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A Triplex PCR Method for Distinguishing the Wild-Type African Swine Fever Virus From the Deletion Strains by Detecting the Gene Insertion

To date, there is no effective vaccine or antiviral therapy available to prevent or treat African swine fever virus (ASFV) infections. ASFV gene deletion strains have been proposed as promising anti-ASFV vaccine candidates. In recent years, most ASFV gene deletion strains worldwide have been recombi...

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Autores principales: Huang, Zhao, Xu, Zhiying, Cao, Haoxuan, Zeng, Fanliang, Wang, Heng, Gong, Lang, Zhang, Shengxun, Cao, Sen, Zhang, Guihong, Zheng, Zezhong
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Frontiers Media S.A. 2022
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9274085/
https://www.ncbi.nlm.nih.gov/pubmed/35836498
http://dx.doi.org/10.3389/fvets.2022.921907
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author Huang, Zhao
Xu, Zhiying
Cao, Haoxuan
Zeng, Fanliang
Wang, Heng
Gong, Lang
Zhang, Shengxun
Cao, Sen
Zhang, Guihong
Zheng, Zezhong
author_facet Huang, Zhao
Xu, Zhiying
Cao, Haoxuan
Zeng, Fanliang
Wang, Heng
Gong, Lang
Zhang, Shengxun
Cao, Sen
Zhang, Guihong
Zheng, Zezhong
author_sort Huang, Zhao
collection PubMed
description To date, there is no effective vaccine or antiviral therapy available to prevent or treat African swine fever virus (ASFV) infections. ASFV gene deletion strains have been proposed as promising anti-ASFV vaccine candidates. In recent years, most ASFV gene deletion strains worldwide have been recombinant strains expressing EGFP or mCherry as markers. Therefore, in this study, a new triplex real-time PCR (RT-PCR) method was established for the broad and accurate differentiation of ASFV wild-type vs. gene deletion strains. We designed three pairs of primers and probes to target B646L, EGFP, and mCherry, and RT-PCR was used to detect these three genes simultaneously. The detection method prevented non-specific amplification of porcine reproductive and respiratory syndrome virus, porcine epidemic diarrhea virus, circovirus type 2, pseudorabies virus, and classical swine fever virus genes. The minimum copy number of standard plasmid DNA detected using triplex RT-PCR was 9.49, 15.60, and 9.60 copies for B646L, EGFP, and mCherry, respectively. Importantly, of the 1646 samples analyzed in this study, 67 were positive for ASFV, all corresponding to the wild-type virus. Overall, our data show that the triplex RT-PCR method established in this study can specifically identify both ASFV wild-type and gene deletion strains.
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spelling pubmed-92740852022-07-13 A Triplex PCR Method for Distinguishing the Wild-Type African Swine Fever Virus From the Deletion Strains by Detecting the Gene Insertion Huang, Zhao Xu, Zhiying Cao, Haoxuan Zeng, Fanliang Wang, Heng Gong, Lang Zhang, Shengxun Cao, Sen Zhang, Guihong Zheng, Zezhong Front Vet Sci Veterinary Science To date, there is no effective vaccine or antiviral therapy available to prevent or treat African swine fever virus (ASFV) infections. ASFV gene deletion strains have been proposed as promising anti-ASFV vaccine candidates. In recent years, most ASFV gene deletion strains worldwide have been recombinant strains expressing EGFP or mCherry as markers. Therefore, in this study, a new triplex real-time PCR (RT-PCR) method was established for the broad and accurate differentiation of ASFV wild-type vs. gene deletion strains. We designed three pairs of primers and probes to target B646L, EGFP, and mCherry, and RT-PCR was used to detect these three genes simultaneously. The detection method prevented non-specific amplification of porcine reproductive and respiratory syndrome virus, porcine epidemic diarrhea virus, circovirus type 2, pseudorabies virus, and classical swine fever virus genes. The minimum copy number of standard plasmid DNA detected using triplex RT-PCR was 9.49, 15.60, and 9.60 copies for B646L, EGFP, and mCherry, respectively. Importantly, of the 1646 samples analyzed in this study, 67 were positive for ASFV, all corresponding to the wild-type virus. Overall, our data show that the triplex RT-PCR method established in this study can specifically identify both ASFV wild-type and gene deletion strains. Frontiers Media S.A. 2022-06-28 /pmc/articles/PMC9274085/ /pubmed/35836498 http://dx.doi.org/10.3389/fvets.2022.921907 Text en Copyright © 2022 Huang, Xu, Cao, Zeng, Wang, Gong, Zhang, Cao, Zhang and Zheng. 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
Huang, Zhao
Xu, Zhiying
Cao, Haoxuan
Zeng, Fanliang
Wang, Heng
Gong, Lang
Zhang, Shengxun
Cao, Sen
Zhang, Guihong
Zheng, Zezhong
A Triplex PCR Method for Distinguishing the Wild-Type African Swine Fever Virus From the Deletion Strains by Detecting the Gene Insertion
title A Triplex PCR Method for Distinguishing the Wild-Type African Swine Fever Virus From the Deletion Strains by Detecting the Gene Insertion
title_full A Triplex PCR Method for Distinguishing the Wild-Type African Swine Fever Virus From the Deletion Strains by Detecting the Gene Insertion
title_fullStr A Triplex PCR Method for Distinguishing the Wild-Type African Swine Fever Virus From the Deletion Strains by Detecting the Gene Insertion
title_full_unstemmed A Triplex PCR Method for Distinguishing the Wild-Type African Swine Fever Virus From the Deletion Strains by Detecting the Gene Insertion
title_short A Triplex PCR Method for Distinguishing the Wild-Type African Swine Fever Virus From the Deletion Strains by Detecting the Gene Insertion
title_sort triplex pcr method for distinguishing the wild-type african swine fever virus from the deletion strains by detecting the gene insertion
topic Veterinary Science
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9274085/
https://www.ncbi.nlm.nih.gov/pubmed/35836498
http://dx.doi.org/10.3389/fvets.2022.921907
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