Cargando…

Development of a Real-time TaqMan PCR Assay for The Detection of Porcine Circovirus 4

INTRODUCTION: Porcine circovirus 4 (PCV4) was first discovered in 2019 in a herd of pigs with porcine respiratory disease, dermatitis and nephropathy syndrome in Hunan Province, China. It has subsequently been detected in other provinces and in South Korea. In consideration of the potential of the v...

Descripción completa

Detalles Bibliográficos
Autores principales: Chen, Wanting, Jiang, Dike, Xiao, Lu, Zhang, Pengfei, Luo, Yan, Yang, Zexiao, Yao, Xueping, Wang, Yin, Wu, Xulong
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Sciendo 2022
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8959683/
https://www.ncbi.nlm.nih.gov/pubmed/35582479
http://dx.doi.org/10.2478/jvetres-2022-0004
_version_ 1784677215936446464
author Chen, Wanting
Jiang, Dike
Xiao, Lu
Zhang, Pengfei
Luo, Yan
Yang, Zexiao
Yao, Xueping
Wang, Yin
Wu, Xulong
author_facet Chen, Wanting
Jiang, Dike
Xiao, Lu
Zhang, Pengfei
Luo, Yan
Yang, Zexiao
Yao, Xueping
Wang, Yin
Wu, Xulong
author_sort Chen, Wanting
collection PubMed
description INTRODUCTION: Porcine circovirus 4 (PCV4) was first discovered in 2019 in a herd of pigs with porcine respiratory disease, dermatitis and nephropathy syndrome in Hunan Province, China. It has subsequently been detected in other provinces and in South Korea. In consideration of the potential of the virus to cause an epidemic, rapid, sensitive, and specific detection of PCV4 is needed, as is the facilitation of further epidemiological research through elucidation of the whole genome of PCV4. This study had those two aims. MATERIAL AND METHODS: Fifty-five blood samples, two pig tissue samples, nine saliva swabs and one semen sample which all originated from Sichuan province pig farms were analysed. The virus’ genome of 1,770 bp was synthesised artificially based on a Chinese reference strain and primers and probes for the ORF2 gene were designed. Then, the amplified target fragment was cloned into the pMD19-T vector and a series of diluted recombinant plasmids were used to generate a standard curve. An optimised real-time TaqMan PCR method was established. RESULTS: The results of this study showed that the established method is specific for PCV4 but not for other viruses, and has amplification efficiency of 99.6%, a regression squared value (R(2)) of 1.000 and a detection limit of 2.2×10 DNA copies. This method was shown to be analytically specific and sensitive with a low intra- and inter-assay coefficient of variation (<1.67 %). Of a total of 67 clinical samples tested using the established method, three were shown to be positive (4%). CONCLUSION: This study confirms the existence of PCV4 in Sichuan and provides a promising alternative tool for rapid detection of PCV4.
format Online
Article
Text
id pubmed-8959683
institution National Center for Biotechnology Information
language English
publishDate 2022
publisher Sciendo
record_format MEDLINE/PubMed
spelling pubmed-89596832022-05-16 Development of a Real-time TaqMan PCR Assay for The Detection of Porcine Circovirus 4 Chen, Wanting Jiang, Dike Xiao, Lu Zhang, Pengfei Luo, Yan Yang, Zexiao Yao, Xueping Wang, Yin Wu, Xulong J Vet Res Research Article INTRODUCTION: Porcine circovirus 4 (PCV4) was first discovered in 2019 in a herd of pigs with porcine respiratory disease, dermatitis and nephropathy syndrome in Hunan Province, China. It has subsequently been detected in other provinces and in South Korea. In consideration of the potential of the virus to cause an epidemic, rapid, sensitive, and specific detection of PCV4 is needed, as is the facilitation of further epidemiological research through elucidation of the whole genome of PCV4. This study had those two aims. MATERIAL AND METHODS: Fifty-five blood samples, two pig tissue samples, nine saliva swabs and one semen sample which all originated from Sichuan province pig farms were analysed. The virus’ genome of 1,770 bp was synthesised artificially based on a Chinese reference strain and primers and probes for the ORF2 gene were designed. Then, the amplified target fragment was cloned into the pMD19-T vector and a series of diluted recombinant plasmids were used to generate a standard curve. An optimised real-time TaqMan PCR method was established. RESULTS: The results of this study showed that the established method is specific for PCV4 but not for other viruses, and has amplification efficiency of 99.6%, a regression squared value (R(2)) of 1.000 and a detection limit of 2.2×10 DNA copies. This method was shown to be analytically specific and sensitive with a low intra- and inter-assay coefficient of variation (<1.67 %). Of a total of 67 clinical samples tested using the established method, three were shown to be positive (4%). CONCLUSION: This study confirms the existence of PCV4 in Sichuan and provides a promising alternative tool for rapid detection of PCV4. Sciendo 2022-03-25 /pmc/articles/PMC8959683/ /pubmed/35582479 http://dx.doi.org/10.2478/jvetres-2022-0004 Text en © 2022 W. Chen et al. published by Sciendo https://creativecommons.org/licenses/by-nc-nd/3.0/This work is licensed under the Creative Commons Attribution-NonCommercial-NoDerivatives 3.0 License.
spellingShingle Research Article
Chen, Wanting
Jiang, Dike
Xiao, Lu
Zhang, Pengfei
Luo, Yan
Yang, Zexiao
Yao, Xueping
Wang, Yin
Wu, Xulong
Development of a Real-time TaqMan PCR Assay for The Detection of Porcine Circovirus 4
title Development of a Real-time TaqMan PCR Assay for The Detection of Porcine Circovirus 4
title_full Development of a Real-time TaqMan PCR Assay for The Detection of Porcine Circovirus 4
title_fullStr Development of a Real-time TaqMan PCR Assay for The Detection of Porcine Circovirus 4
title_full_unstemmed Development of a Real-time TaqMan PCR Assay for The Detection of Porcine Circovirus 4
title_short Development of a Real-time TaqMan PCR Assay for The Detection of Porcine Circovirus 4
title_sort development of a real-time taqman pcr assay for the detection of porcine circovirus 4
topic Research Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8959683/
https://www.ncbi.nlm.nih.gov/pubmed/35582479
http://dx.doi.org/10.2478/jvetres-2022-0004
work_keys_str_mv AT chenwanting developmentofarealtimetaqmanpcrassayforthedetectionofporcinecircovirus4
AT jiangdike developmentofarealtimetaqmanpcrassayforthedetectionofporcinecircovirus4
AT xiaolu developmentofarealtimetaqmanpcrassayforthedetectionofporcinecircovirus4
AT zhangpengfei developmentofarealtimetaqmanpcrassayforthedetectionofporcinecircovirus4
AT luoyan developmentofarealtimetaqmanpcrassayforthedetectionofporcinecircovirus4
AT yangzexiao developmentofarealtimetaqmanpcrassayforthedetectionofporcinecircovirus4
AT yaoxueping developmentofarealtimetaqmanpcrassayforthedetectionofporcinecircovirus4
AT wangyin developmentofarealtimetaqmanpcrassayforthedetectionofporcinecircovirus4
AT wuxulong developmentofarealtimetaqmanpcrassayforthedetectionofporcinecircovirus4