Genomic DNA of seven R. toxicus strains and NTC (water) were used to perform the blind tests at two national (USA) and three international labs (Australia and New Zealand). Blind tests for multiplex endpoint PCR and TaqMan real-time qPCR in four and three labs, respectively were performed by multiple operators at different time periods (Table (Table4).4). All tests were performed with an artificial internal control (AIC-1). All qPCR reactions were performed in three replicates. In two labs (Elizabeth Macarthur Agricultural Institute, Menangle, NSW, Australia and La Trobe University, Bundoora, VIC, Australia), TaqMan real-time qPCR assays were performed in Corbett Rotor-Gene 6000 thermocyclers (Corbett Research, Sydney, Australia) while the other two labs (Kansas State University Manhattan, KS and Plant & Food Research, Lincoln, New Zealand) used Bio-Rad CFX96 engines to perform the qPCR assays. Qiagen Multiplex PCR and Rotor-Gene Multiplex PCR Master Mix kits were used for endpoint and qPCR multiplex assays, respectively.

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