Full Factorial Comparison of the Diagnostic Performance of Three Nucleic Acid Extraction Kits and Three PRRSV RT-qPCR Assays Using Swine Oral Fluids of Known Status

Porcine reproductive and respiratory syndrome (PRRS) is one of the costliest diseases in swine production, causing >$1.2 billion USD in annual losses in the United States. Oral fluids are widely used for PRRS virus (PRRSV) surveillance, accounting for 42% of nearly 480,000 PRRSV RT-qPCR cases submitted to six Midwestern U.S. laboratories between 2020 and 2025. Despite this reliance, few studies have applied appropriate methodological approaches to compare the performance of commercial extraction and PRRSV RT-qPCR protocols for oral fluid specimens. In this study, we evaluated nine extraction-amplification protocols for PRRSV RNA detection, based on three commercial extraction kits and three commercial RT-qPCR assays. For each protocol, performance was evaluated using 314 oral fluid samples of known status (215 positive, 99 negative), collected under controlled conditions from 72 pigs assigned to five groups inoculated with contemporary PRRSV isolates and from one negative control group. The Cq values were normalized as efficiency-standardized Cqs (ECqs) and then analyzed by receiver operating characteristic (ROC) analysis. The mean amplification efficiencies ranged from 67 to 92%, repeatability from 0.98 to 0.99, and overall reproducibility was 0.91. The ROC AUCs ranged from 0.916 to 0.986, with significant pairwise differences (<i>p</i> < 0.05). At optimal ECq cutoffs, sensitivities ranged from 83 to 98.1% with 100% specificity. Normalization enabled objective protocol comparisons and statistically valid diagnostic cutoffs and supports future improvements in PRRSV diagnostics.