This study was approved by the Animal Ethics Committee of the College of Life Science and Engineering, Northwest Minzu University (Approval No. CLSEAEC 2020–003). A total of 174 whole blood samples were collected from free-range, unimmunized cattle herds in five Chinese provinces between 2022 and 2024. The distribution of samples across provinces was as follows: Henan (n = 28), Jilin (n = 36), Gansu (n = 54), Sichuan (n = 24), and Qinghai (n = 32).

Whole blood samples were transported on ice to the laboratory within 1 h of collection. The samples were allowed to clot at room temperature (25 ± 1°C) for 2–3 h, followed by centrifugation at 3,000 × g for 10 min at 4°C. The resulting serum was aliquoted into sterile cryovials and stored at −80°C until further analysis.

Total RNA was extracted from the serum samples using the MagMAX Viral/Pathogen Nucleic Acid Isolation Kit (ThermoFisher Scientific Inc.), following the manufacturer’s protocol. The RNA was then reverse transcribed into complementary DNA (cDNA) using the SuperScript IV First-Strand Synthesis System (ThermoFisher Scientific Inc.). The resulting cDNAs were stored at −20°C for subsequent use in the qPCR assays.

Eight representative BVDV strains were isolated, identified, and preserved at the Laboratory of Biomedical Research Center, Northwest Minzu University. The strains used were as follows: 22Anhui-7 (GenBank: OQ338174.1), 22Gansu-F2 (GenBank: OQ338175.1), 22Sichuan-B8 (GenBank: OQ338177.1), 22NX-69 (GenBank: ON165517.1), 22AH-1 (GenBank: ON624286.1), 21SD-16 (GenBank: ON624287.1), BVDV1/GS (GenBank: PV806860.1), and 21NM-44 (GenBank: ON411191.1).

In consideration of the current epidemiological landscape of various viral pathogens in Chinese cattle herds, we focused on six common pathogens: foot-and-mouth disease virus (FMDV), bovine coronavirus (BCoV), bovine respiratory syncytial virus (BRSV), bovine parvovirus (BPV), classical swine fever virus (CSFV), and bovine parainfluenza virus 3 (BPIV3). Nucleic acids were extracted from clinical serum samples that tested positive for these specific pathogens. Viral DNA/RNA was isolated using the MagMAX Viral/Pathogen Nucleic Acid Isolation Kit, and the extracted nucleic acids were reverse transcribed into cDNA using the SuperScript IV kit. The resulting DNAs and cDNAs were stored at −20°C for subsequent analysis.

To identify relatively conserved regions in the BVDV genome, full genomic sequences of BVDV-1 and BVDV-2 strains, isolated from various countries and over different time periods, were retrieved from the GenBank database (Supplementary Table S1, DOI: 10.6084/m9.figshare.29143592). Multi-sequence alignment analysis was conducted using the ClustalW program within the MegAlign software. Based on the identified conserved regions in the BVDV genome, a pair of universal primers and an MGB probe were designed (Figure 1). All oligonucleotides were commercially synthesized by Tsingke Co., Ltd. (Xi’an, China).

Two representative BVDV strains, 21SD-16 (BVDV-1, GenBank: ON624287.1) and 22Sichuan-B8 (BVDV-2, GenBank: OQ338177.1), were maintained at −80°C in our laboratory. The target sequences derived from these two strains were amplified by PCR and cloned into the pMD18-T vector (TaKaRa, China). After verification by gene sequencing (Tsingke Co., Ltd., Xi’an, China), the two standard positive plasmids were designated as pMD18-T -BVDV1 and pMD18-T -BVDV2. The DNA copy number was calculated by the following formula: DNA copy number = (N × 6.02 × 10²³ × 10⁻⁹)/(l × 660), where N is the amount of DNA in nanograms, and l is length of the plasmid in bp.

The concentrations of the pMD18-T-BVDV1 and pMD18-T-BVDV2 plasmids were determined to be 1.265 × 10¹¹ copies/μL and 8.016 × 10¹⁰ copies/μL, respectively. The standard positive plasmids were serially diluted ten-fold, and the diluted plasmid standards with different copy numbers were used as templates to evaluate the performance of the BVDV TaqMan MGB qPCR assay.

To develop a robust detection assay, two plasmid standards were used as DNA templates in a TaqMan MGB probe-based qPCR assay. The reaction conditions were systematically optimized using response surface methodology (RSM) to establish the final qPCR protocol. Initial optimization involved fixing the MGB probe concentration at 0.2 μmol/L and evaluating the optimal primer concentration (0.35, 0.40, or 0.45 μmol/L). Based on the optimized primer concentration, we then assessed the optimal MGB probe concentration (0.25, 0.30, or 0.35 μmol/L). Finally, the annealing temperature (58°C, 60°C, or 62°C) was optimized using the selected primer and probe concentrations.

To establish the standard curve for the TaqMan qPCR assay with an MGB probe, a series of diluted vector solutions (ranging from 10³ to 10⁸ copies/μL) were used as DNA templates and amplified under the optimized detection conditions. To ensure reliability, each concentration was analyzed in triplicate.

Based on the optimized detection system, we evaluated the sensitivity of TaqMan qPCR with MGB probes using a series of nucleic acid concentrations ranging from 10⁰ copies/μL to 10⁴ copies/μL as DNA templates. Additionally, sensitivity tests were conducted on plasmid pMD18-T-BVDV1, with concentrations ranging from 1.265 × 10⁰ to 1.265 × 10⁶ copies/μL, using a conventional PCR instrument. Deionized water was used as the negative control. To further assess the specificity of the detection method, we used TaqMan qPCR with MGB probes to detect BVDV and tested the cross-reactivity with other non-BVDV pathogens previously mentioned.

Eight representative epidemic BVDV strains—22NX-69, 22AH-1, 21SD-16, 21NM-44, 22Anhui-7, 22Gansu-F2, BVDV1/GS, and 22Sichuan-B8—were selected for inclusivity assessment. Viral RNA was extracted from each strain, reverse transcribed into cDNA, and subsequently analyzed using the established TaqMan MGB qPCR assay.

To evaluate repeatability, the assay was tested using plasmid standards pMD18-T-BVDV1 and pMD18-T-BVDV2 at three concentrations (10³, 10⁵, and 10⁷ copies/μL). Both intra-assay (within-run) and inter-assay (between-run) precision were assessed by performing triplicate measurements. The arithmetic mean (x̄), standard deviation (SD), and coefficient of variation (CV) of Ct values were calculated to assess method stability and repeatability.

To evaluate the clinical applicability of the TaqMan-MGB qPCR assay for BVDV detection, bovine serum samples collected from multiple provinces in China were analyzed. The performance of our assay was compared with a commercial BVDV qPCR kit (IDEXX RealPCR™, IDEXX Laboratories, Shanghai, China) using clinical samples. The positive coincidence rate was calculated as follows: (number of concordant positive results)/ [number of concordant positives + (TaqMan MGB qPCR negatives that were IDEXX method positives)] × 100%.

To determine the prevalent BVDV-1 and BVDV-2 genotypes across five Chinese provinces, one TaqMan MGB qPCR-positive amplicon from each province was randomly selected for direct sequencing (Tsingke Biotechnology Co., Ltd., Xi’an, China). Sequence alignment was performed using the ClustalW algorithm in MEGA 5.0 software, followed by phylogenetic tree construction using the neighbor-joining method with 1,000 bootstrap replicates.

Using Design-Expert 13 software (State-Ease Inc., USA), we optimized and analyzed the reaction conditions through response surface methodology (RSM). The model identified significant parameter interactions (p < 0.05), with the optimal conditions shown in Figure 2. The optimal amplification conditions were determined when the fluorescence signal intensity reached a plateau. The final parameters included a primer concentration of 0.42 μmol/L, a probe concentration of 0.30 μmol/L, and an annealing temperature of 60.2°C.

To validate the detection performance (sensitivity and specificity), we further optimized the TaqMan MGB qPCR assay under practical conditions. The final reaction conditions were as follows: 0.4 μmol/L primer, 0.3 μmol/L MGB probe, and an annealing temperature of 60°C. The 20 μL reaction mixture contained 10 μL of 2 × Pro Taq HS Probe Premix (Accurate Biotechnology, China), 1 μL template DNA, 0.8 μL each of upstream/downstream primers (BVDV-F/R, 10 μM), 0.6 μL MGB probe (BVDV-P, 10 μM), and ddH₂O to adjust the volume. The thermal cycling protocol included an initial denaturation at 95°C for 2 min, followed by 45 cycles of 95°C for 5 s and 60°C for 30 s.

Standard curves were generated using GraphPad Prism version 8.0.1 (GraphPad Software, USA). The logarithm of plasmid copy numbers (x-axis) was plotted against Ct values (y-axis), yielding linear regression equations:y = −3.355x + 41.555 (R² = 0.998) and y = −3.386x + 41.601 (R² = 0.996) for the two standards, respectively (Figure 3). The amplification efficiencies were 98.6 and 97.4%, which meet the MIQE guidelines criteria (R² > 0.98, 90% < E < 110%). These results demonstrate a strong linear relationship between Ct values and logarithmic plasmid copy numbers.

Detection specificity of the TaqMan MGB qPCR assay demonstrated that only BVDV-1(21SD-16) and BVDV-2(22Sichuan-B8) yielded characteristic sigmoidal amplification curves. No cross-reactivity was observed with other bovine pathogens, including FMDV, BCoV, BRSV, BPIV3, CSFV, BPV, or with the negative control (ddH₂O) (Figure 4).

Assay sensitivity was evaluated using serial dilutions of the plasmid standards. The minimum detectable copy numbers were 1.265 copies/μL for pMD18-T-BVDV1 and 8.016 copies/μL for pMD18-T-BVDV2 (Figure 5). Both plasmid standards were tested in 20 replicates at their respective detection limits, yielding a 100% detection rate in both cases (detection rate >95%). Thus, the overall limit of detection (LOD) for the assay was established at 1.265 copies/μL. For comparison, conventional PCR using serially diluted pMD18-T-BVDV1 demonstrated a detection limit of 1.265 × 10³ copies/μL. This result confirms that the TaqMan MGB qPCR assay offers significantly enhanced sensitivity relative to conventional PCR (Figure 6).

The inclusive test (n = 3) demonstrated 100% detection of eight classical genotypic BVDV strains by the TaqMan^® MGB qPCR assay established in this study. The results are shown in Figure 7. This indicates that the newly developed assay is highly inclusive of the eight classical epidemic strains.

Intra-assay and inter-assay repeatability tests were conducted using varying copy numbers of two plasmid standards as templates. The intra-assay coefficients of variation (CVs) ranged from 0.083 to 0.490%, while the inter-assay CVs ranged from 0.873 to 1.167%. All CVs were below 1.5% (Table 1), indicating high precision. These results demonstrate that the established qPCR assay exhibits excellent repeatability and stability.

To further evaluate the clinical applicability of the assay for the differential diagnosis of BVDV-associated viral infections, a total of 174 bovine serum samples were tested using the established TaqMan MGB qPCR method. As shown in the bar chart (Figure 8), the results were fully consistent with those obtained using a commercial BVDV nucleic acid detection kit, yielding a 100% positive agreement rate. The overall positive detection rate among the tested serum samples was 33.9%. Region-specific positive rates were as follows: 25.0% (7/28) in Henan Province, 47.2% (17/36) in Jilin Province, 27.8% (15/54) in Gansu Province, 37.5% (9/24) in Sichuan Province, and 34.4% (11/32) in Qinghai Province. These results demonstrate that the developed qPCR assay possesses excellent clinical applicability for BVDV detection in diverse field settings.

In the analysis of clinical samples, sequence homology and phylogenetic tree construction were performed on BVDV-positive samples collected from different regions. The results revealed that the Henan strain shared 97.44% nucleotide identity with the BVDV-1 HN1736 strain (GenBank accession no. MN442374.1). The Jilin strain exhibited 93.80% homology with the BVDV-1 NM2312 isolate (GenBank accession no. PP992316.2). The Qinghai strain showed 96.58% similarity to the BVDV-1 MRI3543 strain (GenBank accession no. LR822280.1). The Gansu strain demonstrated 98.32% homology with the BVDV-2 B9497 isolate (GenBank accession no. MH231134.1), while the Sichuan strain shared 97.48% identity with the same BVDV-2 isolate.

Phylogenetic analysis further confirmed that the Henan, Jilin, and Qinghai strains clustered within the BVDV-1 genotype, while the Gansu and Sichuan strains grouped within the BVDV-2 genotype. Notably, the Henan and Jilin strains were closely related, forming a distinct phylogenetic branch, suggesting a potential shared lineage or transmission source. Similarly, the Gansu and Sichuan strains clustered together, indicating a close genetic relationship and suggesting possible interregional transmission between Aba Prefecture in Sichuan Province and the Gannan region in Gansu Province (see Figure 9).