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. 2025 Jun 18:12:1589175.
doi: 10.3389/fvets.2025.1589175. eCollection 2025.

Development and implementation of a TaqMan triplex real-time PCR assay for concurrent detection of pseudorabies virus, porcine teschovirus 1, and Streptococcus suis 2

Ranran Lai #  1   2 , Chen Yang #  1   2 , Lili Wu  1   2   3 , Weisheng Wu  4 , Lulu Li  2 , Wei Liu  4 , Zheng Yan  2 , Diankun Yu  4 , Shengzhi Ren  1 , Zhiqiang Hu  5 , Xiaowen Li  1   2   3   4
Affiliations

Development and implementation of a TaqMan triplex real-time PCR assay for concurrent detection of pseudorabies virus, porcine teschovirus 1, and Streptococcus suis 2

Ranran Lai et al. Front Vet Sci. .

Abstract

Introduction: Porcine neurological disorders represent a prevalent clinical condition that leads to significant mortality and economic losses within the swine industry. Pseudorabies virus (PRV), porcine teschovirus 1 (PTV1), and Streptococcus suis 2 (SS2) are key viral and bacterial pathogens implicated in the manifestation of neurological symptoms in pig populations. The overlapping clinical presentations and pathological alterations associated with these pathogens pose challenges in their clinical differentiation. Therefore, it is essential to develop a diagnostic method with high sensitivity and specificity that can simultaneously detect and differentiate these viral and bacterial agents.

Materials and methods: A triplex real-time PCR assay using TaqMan probes was developed to simultaneously detect PRV, PTV1, and SS2. To assess the efficacy of the established assay, 30 clinical samples of animals with nervous symptoms were used to compare the results obtained from the triplex real-time PCR assay with those obtained from commercial singleplex real-time PCR kits. Furthermore, a total of 282 samples were tested and analyzed to validate the utility of the assay.

Results: The triplex real-time PCR assay exhibited high sensitivity, specificity, and repeatability, with a detection limit of 1.0 × 100 copy/μL. The triplex real-time PCR method and commercial singleplex real-time PCR kits showed complete concordance in detecting PRV, PTV1, and SS2. Clinical data indicated single infection rates of 8.16% for PRV, 26.95% for PTV1, and 7.80% for SS2. The observed co-infection rates were 7.45% for PRV + PTV1, 0.71% for PRV + SS2, 1.42% for PTV1 + SS2, and 1.77% for PRV + PTV1 + SS2, respectively.

Conclusion: The triplex real-time PCR method developed in this study effectively distinguishes PRV, PTV1, and SS2 simultaneously, serving as a valuable diagnostic tool. This method is anticipated to play a crucial role in preventing and controlling infectious disease spread and supporting epidemiological investigations.

Keywords: PRV; PTV1; SS2; porcine nervous diseases; triplex real-time PCR.

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Conflict of interest statement

RL, CY, LW, SR, and XL were employed by the Shandong New Hope Liuhe Group Co., Ltd. RL, CY, LW, LL, ZY, and XL were employed by the Juye Xinhao Agriculture and Animal Husbandry Co., Ltd. WW, WL, DY, and XL were employed by the New Hope Liuhe Co., Ltd. The remaining author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

Figure 1
Figure 1
The amplification curves and the standard curve of the single and triplex real-time PCR assay. (A–C) The amplification curves and the standard curve (bottom) of single real-time PCR assay for detection of PRV (A), PTV1 (B), and SS2 (C), respectively, with concentrations of each plasmid standard ranging from 1.0 × 109 copies/μL to 1.0 × 100copies/μL. (D) Amplification curves and standard curves of optimized triplex real-time PCR for simultaneous detection of PRV, PTV1 and SS2. The concentrations of each plasmid standard were from 1.0 × 109 copies/μL to 1.0 × 100 copies/μL.
Figure 2
Figure 2
The amplification curves of specificity tests of triplex real-time PCR. (A) Line 1–3: positive templates of PRV, PTV1 and SS2. Line 4–11: negative control and positive templates of ASFV, PCV2, PCV3, PRRSV, PEDV, CSFV, TGEV. (B) 1–3: positive templates of PRV, PTV1 and SS2. 4–6: templates of clinical samples from healthy pigs.
Figure 3
Figure 3
The detection results of clinical nervous samples using the triplex real-time PCR assay established in this study.
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