African swine fever (ASF) is a devastating disease of pigs. Without a vaccine, early detection and rapid diagnosis of ASF is a crucial step towards effective disease control. In many countries where ASF is endemic, laboratory infrastructure including sampling and sample shipment is inadequate, and a rapid laboratory confirmation would require that the diagnosis is performed at regional laboratories close to the pig farms of concern, or even at the farm‐side. This study intended to evaluate measures including sample preparation methods, dry‐down assay, and a portable, battery‐powered real‐time PCR instrument, to improve molecular diagnosis under field conditions. A simple dilution of blood samples, either in Phosphate‐buffered saline or a commercial buffer, worked similarly to beads‐based nucleic acid extraction using a magnet as the core equipment; the latter method did work as well for those samples with low viral load or high Ct values. The real‐time PCR assay using a Universal ProbeLibrary (UPL) probe tolerated suspected inhibitory substances present in the prepared samples better, whereas a dry‐down assay had a higher diagnostic sensitivity. Additionally, an inhibition control assay proved to be helpful in avoiding false negative results when interpreting negative results of samples that might be of low quality or with inadequate reduction of inhibitory substances. When tested with synthetic DNA standards, the portable instrument performed at a level approaching the stationary thermocycler. In summary, the developments of suitable sample preparation, robust and thermal‐stable real‐time PCR assays with inhibition control, and battery‐powered portable thermocyclers with middle‐throughput offer one way forward to provide rapid, reliable molecular diagnosis under challenging field conditions.

This study characterized the susceptibility and dynamic of porcine deltacoronavirus infection in grower pigs under experimental conditions using a combination of syndromic and laboratory assessments. Seven-week-old conventional pigs (n = 24) were randomly distributed into PDCoV- (n = 12) and mock-inoculated (n = 12) groups. Serum was collected at −7, 0, 3, 7, 10, 14, 17, 21, 28, 35, and 42 days post-inoculation (DPI) to evaluate viremia (RT-qPCR) and antibody response (S1-based ELISA). Viral shedding and potential infectivity were determined using pen-based oral fluids and feces collected every other day between DPI 0 and 42. Pigs showed no clinical signs or viremia throughout the study. Active virus shedding was detected in feces (6-22 DPI) and oral fluids (2-30 DPI), peaking at DPI 10. IgG was first detected at DPI 10, being statistically significant after DPI 14 and increasing thereafter, coinciding with the progressive resolution of the infection. Likewise, a significant increase in proinflammatory IL-12 was detected between DPI 10 and 21 in PDCoV-inoculated pigs, which could enhance innate resistance to PDCoV infection. This study demonstrated that active surveillance based on systematic sampling and laboratory testing combining molecular and serological tools is critical for the accurate detection of subclinical circulation of PDCoV in pigs after weaning.

Foot-and-mouth disease virus (FMDV) is a highly contagious agent that impacts livestock industries worldwide, leading to significant financial loss. Its impact can be avoided or minimized if the virus is detected early. FMDV detection relies on vesicular fluid, epithelial tags, swabs, serum, and other sample types from live animals. These samples might not always be available, necessitating the use of alternative sample types. Meat juice (MJ), collected after freeze-thaw cycles of skeletal muscle, is a potential sample type for FMDV detection, especially when meat is illegally imported. We have performed experiments to evaluate the suitability of MJ for FMDV detection. MJ was collected from pigs that were experimentally infected with FMDV. Ribonucleic acid (RNA) was extracted from MJ, sera, oral swabs, and lymph nodes from the same animals and tested for FMDV by real-time reverse transcription polymerase chain reaction (rRT-PCR). MJ was also tested for FMDV antigen by Lateral Flow Immunoassay (LFI). FMDV RNA was detected in MJ by rRT-PCR starting at one day post infection (DPI) and as late as 21 DPI. In contrast, FMDV RNA was detected in sera at 1–7 DPI. Antigen was also detected in MJ at 1–9 DPI by LFI. Live virus was not isolated directly from MJ, but was recovered from the viral genome by transfection into susceptible cells. The data show that MJ is a good sample type for FMDV detection.

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 (p < 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.