ORIGINAL RESEARCH article

Front. Cell. Infect. Microbiol.

Sec. Veterinary and Zoonotic Infection

Volume 15 - 2025 | doi: 10.3389/fcimb.2025.1616898

This article is part of the Research TopicUnveiling Host-Pathogen Interactions: Insights into Animal Cellular Immunity and Novel Diagnostics - Volume IIView all 8 articles

The updated duplex fluorescence quantitative RT-PCR assay for simultaneous detection of PRRSV-1 and PRRSV-2

Provisionally accepted
Xiaoxiao TianXiaoxiao Tian1Haojie WangHaojie Wang1Zeqing LiuZeqing Liu1Ziyi WeiZiyi Wei1Yong-Bo YangYong-Bo Yang1Haiwei WangHaiwei Wang1Guoqing LiuGuoqing Liu1Hao SongHao Song1Xinyi HuangXinyi Huang1*Tong-Qing AnTong-Qing An1,2*
  • 1Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Harbin, China
  • 2Heilongjiang Provincial Key Laboratory of Veterinary Immunology, Harbin, China

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Porcine reproductive and respiratory syndrome (PRRS) is one of the most economically devastating infectious diseases in the global swine industry. With the continuous mutation and recombination of PRRSV, existing detection methods frequently result in false negatives, further complicating the prevention and control of PRRS. In this study, a duplex real-time quantitative RT-PCR (RT-qPCR) for the simultaneous detection of PRRSV-1 and PRRSV-2 was developed by designing specific primers and probes based on the ORF6 gene, which is different from conventional nucleic acid detection methods that are typically based on the ORF7 gene. The method showed high specificity for exclusively detecting PRRSV-1 and PRRSV-2, with no cross-reactivity observed against other porcine pathogens; showed excellent sensitivity, with a limit of detection (LOD) of 8.42 copies for PRRSV-1 and 7.84 copies for PRRSV-2; showed good repeatability, with intra-assay coefficients of variation (CVs) of 0.22-1.07% and inter-assay CVs of 0.52-1.28%. A total of 356 clinical samples were detected using the developed duplex RT-qPCR and compared to the WOAH-recommended RT-qPCR assay and commercial universal PRRSV RT-qPCR detection kit. The assay established in this study demonstrated higher positivity rates, indicating its superior sensitivity. In summary, an efficient, sensitive, and accurate method for the detection and differentiation of PRRSV-1 and PRRSV-2 was developed and applied to the detection and monitoring of PRRSV.

Keywords: PRRSV-1, PRRSV-2, duplex fluorescence quantitative RT-PCR, Swine, detection

Received: 23 Apr 2025; Accepted: 02 Jun 2025.

Copyright: © 2025 Tian, Wang, Liu, Wei, Yang, Wang, Liu, Song, Huang and An. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

* Correspondence:
Xinyi Huang, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Harbin, China
Tong-Qing An, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Harbin, China

Disclaimer: All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article or claim that may be made by its manufacturer is not guaranteed or endorsed by the publisher.

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