Toolbox

The Level of Fouling (LOF) is a nominal rank scale with six categories and is widely used to rapidly characterize biofouling on submerged surfaces of ships and boats.

Auckland Council recently contracted Cawthron Institute’s Biosecurity Team to develop an updated guideline for application of the LOF scale. This document can be downloaded.

The LOF scale and its application will feature in some of the research associated with the Marine Biosecurity Toolbox programme.

With the establishment of next-generation sequencing technologies, an increasing amount of data can be generated in the pursuit to uncover diversity patterns and detect species of interest. However, the reliability and accuracy of classifying sequencing data depends on the algorithm used, as well as the quality and completeness of reference databases. While curated reference databases are available for commonly used genetic markers in microbial research, curated eukaryotic reference databases are scarce, as are the software tools for generating them. Here, we present CRABS (Creating Reference databases for Amplicon-Based Sequencing), a versatile tool for generating curated reference databases of user-specified genetic markers to aid taxonomy assignment from metagenomic and metabarcoding sequencing data. CRABS is available for download as a conda package and via GitHub.

To address the current need in molecular surveillance techniques that are easy, fast, specific, and usable with minimal equipment and expertise, we have designed a recombinase polymerase amplification (RPA) assay for species-specific Sabella spallanzanii detection. Recombinase polymerase amplification is an isothermal amplification method, that amplifies target DNA within 20 minutes at a temperature range of 25°C to 45°C, which makes it possible to run assays at body temperature (~37°C), i.e., hand-held. With the introduction of labelled probes and primer, RPA can be used for fluorometry-based quantitative real time amplification. Detection is also possible with lateral flow (LF) strips, that provide easy visual read-outs. The combination of RPA and LF (RPA-LF) strip is especially valuable in resource-limited environments. RPA-LF was previously successfully implemented for multiple disease carrying organisms/viruses, including SARS-CoV-2 or for invasive species like the Northern Pacific Seastar, Asterias amurensis. A simplified RPA protocol was developed and tested with different end-user groups.

At our outreach sessions and hands-on activities, we have noticed that micropipette usage is a hurdle for environmental DNA (eDNA) methods application by prospective biosecurity citizen scientists. To facilitate the induction process, we have created a visual pipetting guide, which can be used e.g. in a classroom by teachers when presenting molecular technologies, or added to a point-of-need molecular detection toolkit for biosecurity practitioners on the ground.

The Environmental DNA Protocol Development Guide for Biomonitoring and Environmental DNA test validation guidelines were developed as a collaborative project, funded by the Australian Government Department of Agriculture, Fisheries and Forestry with support from the Marine Biosecurity Toolbox. More than 50 experts from across Australia and New Zealand were involved in the development of these guidelines and provided feedback during various stages of the process. These guidelines harmonise minimum quality standards to develop eDNA-based protocols and tests for biomonitoring applications. Both documents will be reviewed yearly to improve accuracy and clarity with current research and innovation.

A new online tool called the Pest Alert Tool has been developed to screen high-throughput sequencing datasets for species of concern, such as non-indigenous and notifiable and unwanted organisms, in New Zealand’s marine environment, providing a valuable resource for biosecurity decision making. The tool can be adapted for other regions and applications and has the potential to be used for retrospective analysis of existing datasets, research purposes, and educational outreach activities.