Development and Application of a Multiplex Real-Time TaqMan qPCR Assay for the Simultaneous Detection of African Swine Fever Virus, Classical Swine Fever Virus, Porcine Reproductive and Respiratory Syndrome Virus, Pseudorabies Virus, and Porcine Circovirus Type 2
Since its emergence in China in 2018, African swine fever virus (ASFV) has posed a severe threat to the pig farming industry due to its high transmissibility and mortality rate. The clinical signs of ASFV infection often overlap with those caused by other swine viruses such as classical swine fever...
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2025-07-01
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| author | Dongdong Yin Shuangshuang Xu Yayun Liu Hao Guo Mengdie Lan Lei Yin Jieru Wang Yin Dai Xuehuai Shen Kai Zhan Xiaocheng Pan |
| author_facet | Dongdong Yin Shuangshuang Xu Yayun Liu Hao Guo Mengdie Lan Lei Yin Jieru Wang Yin Dai Xuehuai Shen Kai Zhan Xiaocheng Pan |
| author_sort | Dongdong Yin |
| collection | DOAJ |
| description | Since its emergence in China in 2018, African swine fever virus (ASFV) has posed a severe threat to the pig farming industry due to its high transmissibility and mortality rate. The clinical signs of ASFV infection often overlap with those caused by other swine viruses such as classical swine fever virus (CSFV), porcine reproductive and respiratory syndrome virus (PRRSV), pseudorabies virus (PRV), and porcine circovirus type 2 (PCV2), making timely and precise diagnosis a considerable challenge. To address this, we established a TaqMan-based multiplex real-time quantitative PCR (qPCR) assay capable of simultaneously detecting ASFV, CSFV, PRRSV, PRV, and PCV2. Specific primer-probe sets were developed targeting conserved genomic regions: the ASFV <i>P72</i> gene, CSFV <i>5’UTR</i> region, PRRSV <i>ORF6</i>, PCV2 <i>cap</i> gene, and PRV <i>gB</i> gene. After thorough optimization, the assay demonstrated robust analytical performance, exhibiting strong target specificity with no cross-detection of non-target pathogens. The detection threshold was determined to be 10 copies/μL per virus, indicating high assay sensitivity. Repeatability analysis revealed low variability, with intra- and inter-assay coefficient of variation values remaining below 2.3%. When applied to 95 clinical samples, the multiplex assay yielded results that were fully consistent with those obtained using commercially available singleplex qPCR kits. In conclusion, the multiplex TaqMan qPCR method developed in this study is characterized by high specificity, sensitivity, and reproducibility. It provides a reliable and efficient diagnostic tool for the simultaneous detection and differential diagnosis of ASFV and other clinically similar viral infections in swine, thereby offering robust technical support for swine disease surveillance and control. |
| format | Article |
| id | doaj-art-7b129dbb8db349c88828fd4197d47a8b |
| institution | DOAJ |
| issn | 2076-2607 |
| language | English |
| publishDate | 2025-07-01 |
| publisher | MDPI AG |
| record_format | Article |
| series | Microorganisms |
| spelling | doaj-art-7b129dbb8db349c88828fd4197d47a8b2025-08-20T03:08:06ZengMDPI AGMicroorganisms2076-26072025-07-01137157310.3390/microorganisms13071573Development and Application of a Multiplex Real-Time TaqMan qPCR Assay for the Simultaneous Detection of African Swine Fever Virus, Classical Swine Fever Virus, Porcine Reproductive and Respiratory Syndrome Virus, Pseudorabies Virus, and Porcine Circovirus Type 2Dongdong Yin0Shuangshuang Xu1Yayun Liu2Hao Guo3Mengdie Lan4Lei Yin5Jieru Wang6Yin Dai7Xuehuai Shen8Kai Zhan9Xiaocheng Pan10Anhui Provincial Key Laboratory of Livestock and Poultry Product Safety, Institute of Animal Husbandry and Veterinary Science, Anhui Academy of Agricultural Sciences, Livestock and Poultry Epidemic Diseases Research Center of Anhui Province, Hefei 230031, ChinaCollege of Veterinary Medicine, Anhui Agricultural University, Hefei 230036, ChinaAnhui Provincial Key Laboratory of Livestock and Poultry Product Safety, Institute of Animal Husbandry and Veterinary Science, Anhui Academy of Agricultural Sciences, Livestock and Poultry Epidemic Diseases Research Center of Anhui Province, Hefei 230031, ChinaFeixi Xian Agriculture and Rural Affairs Bureau, Feixi 231200, ChinaNingguo City Animal Health Supervision Institute, Ningguo 242300, ChinaAnhui Provincial Key Laboratory of Livestock and Poultry Product Safety, Institute of Animal Husbandry and Veterinary Science, Anhui Academy of Agricultural Sciences, Livestock and Poultry Epidemic Diseases Research Center of Anhui Province, Hefei 230031, ChinaAnhui Provincial Key Laboratory of Livestock and Poultry Product Safety, Institute of Animal Husbandry and Veterinary Science, Anhui Academy of Agricultural Sciences, Livestock and Poultry Epidemic Diseases Research Center of Anhui Province, Hefei 230031, ChinaAnhui Provincial Key Laboratory of Livestock and Poultry Product Safety, Institute of Animal Husbandry and Veterinary Science, Anhui Academy of Agricultural Sciences, Livestock and Poultry Epidemic Diseases Research Center of Anhui Province, Hefei 230031, ChinaAnhui Provincial Key Laboratory of Livestock and Poultry Product Safety, Institute of Animal Husbandry and Veterinary Science, Anhui Academy of Agricultural Sciences, Livestock and Poultry Epidemic Diseases Research Center of Anhui Province, Hefei 230031, ChinaAnhui Provincial Key Laboratory of Livestock and Poultry Product Safety, Institute of Animal Husbandry and Veterinary Science, Anhui Academy of Agricultural Sciences, Livestock and Poultry Epidemic Diseases Research Center of Anhui Province, Hefei 230031, ChinaAnhui Provincial Key Laboratory of Livestock and Poultry Product Safety, Institute of Animal Husbandry and Veterinary Science, Anhui Academy of Agricultural Sciences, Livestock and Poultry Epidemic Diseases Research Center of Anhui Province, Hefei 230031, ChinaSince its emergence in China in 2018, African swine fever virus (ASFV) has posed a severe threat to the pig farming industry due to its high transmissibility and mortality rate. The clinical signs of ASFV infection often overlap with those caused by other swine viruses such as classical swine fever virus (CSFV), porcine reproductive and respiratory syndrome virus (PRRSV), pseudorabies virus (PRV), and porcine circovirus type 2 (PCV2), making timely and precise diagnosis a considerable challenge. To address this, we established a TaqMan-based multiplex real-time quantitative PCR (qPCR) assay capable of simultaneously detecting ASFV, CSFV, PRRSV, PRV, and PCV2. Specific primer-probe sets were developed targeting conserved genomic regions: the ASFV <i>P72</i> gene, CSFV <i>5’UTR</i> region, PRRSV <i>ORF6</i>, PCV2 <i>cap</i> gene, and PRV <i>gB</i> gene. After thorough optimization, the assay demonstrated robust analytical performance, exhibiting strong target specificity with no cross-detection of non-target pathogens. The detection threshold was determined to be 10 copies/μL per virus, indicating high assay sensitivity. Repeatability analysis revealed low variability, with intra- and inter-assay coefficient of variation values remaining below 2.3%. When applied to 95 clinical samples, the multiplex assay yielded results that were fully consistent with those obtained using commercially available singleplex qPCR kits. In conclusion, the multiplex TaqMan qPCR method developed in this study is characterized by high specificity, sensitivity, and reproducibility. It provides a reliable and efficient diagnostic tool for the simultaneous detection and differential diagnosis of ASFV and other clinically similar viral infections in swine, thereby offering robust technical support for swine disease surveillance and control.https://www.mdpi.com/2076-2607/13/7/1573African swine fever virusclassical swine fever viruspseudorabies virusmultiplex qPCRdifferential diagnosisTaqMan assay |
| spellingShingle | Dongdong Yin Shuangshuang Xu Yayun Liu Hao Guo Mengdie Lan Lei Yin Jieru Wang Yin Dai Xuehuai Shen Kai Zhan Xiaocheng Pan Development and Application of a Multiplex Real-Time TaqMan qPCR Assay for the Simultaneous Detection of African Swine Fever Virus, Classical Swine Fever Virus, Porcine Reproductive and Respiratory Syndrome Virus, Pseudorabies Virus, and Porcine Circovirus Type 2 Microorganisms African swine fever virus classical swine fever virus pseudorabies virus multiplex qPCR differential diagnosis TaqMan assay |
| title | Development and Application of a Multiplex Real-Time TaqMan qPCR Assay for the Simultaneous Detection of African Swine Fever Virus, Classical Swine Fever Virus, Porcine Reproductive and Respiratory Syndrome Virus, Pseudorabies Virus, and Porcine Circovirus Type 2 |
| title_full | Development and Application of a Multiplex Real-Time TaqMan qPCR Assay for the Simultaneous Detection of African Swine Fever Virus, Classical Swine Fever Virus, Porcine Reproductive and Respiratory Syndrome Virus, Pseudorabies Virus, and Porcine Circovirus Type 2 |
| title_fullStr | Development and Application of a Multiplex Real-Time TaqMan qPCR Assay for the Simultaneous Detection of African Swine Fever Virus, Classical Swine Fever Virus, Porcine Reproductive and Respiratory Syndrome Virus, Pseudorabies Virus, and Porcine Circovirus Type 2 |
| title_full_unstemmed | Development and Application of a Multiplex Real-Time TaqMan qPCR Assay for the Simultaneous Detection of African Swine Fever Virus, Classical Swine Fever Virus, Porcine Reproductive and Respiratory Syndrome Virus, Pseudorabies Virus, and Porcine Circovirus Type 2 |
| title_short | Development and Application of a Multiplex Real-Time TaqMan qPCR Assay for the Simultaneous Detection of African Swine Fever Virus, Classical Swine Fever Virus, Porcine Reproductive and Respiratory Syndrome Virus, Pseudorabies Virus, and Porcine Circovirus Type 2 |
| title_sort | development and application of a multiplex real time taqman qpcr assay for the simultaneous detection of african swine fever virus classical swine fever virus porcine reproductive and respiratory syndrome virus pseudorabies virus and porcine circovirus type 2 |
| topic | African swine fever virus classical swine fever virus pseudorabies virus multiplex qPCR differential diagnosis TaqMan assay |
| url | https://www.mdpi.com/2076-2607/13/7/1573 |
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