Development of a Real-Time qPCR Method for the Clinical Sample Detection of Capripox Virus
Capripox viruses (CaPVs), including sheep pox virus (SPV), goat pox virus (GPV), and lumpy skin disease virus (LSDV), are the cause of sheep pox (SPP), goat pox (GTP), and lumpy skin disease (LSD) in cattle. These diseases are of great economic significance to farmers, as they are endemic on farms a...
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MDPI AG
2023-10-01
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| Series: | Microorganisms |
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| Online Access: | https://www.mdpi.com/2076-2607/11/10/2476 |
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| author | Jiaxin Wen Xinying Yin Xiaobo Zhang Desong Lan Junshan Liu Xiaohui Song Yu Sun Jijuan Cao |
| author_facet | Jiaxin Wen Xinying Yin Xiaobo Zhang Desong Lan Junshan Liu Xiaohui Song Yu Sun Jijuan Cao |
| author_sort | Jiaxin Wen |
| collection | DOAJ |
| description | Capripox viruses (CaPVs), including sheep pox virus (SPV), goat pox virus (GPV), and lumpy skin disease virus (LSDV), are the cause of sheep pox (SPP), goat pox (GTP), and lumpy skin disease (LSD) in cattle. These diseases are of great economic significance to farmers, as they are endemic on farms and are a major constraint to international trade in livestock and their products. Capripoxvirus (CaPV) infections produce similar symptoms in sheep and goats, and the three viruses cannot be distinguished serologically. In this study, we developed a real-time quantitative polymerase chain reaction (qPCR) method for identifying CaPV in goats, sheep, and cattle. Clinical samples were tested and verified. The developed assay was highly specific for target viruses, including GPVSPV and LSDV, which had no cross-reaction with other viruses causing similar clinical symptoms. An artificially synthesized positive control plasmid using the CaPV 32 gene inserted into the vector pMD19-T was used as a template, and the correlation coefficient of the linear regression curve (<i>R<sup>2</sup></i>) was 0.9916, the estimated amplification efficiency (<i>E</i>) was 96.06%, and the sensitivity (limit of detection, LOD) was 3.80 copies per reaction. Using the clinical samples as a template, the limit of detection (LOD) was 4.91 × 10<sup>−5</sup> ng per reaction (1.60 × 10<sup>−5</sup>–2.13 × 10<sup>−3</sup> ng, 95% confidence interval (CI)), which means that this method was one of the most sensitive detection assays for CaPVs. A total of 85 clinical samples from CaPV-infected animals (goats, sheep, and cattle) and 50 clinical samples from healthy animals were used to test and compare the diagnostic results using the Synergy Brands (SYBR) Green-based PCR method recommended by the World Organization of Animal Health (WOAH). Both diagnostic sensitivity (<i>DSe</i>) (95.8–100%, 95% CI) and diagnostic specificity (<i>DSp</i>) (92.9–100%, 95% CI) results of the real-time quantitative PCR (qPCR) and SYBR Green PCR were 100%, and the kappa value (κ) was 1.0 (1-1, 95% CI). In summary, the assay established based on TaqMan probes was advantageous in high specificity, sensitivity, and general applicability and could be a competitive candidate tool for the diagnosis of CaPV in clinically suspected animals. |
| first_indexed | 2024-03-10T21:01:17Z |
| format | Article |
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| language | English |
| last_indexed | 2024-03-10T21:01:17Z |
| publishDate | 2023-10-01 |
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| series | Microorganisms |
| spelling | doaj.art-1a02e54d11754dbb97a3783fa29d39a32023-11-19T17:27:20ZengMDPI AGMicroorganisms2076-26072023-10-011110247610.3390/microorganisms11102476Development of a Real-Time qPCR Method for the Clinical Sample Detection of Capripox VirusJiaxin Wen0Xinying Yin1Xiaobo Zhang2Desong Lan3Junshan Liu4Xiaohui Song5Yu Sun6Jijuan Cao7Key Laboratory of Biotechnology and Bioresources Utilization of Ministry of Education, Dalian Minzu University, Dalian 116600, ChinaKey Laboratory of Biotechnology and Bioresources Utilization of Ministry of Education, Dalian Minzu University, Dalian 116600, ChinaKey Laboratory of Biotechnology and Bioresources Utilization of Ministry of Education, Dalian Minzu University, Dalian 116600, ChinaLiaoning Center for Animal Disease Control and Prevention, Shenyang 110164, ChinaSchool of Mechanical Engineering, Faculty of Mechanical Engineering, Materials and Energy, Dalian University of Technology, Dalian 116024, ChinaChina Animal Disease Prevention Control Center, Beijing 100125, ChinaChina Animal Disease Prevention Control Center, Beijing 100125, ChinaKey Laboratory of Biotechnology and Bioresources Utilization of Ministry of Education, Dalian Minzu University, Dalian 116600, ChinaCapripox viruses (CaPVs), including sheep pox virus (SPV), goat pox virus (GPV), and lumpy skin disease virus (LSDV), are the cause of sheep pox (SPP), goat pox (GTP), and lumpy skin disease (LSD) in cattle. These diseases are of great economic significance to farmers, as they are endemic on farms and are a major constraint to international trade in livestock and their products. Capripoxvirus (CaPV) infections produce similar symptoms in sheep and goats, and the three viruses cannot be distinguished serologically. In this study, we developed a real-time quantitative polymerase chain reaction (qPCR) method for identifying CaPV in goats, sheep, and cattle. Clinical samples were tested and verified. The developed assay was highly specific for target viruses, including GPVSPV and LSDV, which had no cross-reaction with other viruses causing similar clinical symptoms. An artificially synthesized positive control plasmid using the CaPV 32 gene inserted into the vector pMD19-T was used as a template, and the correlation coefficient of the linear regression curve (<i>R<sup>2</sup></i>) was 0.9916, the estimated amplification efficiency (<i>E</i>) was 96.06%, and the sensitivity (limit of detection, LOD) was 3.80 copies per reaction. Using the clinical samples as a template, the limit of detection (LOD) was 4.91 × 10<sup>−5</sup> ng per reaction (1.60 × 10<sup>−5</sup>–2.13 × 10<sup>−3</sup> ng, 95% confidence interval (CI)), which means that this method was one of the most sensitive detection assays for CaPVs. A total of 85 clinical samples from CaPV-infected animals (goats, sheep, and cattle) and 50 clinical samples from healthy animals were used to test and compare the diagnostic results using the Synergy Brands (SYBR) Green-based PCR method recommended by the World Organization of Animal Health (WOAH). Both diagnostic sensitivity (<i>DSe</i>) (95.8–100%, 95% CI) and diagnostic specificity (<i>DSp</i>) (92.9–100%, 95% CI) results of the real-time quantitative PCR (qPCR) and SYBR Green PCR were 100%, and the kappa value (κ) was 1.0 (1-1, 95% CI). In summary, the assay established based on TaqMan probes was advantageous in high specificity, sensitivity, and general applicability and could be a competitive candidate tool for the diagnosis of CaPV in clinically suspected animals.https://www.mdpi.com/2076-2607/11/10/2476Capripox virus (CaPV)detectionreal-time quantitative PCRclinical validation |
| spellingShingle | Jiaxin Wen Xinying Yin Xiaobo Zhang Desong Lan Junshan Liu Xiaohui Song Yu Sun Jijuan Cao Development of a Real-Time qPCR Method for the Clinical Sample Detection of Capripox Virus Microorganisms Capripox virus (CaPV) detection real-time quantitative PCR clinical validation |
| title | Development of a Real-Time qPCR Method for the Clinical Sample Detection of Capripox Virus |
| title_full | Development of a Real-Time qPCR Method for the Clinical Sample Detection of Capripox Virus |
| title_fullStr | Development of a Real-Time qPCR Method for the Clinical Sample Detection of Capripox Virus |
| title_full_unstemmed | Development of a Real-Time qPCR Method for the Clinical Sample Detection of Capripox Virus |
| title_short | Development of a Real-Time qPCR Method for the Clinical Sample Detection of Capripox Virus |
| title_sort | development of a real time qpcr method for the clinical sample detection of capripox virus |
| topic | Capripox virus (CaPV) detection real-time quantitative PCR clinical validation |
| url | https://www.mdpi.com/2076-2607/11/10/2476 |
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