Specific primers and probes targeting the genomes of bovine kobuvirus (BKoV), bovine astrovirus (BoAstV), and bovine torovirus (BToV) were meticulously designed using Primer Express 3.0.1 software (Applied Biosystems, Foster City, United States). These designs were based on conserved regions identified within all available sequences for the aforementioned viruses sourced from GenBank. The selected genomic regions for amplification included the 3D, open reading frame 1 (ORF1), and nucleocapsid (N) gene segments specific to BKoV, BoAstV, and BToV, respectively. The synthesis of these primers and probes was performed by Shanghai Saiheng Biotech Co., Ltd. Additionally, all primers utilized in conventional reverse transcription polymerase chain reaction (RT-PCR) or polymerase chain reaction (PCR) assays in this study were referenced from previously published reports (Jeoung et al., 2011; Mohamed et al., 2017; Wang et al., 2019). The detailed sequences of primers and probes can be found in Table 1 .

To ensure diverse viral detection, positive samples were screened for BKoV (Jeoung et al., 2011), BoAstV (Mohamed et al., 2017) and BToV (Mohamed et al., 2017), and other related viruses, including bovine viral diarrhea virus (BVDV) (Mohamed et al., 2017), bovine rotavirus (BRV) (Mohamed et al., 2017), bovine parvovirus (BPV) (Wang et al., 2019), bovine norovirus (BNoV) (Mohamed et al., 2017), bovine coronavirus (BCoV) (Mohamed et al., 2017). These screenings employed singleplex conventional RT-PCR or PCR techniques, previously established in our laboratory, and the identity of the viruses was further confirmed through DNA sequencing conducted by Shanghai Saiheng Biotech Co., Ltd.

A total of 80 bovine fecal samples were collected from four dairy farms located in Shanghai in 2024. The sample collection was stratified based on clinical presentation and age: 40 samples were sourced from calves exhibiting diarrhea, while another 40 samples were obtained from healthy, non-diarrheic calves. In terms of age distribution, 40 fecal samples were collected from calves younger than 3 months, and 40 samples were retrieved from older calves aged between 7 to 14 months. The calves presenting diarrhea had shown these symptoms persistently for over three days, with fecal samples being collected during the critical period of days 3 to 5 post-onset of diarrhea. Specific details pertaining to all collected samples are outlined in Table 2 . Upon collection, all fecal specimens were promptly stored at -80°C to preserve nucleic acid integrity until further processing.

For RNA extraction, fecal samples were first homogenized with phosphate-buffered saline (PBS) using a vortex mixer for a duration of a few minutes to ensure uniform suspension. Following homogenization, the mixtures were subjected to centrifugation at 12,000 × g for 10 minutes at 4°C to pellet debris. Nucleic acids were subsequently extracted from the supernatant using the Magnetic Viral DNA/RNA kit (Guanmu Biotechnology, China, Cat#GM10001-16), employing the Automated Nucleic Acid Extractor GM (Guanmu Biotechnology, China) in accordance with the manufacturer’s protocol. The concentration and quality of the extracted viral nucleic acids were assessed using a NanoDrop 2000c spectrophotometer (Thermo Scientific, USA), and all samples were stored at -80°C until further analysis via multiplex real-time quantitative PCR (mRT-qPCR).

To facilitate accurate quantification in our multiplex reverse-transcription quantitative real-time polymerase chain reaction (mRT-qPCR) assay, we synthesized target fragments corresponding to the mRT-qPCR target sequences for bovine astrovirus (BoAstV), bovine kobuvirus (BKoV), and bovine torovirus (BToV). These fragments were subsequently cloned into a pUC57 vector using the TA cloning method. The successful insertion of the target sequences was verified through DNA sequencing conducted by Sangon Biotech (China). The concentration of the constructed standard plasmid was measured using a NanoDrop-2000c spectrophotometer (Thermo Scientific, USA), and the plasmid copy number was calculated using the following Equation 1:

Here, 6.02×10²³ is Avogadro’s constant, the DNA length is the total base pair count of the cloned sequence plus the vector, and 660 is the average molecular weight of a base pair in nucleic acids. Subsequent to the quantification and copy number calculation, the standard plasmid was subjected to a 10-fold serial dilution protocol. This dilution ranged from an initial concentration of 2.4×10⁹ copies/μL down to 2.4×10⁰ copies/μL, thereby creating a comprehensive range of standards for calibration within the mRT-qPCR assays. Each dilution was tested in triplicate to ensure the consistency and reliability of the standard curves, which were essential for quantifying viral load in clinical samples. Standard curves were generated from these dilutions, and the corresponding equations were derived to confirm the reliability and accuracy of the dilution series.

The mRT-qPCR reaction conditions were optimized for the simultaneous detection of BKoV, BoAstV, and BToV. Each 25 µL reaction mixture contained 12.5 µL of 2×GM One-Step Probe RT-qPCR MasterMix (Guanmu Biotechnology, China), 1 µL of 25×GM Enzyme Mix (Guanmu Biotechnology, China), 0.2 µL of dNTPs, 0.2 µL of each primer set (10 µM), and 0.1 µL of each probe (10 µM) corresponding to the target fragment. Additionally, 5 µL of nucleic acid template was added to the mixture, and the final volume was adjusted using enzyme-free water. The mRT-qPCR assay was conducted using the SLAN-96S machine (Hongshi Tech, China) under the following thermal cycling conditions: reverse transcription at 50°C for 15 minutes, followed by an initial denaturation step at 95°C for 2 minutes. This was followed by 40 amplification cycles of 95°C for 15 seconds (denaturation) and 60°C for 30 seconds (annealing). A final step at 25°C for 10 seconds concluded the reaction. During the annealing phase, data acquisition occurred on three fluorescent channels: FAM (blue), HEX (green), and Cy5 (red), corresponding to the probes targeting BKoV, BoAstV, and BToV, respectively.

The sensitivity of the mRT-qPCR assay was determined by testing 10-fold serial dilutions of a standard plasmid ranging from 2.4×10⁹ to 2.4×10⁰ copies/µL. The limit of detection (LOD) was defined as the lowest concentration at which consistent amplification was observed. Standard curves were generated based on the threshold cycle (Ct) values obtained from the serial dilutions, and the experiment was repeated three times to ensure reproducibility. The amplification efficiency (E) was calculated using the formula: E = 10^(-1/slope) - 1, where the slope was derived from the linear regression of the standard curve. The calculated efficiencies for each target were evaluated to ensure that they fell within the acceptable range of 90-110%, confirming the assay’s high sensitivity.

To evaluate the specificity of the mRT-qPCR assay, reactions were performed using viral RNA or DNA templates from both target and non-target viruses. The non-target viruses included bovine viral diarrhea virus (BVDV), bovine rotavirus (BRV), bovine parvovirus (BPV), bovine norovirus (BNoV), and bovine coronavirus (BCoV). Additionally, a negative control consisting of nuclease-free water was included. Each reaction was repeated three times to ensure reliability. The assay demonstrated no cross-reactivity with non-target viruses, confirming its high specificity for BKoV, BoAstV, and BToV, ensuring that only the desired targets were detected.

The repeatability and stability of the mRT-qPCR assay were evaluated by calculating the intra-assay and inter-assay coefficients of variation (CVs). For this purpose, 10-fold serial dilutions of the standard plasmid were tested in at least three replicates in both intra- and inter-assay conditions. The CVs were calculated based on the Ct values obtained for each dilution, ensuring that the variability within and between assays was minimal. Intra-assay CVs were calculated from replicates within a single run, while inter-assay CVs were derived from replicates across multiple runs. Both CVs were consistently below 3%, indicating the assay’s robust repeatability and reliable performance across different experimental conditions.

To evaluate the clinical utility of the developed mRT-qPCR assay, viral RNA was extracted from 80 bovine fecal samples and analyzed using the newly established multiplex assay. These fecal samples were collected from calves presenting both diarrheic and non-diarrheic symptoms. The results of the mRT-qPCR assay were compared with those obtained using previously established singleplex conventional RT-PCR or PCR methods described in earlier studies (Jeoung et al., 2011; Mohamed et al., 2017; Wang et al., 2019).

The mRT-qPCR results for each fecal sample were recorded either as positive or negative for each pathogen and categorized according to calf age (< 3 months and 7~14 months) and clinical symptoms (diarrhea or non-diarrhea). The data were analyzed using the SPSS Statistics 25.0 software package for Windows (SPSS, Chicago, IL, USA). The associations between diarrhea and each pathogen were calculated for all ages together and for each age group separately using Pearson’s chi-squared test or Fisher’s exact test, as appropriate. P value of < 0.05 was considered to be statistically significant.

The development of the mRT-qPCR assay commenced with the design and synthesis of specific primers and probes for the detection of three bovine viruses: bovine kobuvirus (BKoV), bovine astrovirus (BoAstV), and bovine torovirus (BToV). The sequences of these primers and probes are listed in Table 1 . Target-specific probes were labeled with distinct fluorescent dyes—FAM for BKoV, HEX for BoAstV, and Cy5 for BToV—allowing for the simultaneous multiplex detection of these viral agents. Standard plasmids, representing a range of concentrations from 2.4×10⁹ to 2.4×10⁰ copies/µL, were prepared for use as templates in the mRT-qPCR reactions, enabling quantitative analysis of viral loads in subsequent experiments.

To assess the quantitative capability of the mRT-qPCR assay, standard curves were generated through a series of 10-fold serial dilutions of the standard plasmid, ranging from 2.4×10⁹ to 2.4×10² copies/µL. The resulting standard curves exhibited excellent amplification efficiency and high correlation coefficients, validating the effectiveness of the assay. The parameters obtained were as follows: for BKoV, the R² value was 0.9985 with an efficiency (E) of 97.92%; for BoAstV, the R² value was 0.9979 with an E value of 97.58%; and for BToV, the R² was 0.9966 with an E of 99.44% ( Figure 1 ). These high R² values indicate a strong linear relationship between the log of the input copy number and the corresponding Ct values, confirming that the plasmid standards are capable of providing reliable quantification across the specified concentration range.

To determine the limit of detection (LOD) for the mRT-qPCR assay, a range of viral copy numbers (2.4×10⁹ to 2.4×10⁰ copies/µL) were tested in triplicate. The assay demonstrated a high level of sensitivity, with the ability to detect as few as 24 viral copies for each of the three target viruses (BKoV, BoAstV, and BToV). The corresponding quantification cycle (Cq) values ranged from 34 to 37, indicating reliable detection even at low concentrations ( Figures 2A–D ; Table 3 ). This high sensitivity confirms that the mRT-qPCR assay is capable of detecting low viral loads, making it suitable for early-stage diagnosis and epidemiological studies of viral diarrhea in calves.

The specificity of the mRT-qPCR assay was tested by analyzing its performance in the presence of positive samples for BKoV, BoAstV, and BToV, alongside fecal samples from calves diagnosed with diarrhea that tested positive for other viral agents, including bovine viral diarrhea virus (BVDV), bovine coronavirus (BCoV), bovine respiratory virus (BRV), bovine parvovirus (BPV), bovine adenovirus (BAdV), and bovine norovirus (BNoV). The assay successfully detected relevant fluorescent signals exclusively for the targeted viruses, with no cross-reactivity observed with the non-target viruses ( Figure 3 ). These results validate the use of the primers and probes for accurate and specific detection of BKoV, BoAstV, and BToV, making the assay a reliable tool for clinical diagnostics and viral surveillance in cattle populations.

The reproducibility of the mRT-qPCR assay was systematically evaluated using 10-fold serial dilutions of the standard plasmid, ensuring reliability and consistency across experimental runs. The coefficients of variation (CVs) for both intra-assay and inter-assay analyses are summarized in Table 4 . For bovine kobuvirus (BKoV), the CVs between runs ranged from 0.08% to 0.66%; for bovine astrovirus (BoAstV), they ranged from 0.09% to 0.97%; and for bovine torovirus (BToV), the CVs extended from 0.15% to 1.29%. These low CV values indicate that the mRT-qPCR assay demonstrates excellent repeatability and high accuracy for both inter-assay and intra-assay testing, confirming the robustness and reliability of the assay in various laboratory conditions.

To further validate the clinical applicability of the developed mRT-qPCR, 80 bovine fecal samples were tested and the results were compared with those obtained using conventional RT-PCR ( Figure 4 ). Among the 80 samples, 23 were positive for BKoV using mRT-qPCR, while conventional RT-PCR detected BKoV in 22 samples. Notably, the single sample that showed an inconsistent result between the two methods had a higher Ct value (35.87), indicating a lower viral load. This suggests that the mRT-qPCR assay is more sensitive in detecting low amounts of viral RNA compared to conventional RT-PCR. For BoAstV and BToV, the results were fully consistent between the mRT-qPCR and singleplex conventional RT-PCR assays, further demonstrating the accuracy and reliability of the multiplex assay.

Nucleic acid detection for BKoV, BoAstV, and BToV was conducted by mRT-qPCR on all 80 samples from four dairy farms (A-D) in Shanghai ( Figure 5 ). The detection rates of the viral pathogens in the clinical samples are summarized in Table 5 . The mRT-qPCR assay identified 23 samples (28.75%) as positive for BKoV, 7 samples (8.75%) as positive for BoAstV, and 3 samples (3.75%) as positive for BToV. The association between viral pathogens and calf age was investigated ( Figure 6A ). When analyzing the results based on calf age, calves younger than 3 months showed the highest rate of infections. BKoV, BoAstV, and BToV were detected in 11 (27.50%), 5 (12.50%), and 2 (5.00%) samples, respectively, from calves younger than 3 months. For calves aged between 7 to 14 months, BKoV, BoAstV, and BToV were detected in 12 (30.00%), 2 (5.00%), and 1 (2.50%) samples, respectively. There were no statistically significant differences in viral detection based on the age of the calves, suggesting that these pathogens may affect calves across different age groups without a strong age-related prevalence pattern. The BoAstV detection rate in dairy feces was higher in calves aged 3 months compared with calves aged between 7 and 14 months. The differences in the detection rates of viral pathogens according to clinical symptoms (diarrhea or non-diarrhea) were also investigated ( Figure 6B ). Among the 40 fecal samples collected from diarrheic calves, BKoV, BoAstV, and BToV were detected in 15 (37.50%), 5 (12.50%), and 2 (5.00%) samples, respectively. In contrast, among the 40 samples from healthy calves, the detection rates were 8 (20.00%) for BKoV, 2 (5.00%) for BoAstV, and 1 (2.50%) for BToV. These results suggest that while there is a higher detection rate of these viruses in diarrheic calves, there was also no statistically significant association between the presence of these viral pathogens and the occurrence of diarrhea. The diarrheic calves showed the highest rate of infections. In the present study, five BoAstV-positive cases were associated with clinical diarrhea in cattle where calves aged < 3 month accounted for 80% of cases ( Table 5 ). Moreover, BToV can be detected occasionally in healthy calves. As shown in Figure 6 , co-infections with BKoV + BoAstV were detected in two diarrheic calves aged < 3 months. These findings indicate that the mRT-qPCR assay is a highly sensitive and specific method for detecting BKoV, BoAstV, and BToV in clinical samples, providing a useful tool for the rapid diagnosis and monitoring of viral pathogens associated with calf diarrhea.