NDRV, MDRV and RA strains used in this study were isolated from China and kept in our laboratory. Goose Astrovirus. Duck Hepatitis B Virus, Escherichia coli, and Salmonella isolated by our laboratory were used to assess specificity test.

Considering the phylogenetic closeness between NDRV and MDRV, we developed species-specific primers and probes to differentiate these two pathogens without cross-reactivity. We retrieved all available genomic sequences of NDRV and MDRV from GenBank and performed sequence alignment using DNAMAN 9.0 (Lynnon Biosoft, USA). Through a comparative analysis of conserved regions, we identified the sigma B protein-encoding gene (σB) (NDRV: GenBank: PQ456210.1, MDRV: GenBank: KJ569581.1) as the target for detecting both viruses. For RA, the dtxr gene (GenBank: EU541215.1), a conserved regulator of iron metabolism, was selected as the amplification target. Three pairs of different primers and TaqMan probes were designed (Table 1). The TaqMan probe marked the FAM (MDRV), HEX (RA) and Cy5 (HEX) fluorescence reporting groups, respectively.

Use FastPure virus DNA/RNA Mini Kit v2 (Vazyme, Nanjing, China) to extract viral RNA. Use One Step PrimeScript™ RT-PCR kit (Takara, Beijing, China) for follow-up reverse transcription to synthesize complementary DNA (cDNA). Use HiPure bacterial DNA kit (Magen, Guangzhou, China) to isolate genomic DNA from bacterial cultures. All procedures are carried out strictly according to the instructions of the corresponding kit. RNA, cDNA and DNA are stored at −80 °C for subsequent analysis.

Using the cDNA of MDRV and NDRV and the DNA of RA as templates, use the corresponding primer to clone its gene fragments through PCR (Premix Taq; DL2, 000 DNA Marker, Takara, Beijing, China). Following recovery and purification, the amplified genes were individually ligated into the pClon007 vector to construct recombinant plasmids (TSINGKE, Beijing, China). The recombinant plasmids verified as correct through PCR and sequencing were designated as pRA, pNDRV, and pMDRV, respectively, and served as plasmid standards. The concentrations of these recombinant plasmids were measured using a Nanodrop spectrophotometer and subsequently converted to copy number (copies/μL).

Single quantitative PCR (qPCR) assays were conducted utilizing the 2×Q3 Probe qPCR Master Mix (ToloBio, Shanghai, China) in accordance with the manufacturer’s instructions. An orthogonal matrix-based experimental design was employed to optimize the concentrations of primers and probes. Four final primer concentrations (0.1, 0.15, 0.2, and 0.25 pmol/μL) and seven probe concentrations (ranging from 0.1 to 0.4 pmol/μL in increments of 0.05 pmol/μL) were systematically evaluated. Identical plasmid standards (pRA, pNDRV, or pMDRV) served as templates for each experimental group. The thermal cycling protocol comprised an initial denaturation at 95 °C for 5 min, followed by 40 cycles of denaturation at 95 °C for 10 s and annealing/extension at 60 °C for 30 s.

Utilizing the optimal primer-probe concentrations established from single RT-PCR (as detailed in Section 2.5), we integrated the three pairs of primer-probes into a unified triplex reaction system. The initial concentrations of primers and probes were proportionally adjusted to ensure consistency with the single conditions. The thermal cycling parameters adhered to the single protocol.

The three plasmid standards (pRA, pNDRV, pMDRV) underwent a 10-fold serial dilution in nuclease-free water to achieve concentrations ranging from 10⁹ to 10² copies per microliter. Equal volumes of each diluted standard were then combined to formulate a triplex template mixture. Triplex PCR amplification was conducted in triplicate under the optimized reaction conditions detailed in Section 2.6. The cycle threshold (Ct) values were plotted against the logarithm of the template copy numbers (log₁₀ copies/μL) to construct standard curves.

The total RNA of MDRV, NDRV, and Goose Astrovirus was extracted and transcribed into cDNA. Total DNA from RA, Duck Hepatitis B Virus, Escherichia coli, and Salmonella was also extracted for specificity testing, ddH2O as negative controls.

A 1:1:1 mixture of plasmid standards (pMDRV, pNDRV, pRA; see Section 2.4) was subjected to serial 10-fold dilutions in nuclease-free water. Copy numbers were determined using the formula provided in Section 2.4, which is based on plasmid size and concentrations measured by NanoDrop. Triplex TaqMan qPCR amplifications were conducted under optimized conditions as detailed in Section 2.6. To compare sensitivity, the same dilution series was concurrently tested using conventional PCR with pathogen-specific primers (Table 1), and the amplification products were analyzed through 1.5% agarose gel electrophoresis.

To assess the intra- and inter-assay reproducibility of the assay, we systematically conducted repeatability tests using standardized protocols. Three recombinant plasmid standards were uniformly mixed and subjected to serial 10-fold dilutions, resulting in final concentrations of 10⁶, 10⁴, and 10² copies/μL. The repeatability of the assay was evaluated by calculating the standard deviation (SD) and coefficient of variation (CV) of three repeated measurements at different concentration levels.

Cut the duck tissue sample into small pieces and put it into a 2 mL disinfection centrifuge tube. Add an appropriate amount of phosphate buffered brine (PBS) for grinding, and then freeze and thaw three times. Following centrifugation, total RNA and bacterial DNA were extracted from the supernatant. The total RNA was then reverse transcribed into complementary DNA (cDNA), which was analyzed using the established multiplex TaqMan fluorescence quantitative PCR assay.99 liver samples were collected from dead ducks (including Peking duck and Muscovy duck) in Jiangxi province in 2025.

Bacterial isolation was performed from RA-positive samples. Single colonies suspected to be RA were subjected to detection using the described assay. Strains testing positive for RA were further analyzed by PCR using RA-16sRNA primers, and the resulting products were sequenced for definitive identification. After confirmation as RA, the isolates were screened via PCR with primers (Table 2).

The results of optimizing the ratio of primers and probes in a single detection reaction (Table 3) illustrates variations in reaction performance across different probe and primer ratios. Considering both reaction efficiency and cost-effectiveness, the optimized 20 μL qPCR system comprises 10 μL of 2×Q3 probe qPCR Master Mix, 0.4 μL of NDRV-L/R, 0.4 μL of NDRV-probe, 0.3 μL of MDRV-L/R, 0.4 μL of MDRV-probe, 0.4 μL of RA-L/R, and 0.4 μL of RA-probe, with the volume adjusted to 20 μL using nuclease-free water.

The recombinant plasmid standards, pRA, pNDRV, and pMDRV, were subjected to a 10-fold serial dilution and subsequently combined in equal volumes. Plasmids with final concentrations ranging from 10⁹ copies/μL to 10² copies/μL were utilized as templates. The TaqMan-based triplex qPCR amplification curves and standard curves were generated using the methodology developed in this study. The correlation coefficients (R²) of the three standard curves exceeded 0.990. The findings indicated a strong linear relationship between the concentrations of 10⁹ copies/μL to 10² copies/μL and the Ct values of the three plasmid standards (Figure 1).

The results (Figure 2) showed that only RA, NDRV, and MDRV exhibited specific amplification curves, while other pathogens failed to produce amplification. This demonstrates the specificity of the primers and probes.

Sensitivity assessments of both the TaqMan-based triplex qPCR and PCR were performed utilizing serial 10-fold dilutions of plasmid standards (pRA, pNDRV, and pMDRV) combined in equal volume ratios. Quantitative analysis determined the limits of detection to be 10¹ copies/μL for pRA, 10¹ copies/μL for pNDRV, and 10¹ copies/μL for pMDRV when employing our triplex assay. In contrast, the conventional PCR exhibited LODs of 10³ copies/μL, 10³ copies/μL, and 10³ copies/μL, respectively. The TaqMan-based triplex qPCR developed herein demonstrated a 100-fold increase in sensitivity across all targets, underscoring the superior specificity and sensitivity attributes characteristic of TaqMan probe chemistry (Figure 3).

The results show that in the intra-group and inter-group repeatability test, the variation coefficient of the Ct value is less than 2.5% (Table 4), indicating that the assay has good repeatability.

To further substantiate the clinical applicability of this assay, we conducted an analysis of 99 liver samples obtained from ducks with suspected infections across various regions. Pathogen-specific detection yielded positive results in 28 samples (Figure 4), RA (n = 8), NDRV (n = 20), MDRV (n = 0). These results confirm the prevalence of infections in field settings. The assay demonstrated significant diagnostic utility for epidemiological surveillance and clinical management of waterfowl diseases.

The presence of amplified bands indicated potential carriage of the corresponding resistance genes. The detection results of resistance genes in one representative isolate, demonstrating that this strain carries multiple antibiotic resistance genes (Figure 5). These include genes conferring resistance to aminoglycoside antibiotics (RanA, RanB, and aads), macrolide resistance genes [EreD, Erm(35), and ErmF], a chloramphenicol resistance gene (floR), and tetracycline resistance genes [Tet(X3), Tet(X6), and TetX]. And strains carrying a variety of resistance genes accounted for a high proportion in this trial (Table 5).