toxicus, ARGT disease outbreak management, agriculture biosecurity and R. The developed tools will provide capabilities for application in routine detection and discrimination of R. We also designed, developed and compared three kinds of internal controls: two customized artificial internal controls and one to target the host genome. In this research, we developed accurate, reliable and sensitive diagnostic TaqMan probe-based multiplex real time qPCR and multiplex endpoint PCR for reliable detection and discrimination of the Select Agent, R. For diagnostics requiring high confidence in test results, the inclusion of an internal control(s) in each PCR tube is extremely important to avoid the possibilities of false negative results. Therefore, inclusion of a single PCR tube as a positive control cannot determine the reliability and accuracy of PCR results in every reaction tube. Many PCR failures canвЂ™t be detected nor the reason for failure identified using only a single positive control. However, PCR reactions fail for a variety of reasons, including, deviation from the validated SOP, the presence of PCR inhibitors, equipment performance, or operator error 14, 15. Positive controls comprised of a single PCR tube containing the target DNA are commonly used in diagnostic laboratories. Multiplex endpoint PCR methods are cost effective and easy to perform compared to TaqMan probe based multiplex qPCR methods 11.ДЄccuracy and reliability of qPCR and PCR methods are greatly enhanced by using appropriate positive and internal controls 12, 13. Multiplex endpoint PCRs are commonly used to differentiate closely related bacterial strains from the same host based on genomic variation 10 (Strayer et al., 2016). TaqMan probe-based qPCR methods are used to detect target pathogen(s) with high sensitivity and reliability for applications in routine diagnostics, microbial forensics and plant biosecurity 7, 8, 9. Validated diagnostic assays for the reliable detection of R. However, no qPCR-based assay has been developed for this high consequence pathogen. The newly detected population, RT-I, was the predominant population on the Yorke Peninsula, South Australia in 2014вЂ“2016 its prevalence and genetic analyses suggest a more recent emergence 1. toxicus have been described: RT-I and RT-II (South Australia) and RT-III (Western Australia) 1. Currently, three genetic populations of R. To prevent the introduction of potentially damaging plant pathogenic bacteria into new environments and to mitigate the negative impacts following introduction requires an understanding of pathogen population biology. Inspection and certification of ryegrass-based hay is required prior to export from Australia 3.Д®arly detection and accurate diagnosis are essential elements of an effective plant biosecurity system 4, 5. Recurrent livestock deaths due to ARGT outbreaks in Australia have raised biosecurity concerns about the global spread of R. Annual ryegrass toxicity (ARGT) is a disease of livestock and wildlife caused by the ingestion of a toxin excreted by R. (annual beard grass), and Agrostis avenacea J.F. toxicus including, Lolium rigidum Gaudin (annual ryegrass), Polypogon monspeliensis (L.) Desf. Rathayibacter toxicus is a gram-positive bacterial plant pathogen and a Select Agent in the United States since 2008 due to the potential threat to the U.S. Assays were validated by blind ring tests with multiple operators in three international laboratories. To enhance reliability and increase confidence in results, three types of internal controls with no or one extra primer were developed and incorporated into each assay to detect both plant and artificial internal controls.

The endpoint multiplex PCR and TaqMan qPCR assays detected 10В fg and 1В fg of genomic DNA, respectively. Specificity was confirmed with appropriate inclusivity and exclusivity panels no cross reactivity was observed.

toxicus to the population level of discrimination. Highly sensitive and specific TaqMan qPCR and multiplex endpoint PCR assays were developed for the detection and identification of R. toxicus were sequenced (Illumina MiSeq platforms), assembled and genomic regions unique to each population identified. Whole genomes of three genetic populations of R. Accurate diagnostic protocols to support phytosanitary decisions, delineate areas of freedom, and to support research are required to address those concerns.

Biosecurity and phytosanitary concerns arise due to the international trade of seed and hay that harbor the bacterium. A threat to livestock industries globally, the bacterium was designated a U.S. Rathayibacter toxicus is a toxigenic bacterial plant pathogen indigenous to Australia and South Africa.

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