Skip to main content

ORIGINAL RESEARCH article

Front. Vet. Sci.
Sec. Veterinary Infectious Diseases
Volume 11 - 2024 | doi: 10.3389/fvets.2024.1462880

Development of a Triplex Crystal Digital RT-PCR for the Detection of PHEV, PRV, and CSFV

Provisionally accepted
Kaichuang Shi Kaichuang Shi 1*Hu Xin Hu Xin 2Yanwen Yin Yanwen Yin 1Yuwen Shi Yuwen Shi 2*Yi Pan Yi Pan 3*Feng Long Feng Long 1*Shuping Feng Shuping Feng 1*Zongqiang Li Zongqiang Li 2*
  • 1 Guangxi Center for Animal Disease Control and Prevention, Nanning, China
  • 2 Guangxi University, Nanning, Guangxi Zhuang Region, China
  • 3 Youjiang Medical University for Nationalities, Baise, Guangx, China

The final, formatted version of the article will be published soon.

    Porcine hemagglutinating encephalomyelitis virus (PHEV), porcine pseudorabies virus (PRV), and classical swine fever virus (CSFV) are currently prevalent worldwide and cause similar neurological symptoms in infected pigs. It is very important to establish a detection method that can rapidly and accurately detect and differentiate these three viruses. Targeting the PHEV N gene, PRV gB gene, and CSFV 5' untranslated region (5'UTR), three pairs of specific primers and probes were designed, and a triplex crystal digital reverse transcription-PCR (cdRT-PCR) was developed to detect PHEV, PRV, and CSFV. The results indicated that this assay had high sensitivity, and the limitation of detection (LODs) for PHEV, PRV, and CSFV were 4.812, 4.047, and 5.243 copies/reaction, respectively, which was about 50 times higher than that of multiplex real-time quantitative RT-PCR (RT-qPCR). This assay showed good specificity, without cross-reaction with other important swine pathogens, i.e. FMDV, PRRSV, PEDV, SIV, TGEV, PoRV, and PCV2. This assay had high repeatability, with intra-assay coefficients of variation (CVs) of 0.73%-1.87%, and inter-assay CVs of 0.57%-2.95%. The developed assay was used to test 1367 clinical tissue samples from Guangxi province in China, and the positive rates of PHEV, PRV, and CSFV were 3.44% (47/1367), 1.24% (17/1367), and 1.90% (26/1367), respectively, with a coincidence rate of 98.98% and a Kappa value of 0.94 to the reference multiplex RT-qPCR. The established triplex cdRT-PCR was a highly rapid, sensitive, and accurate assay to detect and differentiate PHEV, PRV, and CSFV.

    Keywords: Porcine hemagglutinating encephalomyelitis virus (PHEV), porcine pseudorabies virus (PRV), Classical swine fever virus (CSFV), multiplex crystal digital RT-PCR, Detection method

    Received: 10 Jul 2024; Accepted: 21 Nov 2024.

    Copyright: © 2024 Shi, Xin, Yin, Shi, Pan, Long, Feng and Li. 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:
    Kaichuang Shi, Guangxi Center for Animal Disease Control and Prevention, Nanning, China
    Yuwen Shi, Guangxi University, Nanning, Guangxi Zhuang Region, China
    Yi Pan, Youjiang Medical University for Nationalities, Baise, 533000, Guangx, China
    Feng Long, Guangxi Center for Animal Disease Control and Prevention, Nanning, China
    Shuping Feng, Guangxi Center for Animal Disease Control and Prevention, Nanning, China
    Zongqiang Li, Guangxi University, Nanning, Guangxi Zhuang Region, 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.