Establishment of a TaqMan qPCR method with MGB probe for the specific detection of BVDV field strains circulating in China

Abstract

Bovine viral diarrhea virus (BVDV), a highly mutable pathogen, poses a significant threat to the cattle industry in China. Therefore, the development of a rapid, sensitive, and specific diagnostic assay is essential for effective surveillance and control. In this study, a TaqMan real-time quantitative PCR (qPCR) assay utilizing a minor groove binder (MGB) probe was developed for the detection of BVDV, with a focus on strains currently circulating in China. Universal primers and an MGB probe targeting the conserved 5' untranslated region (5'UTR) of both BVDV-1 and BVDV-2 were designed based on complete genome sequences available in GenBank. Following optimization of the reaction conditions, the assay demonstrated a detection limit of 1.265 copies/μL using a plasmid standard. The method exhibited high specificity for BVDV-1 and BVDV-2, with no cross reactivity observed with other common bovine pathogens. Intra- and inter-assay coefficients of variation were below 1.5%, indicating excellent repeatability and reproducibility. When applied to field serum samples collected from free-range cattle in various regions of China, the assay achieved a 100% concordance rate with a commercial reference kit (IDEXX RealPCR™ BVDV RNA Test). These results suggest that the established TaqMan MGB qPCR assay is a reliable and efficient tool for the detection and epidemiological investigation of BVDV-1 and BVDV-2 infections in cattle herds across China.

Keywords: BVDV-1; BVDV-2; TaqMan MGB qPCR; clinical diagnosis; epidemiological investigation.

Figure 1

Sequence alignment information for primers and probes. The nucleotide bases color-coded as follows: cytosine (C) in blue, thymine (T) in red, adenine (A) in green, and guanine (G) in purple.

Figure 2

Response Surface Methodology (RSM) Optimization of BVDV TaqMan qPCR. (A) Effect of primer-probe concentration interaction on Ct values. (B) Effect of primer-probe concentration interaction on ΔRn. (C) Effect of primer-annealing temperature interaction on Ct values. (D) Effect of primer-annealing temperature interaction on ΔRn. (E) Effect of probe-annealing temperature interaction on Ct values. (F) Effect of probe-annealing temperature interaction on ΔRn.

Figure 3

TaqMan MGB qPCR Standard Curve for BVDV Quantification. (A) Plasmid standard pMD18-T-BVDV1 (1.265 × 10³ ~ 1.265 × 10⁸ copies/μL) was used as the template. (B) Plasmid standard pMD18-T-BVDV2 (8.016 × 10³ ~ 8.016 × 10⁸ copies/μL) was used as the template.

Figure 4

Specificity Test of the BVDV TaqMan MGB qPCR Detection Method. In the presented amplification graph, the x-axis represents the number of cycles, while the y-axis represents the relative fluorescence units (RFU). The amplification conditions for the following viral nucleic acids were tested: BVDV-1 (21SD-16), BVDV-2 (22Sichuan-B8), FMDV, BCoV, BRSV, BPV, CSFV, BPIV3, and a negative control (ddH₂O).

Figure 5

Analytical sensitivity evaluation of TaqMan MGB qPCR for BVDV Detection. The amplification curves for BVDV-1 and BVDV-2 were generated using plasmid standards pMD18-T-BVDV1 and pMD18-T-BVDV2, respectively. In panels (A) and (B), the plasmid concentrations for curves 1 to 5 ranged from 1.265 × 10⁴ to 1.265 × 10⁰ copies/μL for BVDV-1 and from 8.016 × 10⁴ to 8.016 × 10⁰ copies/μL for BVDV-2. Curve 6 represents the negative control.

Figure 6

Sensitivity test for conventional PCR detection of BVDV. The target gene was amplified using plasmid standard pMD18-T-BVDV1 and analyzed by nucleic acid electrophoresis. In the electrophoresis pattern, Lane M: Molecular weight standard (DL 2000 DNA Marker); Lane 1: Negative control; Lane 2 ~ 8: Plasmid standard concentrations ranging from 1.265 × 10⁶ to 1.265 × 10⁰ copies/μL.

Figure 7

Inclusivity analysis of TaqMan MGB qPCR for BVDV detection. In the presented amplification graph, the x-axis represents the number of cycles, while the y-axis represents the relative fluorescence units (RFU). The amplification conditions for the following BVDV strains were tested: 21SD-16, 22NX-69, 21NM-44, 22AH-1, 22Anhui-7, 22Sichuan-B8, 22Gansu-F2, BVDV1/GS, and a negative control (ddH₂O).

Figure 8

Detection of bovine serum clinical samples. (A) Comparison of TaqMan qPCR assay with commercial nucleic acid detection kit. (B) Distribution of BVDV positive clinical samples in different regions of China.

Figure 9

Genetic evolution of BVDV 5’-UTR gene in positive clinical samples from different regions of China.