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
CSIRO Oceans and Atmosphere Hobart
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.
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.