Evaluation of the FMDV 2.1 dry RT-rtPCR prototype assay for detection of foot-and-mouth disease virus in lymph nodes

We evaluated the performance of the FMDV 2.1 dry RT-rtPCR prototype assay (Tetracore) to detect foot-and-mouth disease virus (FMDV) in lymph nodes. Various masses (20-100 mg) of FMDV-negative lymph node tissue were homogenized, diluted, and spiked with FMDV RNA to determine the optimal amount for detection. Optimal amplification was obtained using 20 mg of tissue. Fifteen lymph nodes collected from animals challenged with FMDV that had clinical signs were subjected to manual and automated homogenization and tested using the FMDV 2.1 dry RT-rtPCR prototype assay on a T-COR 8 thermocycler (Tetracore). Additionally, automated homogenates were tested using the World Organisation for Animal Health (WOAH)-recommended RT-rtPCR assay on a CFX real-time thermocycler (Bio-Rad). Strong Pearson correlation was observed between homogenization methods using the FMDV 2.1 dry RT-rtPCR assay (r = 0.74, 95% CI [0.49, 1.00]; n = 15, p < 0.001). The Pearson correlation between the 2 RT-rtPCR assays was weak (r = 0.25, 95% CI [-0.78, 1.00]; n = 15, p = 0.076). When manual and automated homogenization methods were compared using the FMDV 2.1 dry RT-rtPCR prototype assay, 6 of the 15 samples tested positive, 7 tested negative, and 2 had a discrepant result. When the samples homogenized using the automated method were subsequently tested with both the FMDV 2.1 dry RT-rtPCR prototype assay and the WOAH-recommended RT-rtPCR assay, 5 of the 15 samples were positive, 5 were negative, and 5 were discrepant. The Cohen kappa coefficient indicated substantial agreement between homogenization methods (0.74) and fair agreement between assays (0.36). Our findings provide preliminary support for the FMDV 2.1 dry RT-rtPCR prototype assay, but the assay requires further optimization.