore Technologies MinION platforms is currently underway.

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