{"title":"TaqMan三重实时PCR检测伪狂犬病毒、猪teschovirus 1和猪链球菌2的建立和实施","authors":"Ranran Lai, Chen Yang, Lili Wu, Weisheng Wu, Lulu Li, Wei Liu, Zheng Yan, Diankun Yu, Shengzhi Ren, Zhiqiang Hu, Xiaowen Li","doi":"10.3389/fvets.2025.1589175","DOIUrl":null,"url":null,"abstract":"<p><strong>Introduction: </strong>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 <i>Streptococcus suis 2</i> (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.</p><p><strong>Materials and methods: </strong>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.</p><p><strong>Results: </strong>The triplex real-time PCR assay exhibited high sensitivity, specificity, and repeatability, with a detection limit of 1.0 × 10<sup>0</sup> 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.</p><p><strong>Conclusion: </strong>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.</p>","PeriodicalId":12772,"journal":{"name":"Frontiers in Veterinary Science","volume":"12 ","pages":"1589175"},"PeriodicalIF":2.9000,"publicationDate":"2025-06-18","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12213347/pdf/","citationCount":"0","resultStr":"{\"title\":\"Development and implementation of a TaqMan triplex real-time PCR assay for concurrent detection of pseudorabies virus, porcine teschovirus 1, and <i>Streptococcus suis 2</i>.\",\"authors\":\"Ranran Lai, Chen Yang, Lili Wu, Weisheng Wu, Lulu Li, Wei Liu, Zheng Yan, Diankun Yu, Shengzhi Ren, Zhiqiang Hu, Xiaowen Li\",\"doi\":\"10.3389/fvets.2025.1589175\",\"DOIUrl\":null,\"url\":null,\"abstract\":\"<p><strong>Introduction: </strong>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 <i>Streptococcus suis 2</i> (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.</p><p><strong>Materials and methods: </strong>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.</p><p><strong>Results: </strong>The triplex real-time PCR assay exhibited high sensitivity, specificity, and repeatability, with a detection limit of 1.0 × 10<sup>0</sup> 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.</p><p><strong>Conclusion: </strong>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.</p>\",\"PeriodicalId\":12772,\"journal\":{\"name\":\"Frontiers in Veterinary Science\",\"volume\":\"12 \",\"pages\":\"1589175\"},\"PeriodicalIF\":2.9000,\"publicationDate\":\"2025-06-18\",\"publicationTypes\":\"Journal Article\",\"fieldsOfStudy\":null,\"isOpenAccess\":false,\"openAccessPdf\":\"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12213347/pdf/\",\"citationCount\":\"0\",\"resultStr\":null,\"platform\":\"Semanticscholar\",\"paperid\":null,\"PeriodicalName\":\"Frontiers in Veterinary Science\",\"FirstCategoryId\":\"97\",\"ListUrlMain\":\"https://doi.org/10.3389/fvets.2025.1589175\",\"RegionNum\":2,\"RegionCategory\":\"农林科学\",\"ArticlePicture\":[],\"TitleCN\":null,\"AbstractTextCN\":null,\"PMCID\":null,\"EPubDate\":\"2025/1/1 0:00:00\",\"PubModel\":\"eCollection\",\"JCR\":\"Q1\",\"JCRName\":\"VETERINARY SCIENCES\",\"Score\":null,\"Total\":0}","platform":"Semanticscholar","paperid":null,"PeriodicalName":"Frontiers in Veterinary Science","FirstCategoryId":"97","ListUrlMain":"https://doi.org/10.3389/fvets.2025.1589175","RegionNum":2,"RegionCategory":"农林科学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"2025/1/1 0:00:00","PubModel":"eCollection","JCR":"Q1","JCRName":"VETERINARY SCIENCES","Score":null,"Total":0}
Development and implementation of a TaqMan triplex real-time PCR assay for concurrent detection of pseudorabies virus, porcine teschovirus 1, and Streptococcus suis 2.
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.
期刊介绍：
Frontiers in Veterinary Science is a global, peer-reviewed, Open Access journal that bridges animal and human health, brings a comparative approach to medical and surgical challenges, and advances innovative biotechnology and therapy.
Veterinary research today is interdisciplinary, collaborative, and socially relevant, transforming how we understand and investigate animal health and disease. Fundamental research in emerging infectious diseases, predictive genomics, stem cell therapy, and translational modelling is grounded within the integrative social context of public and environmental health, wildlife conservation, novel biomarkers, societal well-being, and cutting-edge clinical practice and specialization. Frontiers in Veterinary Science brings a 21st-century approach—networked, collaborative, and Open Access—to communicate this progress and innovation to both the specialist and to the wider audience of readers in the field.
Frontiers in Veterinary Science publishes articles on outstanding discoveries across a wide spectrum of translational, foundational, and clinical research. The journal''s mission is to bring all relevant veterinary sciences together on a single platform with the goal of improving animal and human health.
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