Development of a TaqMan-Probe-Based Multiplex Real-Time PCR for the Simultaneous Detection of African Swine Fever Virus, Porcine Circovirus 2, and Pseudorabies Virus in East China from 2020 to 2022

文献类型： 外文期刊

第一作者： Liu, Huaicheng

作者： Liu, Huaicheng;Zou, Jianwen;Liu, Rongchao;Chen, Jing;Li, Xiaohan;Zhou, Bin;Zheng, Haixue;Li, Long

作者机构：

关键词： multiplex real-time PCR; African swine fever virus; porcine circovirus 2; pseudorabies virus; detection; mixed infection

期刊名称：VETERINARY SCIENCES （ 影响因子：2.4； ）

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年卷期： 2023 年 10 卷 2 期

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收录情况： SCI

摘要： Simple Summary At present, mixed infection with multiple pathogens is an important reason for the complexity of swine diseases in China. Among them, African swine fever virus (ASFV), porcine circovirus 2 (PCV2), and pseudorabies virus (PRV) are extremely important DNA viruses that cause reproductive abnormalities in sows. Therefore, there is an urgent need for rapid and sensitive detection methods that can diagnose three kinds of DNA viruses in the clinic. Herein, a multiplex real-time PCR (qPCR) assay based on TaqMan probes was developed for the simultaneous determination of ASFV, PCV2, and PRV. To ascertain the use of the multiplex real-time qPCR, 383 field specimens from the pig farms of four provinces in East China were collected. The survey data displayed that the ASFV, PCV2, and PRV single infection rates were 22.45%, 28.46%, and 2.87%, respectively. The mixed infection rates of ASFV + PCV2, ASFV + PRV, PCV2 + PRV, and ASFV + PCV2 + PRV were 5.22%, 0.26%, 1.83%, and 0.26%, respectively. The assay established in this study could be used as a differential diagnostic tool for the monitoring and control of ASFV, PCV2, and PRV in the field. African swine fever virus (ASFV), porcine circovirus 2 (PCV2), and pseudorabies virus (PRV) are important DNA viruses that cause reproductive disorders in sows, which result in huge losses in pig husbandry, especially in China. The multiplex qPCR assay could be utilized as a simultaneous diagnostic tool for field-based surveillance and the control of ASFV, PCV2, and PRV. Based on the conserved regions on the p72 gene of ASFV, the Cap gene of PCV2, the gE gene of PRV, and the porcine endogenous beta-Actin gene, the appropriate primers and probes for a multiplex TaqMan real-time PCR test effective at concurrently detecting three DNA viruses were developed. The approach demonstrated high specificity and no cross-reactivity with major pathogens related to swine reproductive diseases. In addition, its sensitivity was great, with a detection limit of 10(1) copies/L of each pathogen, and its repeatability was excellent, with intra- and inter-group variability coefficients of <2%. Applying this assay to detect 383 field specimens collected from 2020 to 2022, the survey data displayed that the ASFV, PCV2, and PRV single infection rates were 22.45%, 28.46%, and 2.87%, respectively. The mixed infection rates of ASFV + PCV2, ASFV + PRV, PCV2 + PRV, and ASFV + PCV2 + PRV were 5.22%, 0.26%, 1.83%, and 0.26%, respectively. Overall, the assay established in this study provides an effective tool for quickly distinguishing the viruses causing sow reproductive disorders, suggesting its huge clinical application value in the diagnosis of swine diseases.

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