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Development of a triplex real-time quantitative PCR for detection and differentiation of genotypes I and II African swine fever virus
African swine fever virus (ASFV) was first identified in 1921 and is extensively prevalent around the world nowadays, which has a significant negative impact on the swine industry. In China, genotype II ASFV was first discovered in 2018, and has spread quickly to different provinces in a very short...
Autores principales: | , , , , , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
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Frontiers Media S.A.
2023
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Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10620837/ https://www.ncbi.nlm.nih.gov/pubmed/37929278 http://dx.doi.org/10.3389/fvets.2023.1278714 |
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author | Qian, Xinxiu Hu, Liping Shi, Kaichuang Wei, Haina Shi, Yuwen Hu, Xin Zhou, Qingan Feng, Shuping Long, Feng Mo, Shenglan Li, Zongqiang |
author_facet | Qian, Xinxiu Hu, Liping Shi, Kaichuang Wei, Haina Shi, Yuwen Hu, Xin Zhou, Qingan Feng, Shuping Long, Feng Mo, Shenglan Li, Zongqiang |
author_sort | Qian, Xinxiu |
collection | PubMed |
description | African swine fever virus (ASFV) was first identified in 1921 and is extensively prevalent around the world nowadays, which has a significant negative impact on the swine industry. In China, genotype II ASFV was first discovered in 2018, and has spread quickly to different provinces in a very short time; genotype I ASFV was first found in 2020, and has been reported in several provinces since then. To establish an accurate method for detection and differentiation of genotypes I and II ASFV, three primers and probes were designed targeting the ASFV B646L gene for different genotypes, the F1055L gene for genotype I, and the E183L gene for genotype II, and a triplex real-time quantitative PCR (qPCR) for differential detection of genotypes I and II ASFV was developed after optimizing the reaction conditions. The assay showed high sensitivity, and the limits of detection (LOD) of the B646L, F1055L, and E183L genes were 399.647 copies/reaction, 374.409 copies/reaction, and 355.083 copies/reaction, respectively; the coefficients of variation (CVs) of the intra-assay and the inter-assay were 0.22–1.88% and 0.16–1.68%, respectively, showing that this method had good repeatability; the assay could detect only ASFV, without cross-reactivity with other swine viruses including PRRSV, PEDV, PDCoV, CSFV, PRV, and PCV2, showing excellent specificity of this method. A total of 3,519 clinical samples from Guangxi province, southern China, were tested by the developed assay, and 8.16% (287/3,519) samples were found to be positive for ASFV, of which 0.17% (6/3,519) samples were positive for genotype I, 7.19% (253/3,519) samples for genotype II, and 0.80% (28/3,519) samples for genotypes I and II. At the same time, these clinical samples were also tested by a previously reported multiplex qPCR, and the agreement between these two methods was more than 99.94%. In summary, the developed triplex qPCR provided a fast, specific and accurate method for detection and differentiation of genotypes I and II ASFV. |
format | Online Article Text |
id | pubmed-10620837 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2023 |
publisher | Frontiers Media S.A. |
record_format | MEDLINE/PubMed |
spelling | pubmed-106208372023-11-03 Development of a triplex real-time quantitative PCR for detection and differentiation of genotypes I and II African swine fever virus Qian, Xinxiu Hu, Liping Shi, Kaichuang Wei, Haina Shi, Yuwen Hu, Xin Zhou, Qingan Feng, Shuping Long, Feng Mo, Shenglan Li, Zongqiang Front Vet Sci Veterinary Science African swine fever virus (ASFV) was first identified in 1921 and is extensively prevalent around the world nowadays, which has a significant negative impact on the swine industry. In China, genotype II ASFV was first discovered in 2018, and has spread quickly to different provinces in a very short time; genotype I ASFV was first found in 2020, and has been reported in several provinces since then. To establish an accurate method for detection and differentiation of genotypes I and II ASFV, three primers and probes were designed targeting the ASFV B646L gene for different genotypes, the F1055L gene for genotype I, and the E183L gene for genotype II, and a triplex real-time quantitative PCR (qPCR) for differential detection of genotypes I and II ASFV was developed after optimizing the reaction conditions. The assay showed high sensitivity, and the limits of detection (LOD) of the B646L, F1055L, and E183L genes were 399.647 copies/reaction, 374.409 copies/reaction, and 355.083 copies/reaction, respectively; the coefficients of variation (CVs) of the intra-assay and the inter-assay were 0.22–1.88% and 0.16–1.68%, respectively, showing that this method had good repeatability; the assay could detect only ASFV, without cross-reactivity with other swine viruses including PRRSV, PEDV, PDCoV, CSFV, PRV, and PCV2, showing excellent specificity of this method. A total of 3,519 clinical samples from Guangxi province, southern China, were tested by the developed assay, and 8.16% (287/3,519) samples were found to be positive for ASFV, of which 0.17% (6/3,519) samples were positive for genotype I, 7.19% (253/3,519) samples for genotype II, and 0.80% (28/3,519) samples for genotypes I and II. At the same time, these clinical samples were also tested by a previously reported multiplex qPCR, and the agreement between these two methods was more than 99.94%. In summary, the developed triplex qPCR provided a fast, specific and accurate method for detection and differentiation of genotypes I and II ASFV. Frontiers Media S.A. 2023-10-19 /pmc/articles/PMC10620837/ /pubmed/37929278 http://dx.doi.org/10.3389/fvets.2023.1278714 Text en Copyright © 2023 Qian, Hu, Shi, Wei, Shi, Hu, Zhou, Feng, Long, Mo and Li. https://creativecommons.org/licenses/by/4.0/This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. |
spellingShingle | Veterinary Science Qian, Xinxiu Hu, Liping Shi, Kaichuang Wei, Haina Shi, Yuwen Hu, Xin Zhou, Qingan Feng, Shuping Long, Feng Mo, Shenglan Li, Zongqiang Development of a triplex real-time quantitative PCR for detection and differentiation of genotypes I and II African swine fever virus |
title | Development of a triplex real-time quantitative PCR for detection and differentiation of genotypes I and II African swine fever virus |
title_full | Development of a triplex real-time quantitative PCR for detection and differentiation of genotypes I and II African swine fever virus |
title_fullStr | Development of a triplex real-time quantitative PCR for detection and differentiation of genotypes I and II African swine fever virus |
title_full_unstemmed | Development of a triplex real-time quantitative PCR for detection and differentiation of genotypes I and II African swine fever virus |
title_short | Development of a triplex real-time quantitative PCR for detection and differentiation of genotypes I and II African swine fever virus |
title_sort | development of a triplex real-time quantitative pcr for detection and differentiation of genotypes i and ii african swine fever virus |
topic | Veterinary Science |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10620837/ https://www.ncbi.nlm.nih.gov/pubmed/37929278 http://dx.doi.org/10.3389/fvets.2023.1278714 |
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