Quantitative reverse transcription polymerase chain reaction (qRT-PCR) is a powerful technique for studying gene expression. The key to quantitative accuracy depends on the stability of the reference genes used for data normalization under different experimental conditions. Pine wood nematode (PWN, Bursaphelenchus xylophilus) is the causal agent of the devastating pine wilt disease (PWD). Extensive and prompt research is needed to understand the molecular mechanism of PWD, but identification of the reference PWN genes for standardized qRT-PCR has not been reported yet. We have analyzed eight candidate reference genes of PWN across different temperature conditions and developmental stages. Delta Ct method, GeNorm, NormFinder, BestKeeper, and RefFinder algorithms were used to evaluate the stability of expression of these genes. Finally, we use heat shock protein 90 (HSP90) in different temperatures and arginine kinase gene (AK) in different developmental stages to confirm the stability of these genes. UBCE and EF1γ were most stable across different temperature treatments, whereas EF1γ and Actin were most stable across different developmental stages. In general, these results indicate that EF1γ is the most stable gene for qRT-PCR under different conditions. The systematic analysis of qRT-PCR reference gene selection will be helpful for future functional analysis and exploration of B. xylophilus genetic resources.

Co-infections involving Actinobacillus pleuropneumoniae (APP) and influenza A (H1N1) virus present a serious diagnostic challenge in swine respiratory disease, complicating effective outbreak management and control. This study reports the development and validation of a novel duplex TaqMan real-time PCR assay, optimized for the Coyote Flash10 portable automated detection system, for the simultaneous identification and quantification of both pathogens. The assay employs primers and probes specific to the apxIVA virulence gene of APP and the conserved matrix (M) gene of H1N1, with porcine RNase P as an internal control. Validation using recombinant plasmid standards demonstrated a sensitivity of 1 copy/μL for each target, with strong linear correlation ( R ² > 0.99). Calculated amplification efficiencies (APP: 121.6%; H1N1: 118.6%) were marginally above the typical 90–110% range. However, the assay exhibited robust and consistent quantification without evidence of reaction competition. In a simulated clinical matrix, detection limits corresponded to 10,000-fold and 100-fold dilutions for APP and H1N1, respectively. The method showed excellent repeatability (intra-assay CV <3%) and high specificity, with no cross-reactivity against six other common porcine respiratory pathogens. This rapid, closed-tube assay provides a complete result within one hour and offers a practical, point-of-care-compatible solution for on-farm surveillance and the differential diagnosis of complex respiratory co-infections in swine populations. A key limitation of this study is that validation was performed primarily using simulated samples; future validation with authentic clinical samples will further confirm the method’s clinical utility.

Porcine epidemic diarrhea virus (PEDV) and porcine deltacoronavirus (PDCoV) cause severe diarrhoea in piglets, requiring rapid laboratory differentiation. In this technical note, we evaluated primer–probe sets from Chinese quarantine industry standards on the Coyote Flash10 cartridge-based POCT combined primer–probe RT qPCR platform. Assays were characterised using a digitally PCR-quantified single-target in vitro transcribed viral RNA and RNA-spiked porcine faecal matrix. Exploratory low-concentration amplifications were observed at 3.222 copies/μL (PEDV) and 2.28 copies/μL (PDCoV); however, the corresponding Ct values fell within an indeterminate range, and no statistically validated limits of detection were established. The PEDV standard curve included concentrations with mean Ct values in the indeterminate and negative ranges, so its apparent efficiency was not considered a validated performance metric. PDCoV efficiency was sub-optimal (~77.7%). Low intra run Ct variability (CV < 3%) was observed in spiked matrix tests. No cross amplification was detected against four selected swine pathogens under the tested conditions. Notably, mixed-ratio co-template viral RNA experiments simulating co-infection were not performed. The proprietary internal control (IC) was recorded descriptively and was not used to assess extraction efficiency or sample validity. This work demonstrates preliminary analytical feasibility of this primer–probe combination on the Flash10 platform; full clinical diagnostic performance remains to be validated in further studies.