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Aquatic Animal Health Subprogram: Development of stable positive control material and development of internal controls for molecular tests for detection of important endemic and exotic pathogens

Project number: 2014-002
Project Status:
Completed
Budget expenditure: $172,185.00
Principal Investigator: Nicholas J. Moody
Organisation: CSIRO Oceans and Atmosphere Hobart
Project start/end date: 30 Jun 2014 - 29 Jun 2016
Contact:
FRDC

Need

Quality assured positive control material is critical to demonstrate an assay has performed as expected. Similarly, testing for internal control material ensures extraction procedures produced template of acceptable quality, free of test inhibitors. Both types of controls are particularly important where the samples are being tested to demonstrate freedom from disease (i.e. are negative).

One internal control target is the 18S ribosomal RNA gene. However, assays targeting 18S need to be optimised if multiplexed, 18S is ubiquitous (negative control reactions can test positive) and 18S is not applicable for crustacean samples. Other targets include genes of the host organism, which are often limited to a single species, and are an option that will be investigated for priority species. Plant viruses enable development of one internal control assay for RNA and one for DNA. Optimisation of each assay would still be required if multiplexed, one internal control and one set of primers/probe for any assay reduces costs, eliminates 18S contamination issues and is applicable for all hosts (i.e. finfish, mollusc, crustacean).

Synthetic RNA and plasmid DNA positive controls enable accurate quantification of targets, control over the level of positive template (i.e. added at levels approaching the limits of detection of the assay), are consistent and greatly aid troubleshooting when contamination occurs. They also eliminate the need to source infected animals for positive control material. AFDL implements OIE, EU and/or ANZSDPs for diagnostic assays, reducing the risk of test changes requiring redevelopment of positive controls, which are limitations of synthetic and plasmid controls.

Objectives

1. Produce quantified synthetic RNA positive control material for conventional and real-time RT-PCR assays, available on request.
2. Produce quantified plasmid DNA positive control material for conventional and real-time PCR assays, available on request.
3. Optimised universal internal control based on plant viral RNA and DNA and/or species-specific genes for use in molecular assays developed and implemented
4. Technology transferred and adopted by participating laboratories.

Final report

ISBN: 978-1-925994-19-3
Authors: Moody NJG Cummins DM Mohr PM Williams LM Hoad J Valdeter S Klein R Slater J and Crane MStJ
Final Report • 1.94 MB
2014-002-DLD.pdf

Summary

This project has resulted in the production of a bank of quality-assured, non-infectious, quantifiable, molecular test controls that can be provided to any diagnostic laboratory in a ready-to-use form to assist them with the implementation of specific aquatic animal disease diagnostic tests. In addition, these controls will be useful in the diagnostic laboratory quality systems to demonstrate laboratory competency.

Thirty-two positive control plasmids (22 for real-time assays and 10 for conventional assays) have been prepared and are in routine use. A further 10 plasmid positive controls (8 for real-time assays and 2 for conventional assays) are undergoing final quality checks prior to release for routine use. Therefore, a total of 42 plasmid positive controls for 25 different pathogens have been generated as a result of this project.

Their most important use is as positive controls during diagnostic testing. Because these controls are distinguishable from the pathogens’ genomic nucleic acid, they will assist in identification of cross-contamination between the positive control samples and the diagnostic samples and thus will mitigate against the reporting of false-positive results that occur due to contamination of test samples with positive controls.

In addition, T4 and QBeta phages have been evaluated as heterologous internal positive controls for DNA and RNA targets, respectively, for use in establishing that generic aspects of PCR testing (e.g. nucleic acid extraction and absence of PCR inhibitors) are performing as expected. Implementation of the use of the T4 and QBeta phages as internal positive controls has improved the quality of molecular testing, through more sensitive assessment of the effect of PCR inhibitors and confidence in results generated when testing atypical samples (i.e. plankton, dirt, feed).

The use of these controls in diagnostic testing will assist diagnostic laboratories to monitor the performance of current methods and assist with technology transfer of new methods. This will, in turn, provide laboratories, industry, regulators (managers and policy makers), the general public and trade partners with enhanced confidence in Australia’s diagnostic capability for important exotic and endemic aquatic pathogens.
People
Industry

SCRC: SCRC RTG: Study Tour to Norway (Richard Taylor)

Project number: 2010-762
Project Status:
Completed
Budget expenditure: $0.00
Principal Investigator: Richard Taylor
Organisation: CSIRO Oceans and Atmosphere Hobart
Project start/end date: 8 Sep 2010 - 31 Oct 2010
Contact:
FRDC

Final report

ISBN: 978-1-925982-98-5
Author: Richard Taylor
Final Report • 2010-11-01 • 989.25 KB
2010-762-DLD-RTG.pdf

Summary

The primary reason for this travel was to attend the Fish Breeders' Round Table in Stavanger, Norway. This is an international forum, where knowledge and experience is exchanged between fish breeding researchers and those involved in applied genetic improvement work on a commercial basis.

The forum included over 30 presentations on genomics, genetic models and commercial application of fish breeding. Following the meeting the author visited research institutions and Atlantic Salmon breeding installations.

Industry
PROJECT NUMBER • 2011-761
PROJECT STATUS:
COMPLETED

Seafood CRC: optimisation of viral clearance from broodstock prawns using targeted RNA interference

Economic losses due to diseases mostly caused by viruses remain a major obstacle to realizing the production potential of prawn aquaculture industries in many parts of the world. Broodstock used in prawn hatcheries in Australia are generally managed quite intensively and numbers used are generally...
ORGANISATION:
CSIRO Oceans and Atmosphere Hobart
Environment
PROJECT NUMBER • 2016-015
PROJECT STATUS:
COMPLETED

Proposed northern Australia water developments pertinent to the Northern Prawn Fishery: collation and review

The project reviewed the legislation dealing with Water Resource Management in each of Queensland, the Northern Territory, and Western Australia that effects the management of overland flow in catchments that empty into water managed as part of the Northern Prawn Fishery. The project...
ORGANISATION:
CSIRO Oceans and Atmosphere Hobart
Environment
PROJECT NUMBER • 2016-047
PROJECT STATUS:
COMPLETED

Addressing knowledge gaps for studies of the effect of water resource development on the future of the Northern Prawn Fishery

Overview The objectives of this project were to use historical data and derived knowledge from banana prawn research in the Gulf of Carpentaria (GoC) to identify knowledge gaps and examine estuarine juvenile banana prawn abundance in a subset of Gulf estuaries where water development is...
ORGANISATION:
CSIRO Oceans and Atmosphere Hobart
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