Status: Completed
Start date: 28 March 2019
Completion date: 1 June 2022
Project code: A-019
Species/Threats: Multiple
This project focused on how we can quickly and easily detect biosecurity threats through the importation of 12-14 million ornamental fish into Australia each year, with an estimated trade value of about $350 million.
Of the 37 established alien fish species, 25 are thought to have arrived via the ornamental fish trade and species used in this industry are now the main source of new introductions.
The aims of this project were to:
Environmental DNA (eDNA) samples have been collected from an international aquarium trade wholesaler and sequenced and analysed using the Illumina Miseq and the Oxford Nanopore MinION portable sequencer, which is forming the basis of a DNA reference database. Standard operating procedures have been developed to prepare metabarcoding libraries for Illumina and Nanopore sequencing platforms applicable to high priority species.
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 usingthe 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.
