Scientific Abstract

The COVID-19 pandemic clearly illustrated the value of having diversified and scalable supply chains of the testing reagents and consumables used in diagnostic testing; effective private sector, state, and federal partnerships; and fit for purpose diagnostic sample types that are well suited for high throughput testing environments. This study involved leveraging the capabilities and expertise of three swine interest veterinary diagnostic laboratories (VDLs) in the USDA National Animal Health Lab Network (NAHLN) working together with USDA colleagues and industry partners to enhance Classical Swine Fever (CSF) PCR testing capacities and foreign animal disease preparedness across the US. The purpose of this study was to more fully evaluate the specificity of three different CSF PCR assays on two aggregate sample types (oral fluid and processing fluid) obtained from swine herds located throughout the US.  Due to the notable implications of dealing with false positive test results in foreign animal disease testing use cases, very clearly understanding assay specificity across a broad range of farms, regions, and sample types is especially important. Each of the three participating VDLs were responsible for conducting CSF PCR testing utilizing the three different assays on 250 swine oral fluid samples and 250 processing fluid samples. Each of the 500 swine samples were tested by the custom-made single-plex CSF PCR assay currently utilized across the USDA NAHLN, as well as by the single-plex CSF PCR assays provided by the two largest suppliers of commercial PCR reagents (i.e., Tetracore® and Thermo Fisher®) to USDA NAHLN labs. Thus, each swine oral fluid and processing fluid sample tested had nine individual PCR assays performed on them, for a total of 4,500 PCR tests conducted. Each laboratory tested each sample using the nucleic acid extraction protocol (first step in the PCR testing process) they routinely use in the PCR testing of oral fluid and processing fluid samples. PCR was run on the extracts, using the current NAHLN CSF PCR assay, the Tetracore® CSF PCR assay, and the Thermo Fisher® CSF PCR assay. All 4,500 tests yielded a valid CSF PCR negative result, suggesting each of the CSF PCR assays evaluated have an estimated diagnostic specificity of 100% on oral fluid and processing fluid samples from US swine. In addition, this study indicated that the extraction techniques used by each of the participating laboratories, which were consistent with what is implemented in each of the laboratories’ routine testing, consistently produced valid test results. This supports the suitability of the respective extraction procedures for CSF PCR testing on the sample types tested. Being able to use the same extraction procedure for CSF PCR testing as is being used for the other routine PCR testing on these sample types would allow for streamlined and efficient testing, as this foreign animal disease testing would fit very well into a laboratory’s existing PCR workflow and processes.  This in turn would enable much higher laboratory testing capacities than if a different extraction process were required. In total, this study made a substantive contribution to the larger set of highly collaborative efforts being made towards expanding the number of high-quality CSF PCR assays and fit for purpose sample types available for use in the USDA NAHLN laboratories. Such efforts are foundational towards enhancing testing capacities and the overall foreign animal disease preparedness across the network.