Cargando…
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...
Autores principales: | , , , , , , , |
---|---|
Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
MDPI
2023
|
Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10608805/ https://www.ncbi.nlm.nih.gov/pubmed/37894134 http://dx.doi.org/10.3390/microorganisms11102476 |
_version_ | 1785127864670093312 |
---|---|
author | Wen, Jiaxin Yin, Xinying Zhang, Xiaobo Lan, Desong Liu, Junshan Song, Xiaohui Sun, Yu Cao, Jijuan |
author_facet | Wen, Jiaxin Yin, Xinying Zhang, Xiaobo Lan, Desong Liu, Junshan Song, Xiaohui Sun, Yu Cao, Jijuan |
author_sort | Wen, Jiaxin |
collection | PubMed |
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 (R(2)) was 0.9916, the estimated amplification efficiency (E) 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(−5) ng per reaction (1.60 × 10(−5)–2.13 × 10(−3) 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 (DSe) (95.8–100%, 95% CI) and diagnostic specificity (DSp) (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. |
format | Online Article Text |
id | pubmed-10608805 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2023 |
publisher | MDPI |
record_format | MEDLINE/PubMed |
spelling | pubmed-106088052023-10-28 Development of a Real-Time qPCR Method for the Clinical Sample Detection of Capripox Virus Wen, Jiaxin Yin, Xinying Zhang, Xiaobo Lan, Desong Liu, Junshan Song, Xiaohui Sun, Yu Cao, Jijuan Microorganisms Article 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 (R(2)) was 0.9916, the estimated amplification efficiency (E) 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(−5) ng per reaction (1.60 × 10(−5)–2.13 × 10(−3) 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 (DSe) (95.8–100%, 95% CI) and diagnostic specificity (DSp) (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. MDPI 2023-10-02 /pmc/articles/PMC10608805/ /pubmed/37894134 http://dx.doi.org/10.3390/microorganisms11102476 Text en © 2023 by the authors. https://creativecommons.org/licenses/by/4.0/Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/). |
spellingShingle | Article Wen, Jiaxin Yin, Xinying Zhang, Xiaobo Lan, Desong Liu, Junshan Song, Xiaohui Sun, Yu Cao, Jijuan Development of a Real-Time qPCR Method for the Clinical Sample Detection of Capripox Virus |
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 | Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10608805/ https://www.ncbi.nlm.nih.gov/pubmed/37894134 http://dx.doi.org/10.3390/microorganisms11102476 |
work_keys_str_mv | AT wenjiaxin developmentofarealtimeqpcrmethodfortheclinicalsampledetectionofcapripoxvirus AT yinxinying developmentofarealtimeqpcrmethodfortheclinicalsampledetectionofcapripoxvirus AT zhangxiaobo developmentofarealtimeqpcrmethodfortheclinicalsampledetectionofcapripoxvirus AT landesong developmentofarealtimeqpcrmethodfortheclinicalsampledetectionofcapripoxvirus AT liujunshan developmentofarealtimeqpcrmethodfortheclinicalsampledetectionofcapripoxvirus AT songxiaohui developmentofarealtimeqpcrmethodfortheclinicalsampledetectionofcapripoxvirus AT sunyu developmentofarealtimeqpcrmethodfortheclinicalsampledetectionofcapripoxvirus AT caojijuan developmentofarealtimeqpcrmethodfortheclinicalsampledetectionofcapripoxvirus |