Introduction

With the continuous intensification of goose farming in China, the epidemiological characteristics of viral diseases have become increasingly complex. Clinical epidemiological investigations indicate that GPV, GoCV, TMUV, and GAstV have emerged as the four major pathogens in the current goose industry1,2,3,4. These viruses exhibit significant differences in molecular biological characteristics, pathogenic mechanisms, and epidemiological patterns. Moreover, they often cause complex mixed infections, posing serious challenges to clinical diagnosis and comprehensive prevention and control efforts. GPV is a member of the family Parvoviridae. Its genome consists of linear single-stranded DNA, approximately 5.2 kb in length, which encodes structural proteins including NS1, NS2, and VP1–VP35. The virus primarily affects goslings under three weeks of age, targeting mainly the digestive and immune systems, and can cause characteristic fibrinous enteritis and myocarditis6. GPV is transmitted via the fecal–oral route. Following infection, the incubation period typically ranges from 3 to 5 days, with clinical manifestations including severe diarrhea, ataxia, and acute death. Molecular epidemiological studies indicate that currently circulating GPV strains in China can be classified into three genotypes, with genotype II exhibiting significantly enhanced pathogenicity.

GoCV, a member of the family Circoviridae, possesses a circular single-stranded DNA genome with a length of approximately 1.8–2.0 kb, which encodes the replication-associated protein (Rep) and the capsid protein (Cap)7. The virus exhibits marked lymphotropism, primarily infecting B lymphocytes in the bursa of Fabricius and the spleen, leading to atrophy and functional impairment of immune organs8. Infected geese often exhibit growth retardation, abnormal feather development, and increased susceptibility to subsequent infections. It is important to emphasize that GoCV infection significantly enhances the host′s susceptibility to other pathogens and frequently plays a critical role in mixed infections9. TMUV is a single-stranded, positive-sense RNA virus belonging to the family Flaviviridae, with a genome approximately 11 kb in length that encodes three structural proteins and seven nonstructural proteins3. The virus exhibits broad cellular tropism, enabling it to infect various tissues and organs, including the nervous, reproductive, and immune systems10. Based on variations in the E gene sequence, TMUV can be classified into three genotypes, with genotype 3 currently being the most prevalent in China3,11. Notably, TMUV is not solely dependent on mosquito vectors for transmission; it can also spread through direct contact and aerosol routes, significantly complicating its control and prevention12. GAstV-Ⅱ, a newly identified pathogen belonging to the Astroviridae family, possesses a single-stranded positive-sense RNA genome of approximately 6.8 kb, encoding RNA-dependent RNA polymerase and capsid proteins13,14. The virus primarily affects the kidneys and joint tissues of goslings, inducing characteristic visceral urate deposition and arthritis15,16.

The clinical manifestations of these four pathogens are similar, including anorexia, growth retardation, and neurological symptoms, yet their pathogenic mechanisms and control strategies differ markedly. Current single-pathogen detection methods are not only inefficient but also inadequate for accurately identifying complex mixed infections. Therefore, establishing a highly efficient, specific, and multiplex qPCR assay17 capable of simultaneously differentiating these four pathogens holds profound theoretical and practical significance for achieving precise diagnosis, guiding scientific control measures, and ensuring the sustainable development of the goose industry. This study aims to develop detection technology based on the specific gene sequences of four pathogens, providing reliable technical support for clinical disease diagnosis.

Materials and methods

Virus strains and clinical samples

The viral reference strains used in this study included: classical GPV strain (GenBank No.: NC001701 & MF405917), GoCV strain (GenBank No.: MN756799 & KT808660), TMUV strain (GenBank No.: KM102539 & OP186478), GAstV-Ⅱ strain (GenBank No.: ON205965 & MN099162).

The clinical samples consisted of 72 collected from diseased goose flocks in Sichuan province between 2023 and 2024. The samples included: tissues (including heart (n = 4), liver (n = 8), spleen (n = 7), lung (n = 7), and kidney (n = 6)), bursa of Fabricius (n = 25), and fecal specimens (n = 15). Sample collection was performed following standard operating procedures. All samples were preserved at − 80℃ in our laboratory.

Primers and hydrolysis probes

Based on the viral reference sequences published in the GenBank database, a quadruplex qPCR assay was developed in this study for the simultaneous detection of four major viral pathogens in geese. The selected target genes were the VP3 gene of GPV, the Rep gene of GoCV, the E gene of TMUV, and the ORF2 gene of GAstV-Ⅱ. Four pairs of specific primers and corresponding hydrolysis probes were designed using the NCBI Primer-BLAST online tool (Primer designing tool (nih.gov)) and synthesized by Sangon Biotech (Shanghai) Co., Ltd. The 5′ ends of the hydrolysis probes were labeled with FAM (GPV), HEX (GoCV), Texas Red (TMUV), and CY5 (GAstV-Ⅱ) fluorescent reporter groups, respectively, while the 3′ ends were all labeled with the BHQ1 quencher group. Detailed sequences of the primers and hydrolysis probes are provided in Table 1.

Table 1 Primer and probe sequences.
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Viral RNA/DNA extraction and reverse transcription

All clinical samples were resuspended in 5 mL of physiological saline, vortexed thoroughly, and centrifuged at 4 °C and 12,000 rpm for 15 min. 200 µL of the supernatant was carefully collected and transferred to sterile EP tubes for subsequent nucleic acid extraction. RNA/DNA extraction was performed using the Viral RNA/DNA Extraction Kit (Product No.: RC202-01) from Vazyme Biotech Co., Ltd. (Nanjing), strictly following the manufacturer′s standard operating procedures. The specific steps were as follows: 200 µL of supernatant was mixed with lysis buffer, vortexed thoroughly, and incubated at room temperature for 10 min; anhydrous ethanol was added to precipitate nucleic acids; the mixture was transferred to an adsorption column, washed twice, and finally eluted with 50 µL of nuclease-free water. All procedures were conducted inside a biosafety cabinet to prevent cross-contamination.

Reverse transcription was carried out using the HiScript III All-in-one RT SuperMix Perfect for qPCR (Product No.: R333-01) from Vazyme Biotech Co., Ltd. (Nanjing). The 20 µL reaction system consisted of: 4 µL of 5× HiScript II Buffer, 1 µL of HiScript II Enzyme Mix, 1 µL of random hexamer primers, 2 µL of dNTP mix (10 mM), 5 µL of RNA template, and 7 µL of nuclease-free water. The reaction protocol was set as follows: 5 min of annealing at 25 °C, 15 min of reverse transcription at 50 °C, and 5 min of enzyme heat inactivation at 85 °C. The resulting cDNA was either used immediately in subsequent PCR amplification or stored at − 20 °C for use.

Construction of Recombinant plasmids for standard curves

Using DNA from GPV and GoCV, and cDNA from TMUV and GAstV-Ⅱ as templates, target gene fragments of each virus were amplified by PCR with the corresponding primers listed in Table 1. The amplification products were purified using a PCR purification kit, ligated into the pMD18-T vector (Vazyme, Nanjing, China). The recombinant plasmids were transformed into E. coli DH5α (Vazyme, Nanjing, China) competent cells via heat-shock transformation. Positive clones were identified by PCR screening and confirmed by DNA sequencing. Plasmids were purified using a plasmid extraction kit (Vazyme, Nanjing, China). The purified plasmids were aliquoted and stored long-term at −80 °C.

To establish standard curves, the concentrations of the four recombinant plasmids mentioned above were determined, and their copy numbers were calculated based on the formula. The copy number of each recombinant plasmid was calculated using the following formula: Copies/µL=[(Plasmid concentration ng/µL×10− 9)×(6.022 × 1023)]/[Plasmid length (bp)×660]. The four plasmid solutions were mixed at equal copy number ratios to prepare a mixed standard stock solution containing all targets. Starting from this stock solution, a 10-fold serial dilution series (typically from 1010 to 104 copies/µL) was prepared using nuclease-free water to establish the standard curve. Each dilution was assayed in triplicate. The standard curve was used to determine the amplification efficiency, linear range (R²), and limit of detection of the reaction.

Optimization of reaction conditions for quadruplex qPCR

A 20 µL multiplex qPCR reaction system was established in this study. The reaction mixture consisted of the following components: 4 µL of 5×T5 Fast qPCR Mix, four specific primers (0.4 µL each at 10 µM/L), hydrolysis probe (0.2 µL at 10 µM/L), 2.5 µL of template DNA, and nuclease-free water added to a final volume of 20 µL. To determine the optimal reaction conditions, systematic optimization of primers and hydrolysis probes was performed across a 10 µM concentration gradient. The thermal cycling conditions were as follows: a pre-denaturation at 95 °C for 30 s; followed by 40 cycles of denaturation at 95 °C for 10 s, and combined annealing/extension at 60 °C for 30 s with fluorescence signal acquisition. All reactions were performed on a Roche LightCycler® 96 real-time PCR instrument using the standard mode.

The experimental results indicated that the highest detection efficiency was achieved when the final concentrations of primers and hydrolysis probe were 300–500 nM and 100–300 nM, respectively. All optimization experiments were conducted using a plasmid standard with a concentration of 1 × 104 copies/µL as the template.

Specificity of quadruplex qPCR

To validate the specificity of the multiplex qPCR assay established in this study, cross-reactivity evaluations were performed using several major waterfowl viruses, including GPMV, FADV, NDV, E. coli, and P. multocida. Positive controls consisted of recombinant plasmids containing the VP3 gene of GPV, the Rep gene of GoCV, the E gene of TMUV, and the ORF2 gene of GAstV-Ⅱ, with nuclease-free water serving as the negative control. All tests were conducted under the optimized reaction conditions of this study. The results demonstrated that the assay generated specific amplification signals exclusively for the four target viruses (GPV, GoCV, TMUV, and GAstV-Ⅱ), with no cross-reactivity observed with other common goose pathogens, confirming the high specificity of this detection system.

Sensitivity of quadruplex qPCR

To determine the limit of detection (LOD) of the multiplex qPCR assay, the following systematic evaluation protocol was implemented: Standard plasmids were serially diluted (10-fold gradient) to generate templates with concentrations ranging from 1 × 108 to 1 × 101 copies/µL. For the low-concentration range (1 × 103 to 1 × 101 copies/µL), 10 independent replicate tests were performed. The LOD was defined as the lowest concentration demonstrating ≥ 90% detection positivity rate through statistical analysis.

Repeatability of quadruplex qPCR

To evaluate the reproducibility, ten-fold serially diluted standard plasmids ranging from 1 × 108 copies/µL to LOD were used for the determination of the coefficient of variation (CV%) in multiplex qPCR. For intra-assay repeatability validation, each sample was tested in triplicate. To assess inter-assay reproducibility, the experiment was independently repeated three times using different batches of standard plasmids.

Clinical sample detection

This study employed an established quadruplex qPCR assay to detect four viruses—GPV, GoCV, TMUV, and GAstV-Ⅱ—in 72 clinical specimens (including heart, liver, spleen, lungs, kidneys, bursa of Fabricius, and feces) collected between 2023 and 2024. RNA/DNA extraction and cDNA synthesis for all samples were performed according to the standardized protocols described previously. To verify the reliability of the detection results, conventional PCR was employed for parallel testing, following the established technical procedure described by Zhang et al. and Lin et al18,19.. Furthermore, five samples were randomly selected from the positive samples for further validation by DNA sequencing. Data analysis was performed using GraphPad Prism software (version 8.0). Comparisons of different virus positivity rates were conducted using the Chi-square test. All statistical tests were two-sided, with a P-value of less than 0.05 considered statistically significant.

Results

Establishment of qPCR standard curves

The recombinant plasmids, ranging from 1 × 1010 to 1 × 104 copies/µL, we reused to create standard curves. The standard curves showed a good amplification efficiency and correlation coefficient: GPV (R2 = 0.9984; Efficiency = 99.99%), GoCV (R2 = 0.9988; Efficiency = 99.66%), TMUV (R2 = 0.9972; Efficiency = 100.41%), and GAstV-Ⅱ (R2 = 0.9978; Efficiency = 104.48%); all standard curves were generated using GraphPad Prism 8.0 software. The results showed that our standard plasmids were reliable and that primers and hydrolysis probes were qualified (Fig. 1).

Fig. 1
figure 1

Establishment of amplification curves and standard curves. (A-D) Amplification and standard curves of GPV, GoCV, TMUV, and GAstV-Ⅱ, respectively.

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Establishment and optimization of the quadruplex qPCR reaction method

During the establishment and optimization of the multiplex qPCR method due to mutual interference between different fluorophores, various concentrations of primers and hydrolysis probes can result in different amplification efficiencies. In this regard, we designed a combination of different concentrations of hydrolysis probes and primers, the hydrolysis probe ranging from 100 nM to 300 nM and the primer ranging from 300 nM to 500 nM, and then selected the optimal amplification curve. The results showed that the optimal concentration of hydrolysis probes and primers was a combination of 100 nM and 300 nM.

Specificity of the quadruplex qPCR

In order to evaluate the specificity of qPCR method, five other common pathogens in goose were used as detection templates, including GPMV, FADV, NDV, E. coli, and P. multocida. Then, the recombinant plasmids of GPV, GoCV, TMUV, and GAstV-Ⅱ were used as the positive control, and the concentration of the plasmid was uniformly selected to be 1 × 105 copies/µL. Meanwhile, nuclease-free water was used as the negative control for amplification. The results showed that only four positive amplification curves were presented, indicating that only all four target viruses in this study were detected (Fig. 2), proving that the specificity of the multiplex method was reasonable.

Fig. 2
figure 2

Specificity assays of the quadruplex qPCR. The four amplification curves represent the four positive controls for GPV, GoCV, TMUV, and GAstV-Ⅱ. No fluorescent signal was observed for other goose pathogen samples and the negative control (NC).

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Sensitivity of the quadruplex qPCR

To determine the sensitivity of qPCR method, four recombinant plasmids were added to a reaction system after 10-fold serial dilution ranging from 1 × 108 copies/µL to 1 × 101 copies/µL, the results showed that the method for every viral pathogen was effectively established at the LOD at 1 × 101 copies/µL. However, three recombinant-plasmid-positive detection rate of 1 × 101 copies/µL was found to be less than 90% in follow-up experiences, so there liable LOD was judged to be 1 × 102 copies/µL (Table 2; Fig. 3).

Table 2 Sensitivity results of the quadruplex qPCR.
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Fig. 3
figure 3

Sensitivity assay of the quadruplex qPCR. (A-D) Amplification curves are GPV, GoCV, TMUV, and GAstV-Ⅱ, respectively.

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Repeatability test of the quadruplex qPCR

The four standard plasmids were used as templates for qPCR amplification after 10-folddilution, and both the intra- and the inter-assay were repeated three times; the results showed that the variation coefficients (CV%) of CT values in the intra-assay and inter-assay tests ranged from 0.03% to 3.3% and 0.26% to 2.02%, respectively, indicating that this multiplex qPCR method is repeatable and stable (Table 3).

Table 3 Repeatability results (CT value) of the quadruplex qPCR.
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Detection of the clinical samples

In this study, an established quadruplex qPCR assay was systematically employed to analyze 72 goose-derived clinical samples collected from Sichuan province. The detection results from 72 clinical samples (Fig. 4) revealed significant differences in the positive rates of the four target viruses. Among them, GAstV-II exhibited the highest detection rate (52.78%, 38/72), which was significantly higher than that of GPV (23.61%, 17/72; χ² = 12.64, P < 0.001), GoCV (13.89%, 10/72; χ² = 22.15, P < 0.001), and TMUV (9.72%, 7/72; χ² = 28.07, P < 0.001). The positive rate of GPV (23.61%) was also significantly higher than that of TMUV (9.72%) (χ² = 4.50, P = 0.034). However, no statistically significant differences were observed between the positive rates of GPV and GoCV (23.61% vs. 13.89%; χ² = 2.05, P = 0.152), or between GoCV and TMUV (13.89% vs. 9.72%; χ² = 0.57, P = 0.450).

Fig. 4
figure 4

Results of multiplex qPCR for clinical samples. This Venn diagram, plotted via the ((bioinformatics.com.cn)) website, shows individual infections and co-infections in detected.

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Mixed infections were common in the samples. The predominant dual infection combination was GPV/GAstV-II (9.72%, 7/72), followed by GPV/GoCV and GoCV/TMUV (both 4.17%, 3/72), as well as GPV/TMUV (2.78%, 2/72) and GAstV-II/GoCV (1.39%, 1/72). Notably, triple infection cases involving GPV, GoCV, and GAstV-II were detected for the first time in this study, with a detection rate of 2.78% (2/72). Furthermore, 29.17% (21/72) of the samples tested negative for all target viruses.

To validate the reliability of the detection results, rigorous comparative experiments were conducted: First, all sample results were compared with conventional single RT-PCR assays, demonstrating 100% concordance between both methods. Second, five samples positive for co-infection with both viruses were (covering all four viruses) selected and subjected to Sanger sequencing for validation, with all sequences showing > 99% similarity to the corresponding target viral gene fragments (The sequencing results have been uploaded to Supplementary Material 2). These results conclusively demonstrate that the established quadruplex qPCR assay possesses high accuracy and reliability, providing robust technical support for both clinical diagnosis and epidemiological investigations of goose viral diseases.

Discussion

China has the largest waterfowl farming scale in the world. Waterfowl farming often relies on natural or artificial water environments. As natural hosts for multiple viruses, waterfowl such as geese face significantly higher risks of viral exposure and transmission compared to terrestrial poultry. With the increasing intensification of farming and insufficient implementation of disease prevention measures, the prevalence of waterfowl viruses continues to rise, with emerging variants making disease control increasingly challenging. As the world’s largest goose-producing country, China has witnessed frequent outbreaks of viral diseases in geese in recent years, causing substantial economic losses and posing significant threats to the poultry industry20. Among these pathogens, GPV, GoCV, TMUV, and GAstV-Ⅱ have demonstrated particularly severe epidemic trends. These viruses not only lead to high morbidity and mortality rates in goose populations but also present potential public health risks to waterfowl farming and other animal species due to their notable cross-species transmission capacity21,22,23,24,25.

Currently, the mainstream diagnostic methods for viruses primarily include the following three categories: first, virus isolation, which serves as the '‘gold standard’' and provides reference value but is time-consuming and has limited sensitivity26; second, serological methods, which are suitable for antibody monitoring but cannot be used for early diagnosis27; and third, conventional PCR/RT-PCR, which is cost-effective but exhibits relatively low sensitivity and is prone to contamination28. Given the current lack of a comprehensive molecular diagnostic method capable of simultaneously detecting GPV, GoCV, TMUV, and GAstV-II, significant challenges remain in the differential diagnosis and epidemiological surveillance of these pathogens in waterfowl. To address this gap, a quadruplex qPCR assay was developed, targeting conserved regions within the VP3 gene of GPV, the Rep gene of GoCV, the E gene of TMUV, and the ORF2 gene of GAstV-II. This method demonstrated high specificity, sensitivity, and reproducibility, with a detection limit of 1 × 10² copies/µL for virus, outperforming conventional PCR in sensitivity.

In terms of sensitivity, this method is comparable to the TaqMan probe-based triple detection method reported by Yu et al. (both at 10² copies)29. However, compared to the triple qPCR method established by Wang et al. for GPV, H5 subtype Avian Influenza Virus (AIV), and GAstV (with sensitivity reaching 10¹ copies/µL for GPV and GoAstV)30, and the reported dual qPCR method for GAstV-II (sensitivity of 10¹ copies/µL)31, the LOD of this method is slightly higher. Furthermore, SYBR Green I qPCR methods have also demonstrated higher sensitivity (minimum 57 copies)32. The aforementioned differences primarily stem from design trade-offs inherent to multiplex detection systems: compared to multiplex methods with fewer targets, the quadruplex reaction system established here is more complex, with more pronounced competition among primers and probes, which consequently limits the ultimate sensitivity for individual targets to some extent. Nevertheless, while maintaining the capability for simultaneous multi-target detection, the sensitivity of this method remains significantly superior to that of conventional PCR. It achieves a favorable balance between detection breadth and depth, providing a reliable tool for the joint screening and epidemiological surveillance of common waterfowl viruses.

Through systematic detection of clinical samples, this study preliminarily untangled the prevalence of four major goose-origin viruses in the current farming environment. Among them, the infection rate of GAstV-II (52.78%) was significantly higher than that of the other three viruses, suggesting it has become the dominant pathogen in local goose flocks. This finding aligns with the epidemiological trends reported from multiple regions in China. For instance, in studies from Jiangsu and Anhui, the detection rates of GAstV-II were 48.6% and 51.3%, respectively29,31,33,34,35. However, the intensity of its prevalence exhibits significant regional variation. The occurrence rate in Guangdong Province during 2019–2020 was 31.2%36, whereas in Taiwan during the same period, the infection rate was as high as 83.3%–94.6%9. Some provinces have even reported a positivity rate of 86.1%, accompanied by a mortality rate exceeding 80%1. These data indicate that the transmission and pathogenicity of GAstV-II possess distinct geographical heterogeneity. Furthermore, given the current lack of effective vaccines or therapeutic drugs, its prevention and control remain a formidable challenge.

The detection rate of GPV (23.61%) ranked second, which is close to the recently reported infection rate of 27.4% in Guangdong37,38. Although GPV vaccination has been widely implemented and effectively controlled the epidemic, with a national occurrence rate of approximately 12.74% in 202133, continuous surveillance remains necessary to guard against potential declines in vaccine protection or the emergence of new variant strains.

Furthermore, the rates of GoCV (13.89%) and TMUV (9.72%) observed in this study were significantly lower than those reported in Guangdong (GoCV 52.90%)36 and Taiwan (GoCV 83.3–94.6%)9. The virus exhibits immunosuppressive properties and may facilitate subsequent infections by other pathogens. However, the specific mechanisms underlying its role in co-infection processes require further investigation. Similarly, the detection rate of TMUV genotype 3 in domestic waterfowl populations continues to increase3,10. This virus also demonstrates immunosuppressive capabilities and may exacerbate the severity of disease following mixed infection. Currently, there are no commercial vaccines available against TMUV genotype 3, which poses additional challenges for the control and prevention of this virus.

Notably, co-infections are relatively common, reflecting the complexity of viral transmission within goose flocks. Among all dual mixed infections, the co-infection of GPV and GAstV-II was the most prevalent, a result consistent with the findings reported previously by Wang et al.30. However, the overall co-infection rate detected in this study was significantly lower than that reported in some previous studies, such as the 28.24% reported by Yu et al29. This discrepancy may be attributed to differences in geographical location, sampling time, or farming management practices among the studied populations. It should also be noted that 21 samples (29.17%) tested negative for all four target viruses. To exclude the possibility of false negatives due to insufficient sensitivity of the detection method, these samples were further validated using conventional PCR, which confirmed the absence of all four pathogens. Therefore, the clinical manifestations associated with these samples may have been caused by infections from other pathogens not covered in this assay or by non-infectious factors.

In summary, the quadruplex qPCR method established in this study provides a high-throughput and rapid detection of four important waterfowl viruses, facilitating timely clinical diagnosis and investigation of infection dynamics. The high prevalence of GAstV-II and the common occurrence of co-infections further highlight the necessity of strengthening systematic surveillance and developing targeted prevention and control strategies.