BRSV and BVDV are both preserved in the Parasitology Laboratory of the Jilin Academy of Animal Science and Veterinary Medicine. DH5α competent cells were purchased from Sangon Biotech (Shanghai) Co., Ltd.

The pET-28a(+) vector, QuickCut™ BamH I, QuickCut™ Hind III, and DL2000 DNA Marker were purchased from Toyobo Bio-Engineering (Dalian) Co., Ltd. The virus DNA/RNA extraction Kit was purchased from Xi’an Tianlong Technology Co., Ltd. The 2×Taq Plus PCR Master Mix, FastKing One Step RT-PCR Kit, Plasmid Mini Kit, and DNA Gel Extraction Kit were purchased from Tiangen Biotech (Beijing) Co., Ltd. The 4S Green Plus Nucleic Acid Stain, Seamless Cloning Kit, and T7 RNA In Vitro Transcription Kit were purchased from Sangon Biotech (Shanghai) Co., Ltd. The TaqMan™ Fast Virus One-Step Multiplex Master Mix was purchased from Thermo Fisher Scientific (China) Co., Ltd. The Basic Nucleic Acid Amplification Kit (ERA), RT-Basic Nucleic Acid Amplification Kit (ERA), Strip-Based Nucleic Acid Amplification Kit (ERA), RT-Strip-Based Nucleic Acid Amplification Kit (ERA), and lateral flow test strips were purchased from Suzhou GenDx Biotech Co., Ltd. Infectious bovine rhinotracheitis virus (IBRV), BRSV, BVDV, Bovine Coronavirus (BCoV), Pasteurella multocida, and Toxoplasma gondii genomes are preserved in the Parasitology Laboratory of the Jilin Academy of Animal Science and Veterinary Medicine, and 46 bovine nasal swabs were obtained from cattle farms in Changchun, Jilin Province. All clinical samples were collected from July 2023 to October 2024 and stored at 4 °C temporarily.

The viral genomes from BVDV, BRSV, and clinical samples were extracted by the Automated Nucleic Acid Extraction System. Total RNA extracted from BRSV and BVDV was reversed-transcribed into cDNA using the RevertAid First Strand cDNA Synthesis Kit (Thermo Fisher Scientific) following the manufacturer’s instructions.

Following the kit’s instruction, reverse-transcribed BVDV cDNA and BRSV cDNA were used as templates for PCR. The 25-μL reaction contained 12.5 μL 2×Taq Plus PCR Mix, 1 μL each of primers BRSV-N-28aF (or BVDV-5UTR-28aF) and BRSV-N-28aR (or BVDV-5UTR-28aR), 3 μL of templates, and nuclease-free water to volume. Cycling conditions were: 94 °C 3 min; 30 cycles of 94 °C 30 s, 55 °C 30 s, 72 °C 1 min; final extension 72 °C 5 min; hold at 4 °C. Specific primers targeting the conserved regions of the BRSV N gene (GenBank No. NC_001989.1) and the BVDV 5′-UTR (GenBank No. KP749796.1) were designed. BamHI and HindIII restriction sites were appended to the 5′ and 3′ ends of the primers, respectively. Details are provided in Table 1.

The pET-28a vector was double-digested with BamHI and HindIII. PCR products and linearized vector were separated on 1% agarose gels, excised and purified. Insert fragment and vector were joined in a 20-μL reaction containing 1 μL of linearized vector, 3 μL of insert fragments, 10 μL of 2×Seamless Cloning MasterMix, and sterile ddH₂O to volume, incubated at 50 °C for 20 min and chilled on ice for 2 min. The assembly mixture was transformed into E. coli DH5α competent cells; recombinants were selected by kanamycin. The pET28a-BRSV-N and pET28a-BVDV-5UTR plasmids were verified by PCR and sequencing, then quantified and stored as standard positive controls for the BVDV ERA and BRSV ERA assays.

After the concentration of the positive standard was determined with a UV–vis spectrophotometer, the copy number was calculated using the formula: Copy number (copies/μL) = (6.02 × 10²³) × (ng/μL × 10⁻⁹) / (RNA length × 340). Homogeneity of the same batch of BRSV and BVDV positive standards was assessed by random sampling. The accurately diluted positive standard was dispensed into 100 identical aliquots; five aliquots were randomly selected and tested by qPCR. Consistent Ct values indicate good homogeneity, whereas large deviations suggest heterogeneity.

Stability under different storage temperatures was evaluated by storing the aliquoted positive standards at −80 °C and testing aliquots on days 7, 30, and 180 by qPCR. The cycle threshold (Ct) value reflecting the fluorescence signal was used to quantify the nucleic acid content and thus the stability of the standards under the specified storage conditions.

Based on the conserved regions of the BRSV N gene (NC_001989.1) and the BVDV 5’-UTR sequence (KP749796.1), and according to the primer design principles of the ERA method, three forward primers, four reverse primers, and two probes were designed for BRSV, and two forward primers, four reverse primers, and three probes were designed for BVDV. The synthesis of primers and probes was completed by Sangon Biotech (Shanghai) Co., Ltd., and the sequence information is shown in Table 2. According to the instructions for the RT-ERA Basic Nucleic Acid Amplification Kit, the reaction system was set up as follows: 20 μL of solubilizing agent, 2.5 μL each of the forward and reverse primers (10 μmol/L), 2 μL of template, and ddH₂O added to a final volume of 48 μL. After brief mixing, the mixture was added to each tube containing the basic amplification reagent. Then, 2 μL of activator was added to the inside of the tube cap. Following a quick centrifugation, the tubes were briefly vortexed and centrifuged again. The reaction conditions for BRSV and BVDV primer screening were set at 40 °C for 20 min. Next, 10 μL of the amplification product was mixed with 1.5 μL of the 6× Loading Buffer provided with the kit, incubated at 56 °C for 5 min, and then subjected to 1.5% agarose gel electrophoresis. Amplification results were observed to select optimal primer pairs. The above procedure was repeated to confirm the best primer pairs for the BRSV and BVDV RT-ERA assays.

The selected optimal forward and reverse primers were synthesized by Sangon Biotech, with a Biotin group added to the 5′ end of the reverse primer. Probe screening was then performed separately for BRSV and BVDV using the RT-ERA Dipstick Nucleic Acid Amplification Kit, following the manufacturer’s instructions. The previously prepared positive standards for BRSV and BVDV were used as templates, and a negative control was included in each assay. The reaction mixture consisted of 20 μL solubilizing agent, 2.1 μL each of forward and reverse primers (10 μmol/L), 0.6 μL of probe, 2 μL of template, and ddH₂O added to a final volume of 48 μL. After brief mixing, the mixture was added to the RT-dipstick amplification reagent. Then, 2 μL of activator was carefully pipetted into the tube cap. The tubes were subjected to a quick spin, briefly vortexed, and centrifuged again. The reaction conditions for probe screening were 40 °C for 30 min. After amplification, each product was diluted 80-fold in a buffer containing 0.1% Tween-20. An 100 μL aliquot of the diluted product was applied to the sample pad of a lateral-flow dipstick (Suzhou GenDx Biotech) and incubated at room temperature (22 °C–25 °C). Results were read at 5 min; strips interpreted after 15 min were considered invalid. A valid test was scored as follows: positive: both control (C) and test (T) lines clearly visible; negative: only C line visible; invalid: C line absent, requiring retesting.

For both the BRSV RT-ERA-LFD and BVDV RT-ERA-LFD assays, the reaction temperature was systematically varied—39 °C, 40 °C, 41 °C, 42 °C and 43 °C—while the incubation time was kept at 30 min. After amplification, each product was diluted 80-fold; 100 μL of the diluted solution was transferred to a 1.5 mL microtube, a lateral-flow dipstick was inserted, and the result was read within 10 min. By comparing the LFD signals across the temperature gradient, the optimal reaction temperature for each ERA-LFD method was selected.

For both the BRSV and BVDV RT-ERA-LFD assays, the incubation time was tested at 10, 15, 20, 25, 30, 35 and 40 min while the reaction temperature was fixed at the optimum value determined previously. After amplification, each product was diluted 80-fold; 100 μL of the diluted solution was transferred to a 1.5 mL microtube, a lateral-flow dipstick was inserted, and the result was read within 10 min. By comparing the LFD signals across the time-course, the optimal reaction time for each ERA-LFD method was selected.

The specificity of the established ERA-LFD assays was evaluated using nucleic acids from IBRV, BRSV, BVDV, BCoV, Pasteurella multocida (P. multocida) and Toxoplasma gondii (T. gondii), with ddH₂O as the blank control, to confirm that both the BRSV and BVDV RT-ERA-LFD methods recognize only their intended targets.

The BRSV and BVDV positive standards were first adjusted to 10⁸ copies/μL and then serially diluted 10-fold. Each dilution was tested with the newly established BRSV RT-ERA-LFD and BVDV RT-ERA-LFD assays. After amplification, the lateral-flow dipsticks were read and the lowest dilution that still gave a visible positive signal was taken as the limit of detection (LOD) for each method. Using BVDV and BRSV positive standards as templates, amplification was carried out with the PCR method already established in the laboratory. The products were analyzed by 1% agarose gel electrophoresis to compare the sensitivity of the methods. The specific primers are listed in Table 3. The RT-PCR for BRSV and BVDV was carried out in a 50 μL mixture containing 25 μL of 2× FastKing One-Step RT-PCR MasterMix, 2 μL of 25× RT-PCR Enzyme Mix, 1.25 μL each of forward and reverse primers (10 μM), 2 μL of template RNA, and RNase-free ddH₂O to volume. Cycling conditions were: 42 °C for 30 min (reverse transcription); 95 °C for 3 min (initial denaturation); 35 cycles of 94 °C for 30 s, 55 °C for 30 s, and 72 °C for 30 s; final extension at 72 °C for 5 min; hold at 4 °C.

A total of 26 nasal-swab specimens collected from cattle herds in Changchun, suspected of BRSV/BVDV infection, were tested in parallel with the newly developed ERA-LFD assay and the conventional RT-PCR protocol already established in our laboratory. Concordance between the two methods was calculated from Table 4 as: Concordance (%) = (a + d)/(a + b + c + d) × 100. The two-by-two table was defined with RT-PCR as the gold standard: a) both ERA-LFD and RT-PCR positive; b) ERA-LFD positive but RT-PCR negative; c) ERA-LFD negative but RT-PCR positive; d) both negative. RT-PCR positivity required Ct ≤ 35 with a single melting-curve peak, while ERA-LFD positivity was defined by the visibility of both the test (T) and control (C) lines. Concordance was assessed using the overall percent agreement [OPA = (a + d)/N × 100%] and Cohen’s κ coefficient [κ = (Po–Pe)/(1–Pe)], with κ > 0.80 indicating excellent agreement. All amplicons that gave a positive signal in the ERA-LFD assay were sent to Sangon Biotech for sequencing to confirm their identity and accuracy.

A total of 920 cattle from farms and slaughterhouses in five areas of Jilin Province—Nong’an, Baicheng, Dunhua, Da’an, and Changchun—were sampled at 5%, yielding 46 nasal swabs. We determined a sampling rate of 5% (46 out of 920 head) by considering the available on-site manpower, which included two veterinarians capable of collecting up to 20 samples per day. This rate also met the minimum requirement of 45 samples to achieve an expected prevalence of 8%–11% with 95% confidence, as calculated using Epitools. Additionally, this approach was chosen to minimize stress associated with transportation. Nucleic acids were extracted and tested with the field-assembled BRSV RT-ERA-LFD and BVDV RT-ERA-LFD kits, and every result was cross-verified with the laboratory-established qPCR assay (see Table 5).

Using the cDNA reverse-transcribed from BRSV and BVDV RNA as templates, two pairs of gene-specific primers (BRSV-N-28aF/R and BVDV-5UTR-28aF/R) were employed in independent PCRs. Amplicons of 1,176 bp and 290 bp, corresponding to the predicted sizes, were obtained (Figures 1A,B). The purified BRSV-N and BVDV-5UTR fragments were ligated into pET-28a previously digested with the appropriate restriction enzymes. After transformation into E. coli, single colonies were picked and plasmid DNA was extracted. PCR screening of the resulting clones yielded products identical in size to the original inserts (Figure 1C). Sanger sequencing confirmed 100% nucleotide identity with the target sequences.

In-vitro-transcribed RNAs corresponding to the BVDV-5UTR and BRSV-N genes were quantified spectrophotometrically. The BRSV standard contained 36.864 ng/μL, whereas the BVDV standard contained 15.130 ng/μL. Using the molecular weight of single-stranded RNA, these concentrations correspond to 5.55 × 10¹⁰ copies/μL and 9.23 × 10¹⁰ copies/μL, respectively. Each standard was divided into 100 aliquots of equal volume; five aliquots were then selected at random and tested by one-step qPCR to assess homogeneity. Ct values ranged from 11.17 to 13.05 for the BRSV standard (Figure 1D) and from 15.12 to 16.03 for the BVDV standard (Figure 1E). The coefficient of variation (CV) was 6.35% for BRSV and 2.53% for BVDV, indicating low inter-aliquot variability and high uniformity. Thus, both RNA standards are sufficiently homogeneous for use as quantitative reference materials in downstream assays.

To assess the long-term stability of the positive standards, aliquots stored at −80 °C were analyzed by qPCR on days 7, 30, and 180 post-freezing. No significant differences were observed in the amplification curves across the tested time points. The CV for Ct values were 4.17% for the BRSV standard and 3.31% for the BVDV standard (Supplementary Table 1). These results demonstrate that both BRSV and BVDV positive standards maintain good stability when stored at −80 °C for up to 6 months.

Using the previously prepared BRSV-positive standard as template, 12 primer pairs (BRSV-ERA-F1/R1–F3/R4) were screened by RT-ERA; the two that yielded visible amplicons—BRSV-ERA-F1/R4 and F2/R4 —were retested (Figures 2A,B), and BRSV-ERA-F1/R4 produced the brightest, clearest band, designating F1/R4 as the optimal BRSV pair. Similarly, with the BVDV-positive standard, eight primer pairs (BVDV-ERA-F1/R1–F2/R4) were evaluated; BVDV-ERA-F1/R2, F1/R3 and F1/R4 gave visible products (Figure 2C), and subsequent retesting (Figure 2D) showed BVDV-ERA-F1/R4 to be the brightest and most specific, establishing BVDV-ERA-F1/R4 as the optimal BVDV primer pair. Using BRSV-positive RNA and primers F1/R4, dipstick screening (Figure 2E) showed P1 and P2 both positive; however, P1 produced a false-positive in the negative control, so P2 was selected. For BVDV (Figure 2F), P1–P3 all generated positive signals, yet only P1’s negative control remained clean, so P1 was chosen as the optimal BVDV probe.

For both the BRSV and BVDV RT-ERA-LFD assays, reactions were run at 39 °C, 40 °C, 41 °C, 42 °C, and 43 °C. As shown in Figure 3A (BRSV) and Figure 3B (BVDV), distinct control (C) and test (T) lines were observed at every temperature. Because the kit specifications recommend 40 °C–42 °C and a lower temperature reduces energy consumption, 40 °C was selected as the optimal reaction temperature. Using this temperature, amplification times of 10, 15, 20, 25, 30, 35 and 40 min were then compared (Figures 3C,D). All durations produced clear C and T lines; however, to meet the requirements of rapid clinical testing, 20 min was chosen as the standard reaction time for both assays.

To assess specificity, the established BRSV and BVDV RT-ERA-LFD assays were challenged with IBRV, BCoV, P. multocida, T. gondii and the homologous viruses. Only the respective target virus (BRSV or BVDV) generated a red test line (T); all other samples showed only the control line (C) (Figures 4A,B), confirming high specificity for both methods.

To evaluate sensitivity, serial 10-fold dilutions of BRSV and BVDV RNA (10⁸–10⁰ copies/μL) were tested by the established RT-ERA-LFD assays and conventional RT-PCR. As shown in Figures 4C,D, the lower limits of detection were 10¹ copies/μL for BRSV RT-ERA-LFD and 10² copies/μL for BVDV RT-ERA-LFD, whereas RT-PCR detected 10³ copies/μL for both viruses. Thus, the BRSV and BVDV RT-ERA-LFD methods exhibited 100-fold and 10-fold higher sensitivity than RT-PCR, respectively.

To determine the concordance of the assays, 26 nasal swabs from suspected cases were simultaneously tested by BRSV RT-ERA-LFD, BVDV RT-ERA-LFD, and conventional RT-PCR. BRSV RT-ERA-LFD identified 4 positives (15.38%, 4/26), in full agreement with the 4 detected by RT-PCR (Figure 4E), yielding 100% concordance. Similarly, BVDV RT-ERA-LFD detected 5 positives (19.23%, 5/26), matching exactly the 5 found by RT-PCR (Figure 4F), again with 100% concordance. For BRSV, 5 samples were true-positive and 41 were true-negative, with no false-positive or false-negative results, yielding an OPA of 100% and a Cohen’s κ of 1.00 (95% CI: 1.00–1.00). Likewise, for BVDV, 4 true-positive and 42 true-negative samples were identified, giving identical agreement statistics (OPA = 100%, κ = 1.00; 95% CI: 1.00–1.00), indicating perfect concordance between the ERA-LFD assay and qPCR. All RT-ERA-LFD amplicons from positive samples were sequenced and confirmed as BRSV or BVDV, respectively.

The established BRSV and BVDV RT-ERA-LFD assays were used to test 46 nasal swabs collected from five prefectures in Jilin Province (Nong’an, Baicheng, Dunhua, Da’an and Changchun). Results were compared with laboratory-developed BRSV and BVDV real-time RT-PCR. BRSV RT-ERA-LFD identified five positive samples (10.87%; Figure 5A), identical to the real-time RT-PCR result (Figure 5B). BVDV RT-ERA-LFD detected four positives (8.7%; Figure 5C), again in complete agreement with the real-time RT-PCR result (Figure 5D). These data demonstrate that the newly developed RT-ERA-LFD protocols are reliable for routine clinical detection of both viruses.