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Development and Implementation of a Quadruple RT-qPCR Method for the Identification of Porcine Reproductive and Respiratory Syndrome Virus Strains

Background: Porcine reproductive and respiratory syndrome virus (PRRSV) causes porcine reproductive and respiratory syndrome (PRRS), leading to abortion in sows and respiratory distress in breeding pigs. In China, PRRSV1 and PRRSV2 are the two circulating genotypes in swine herds, with distinct viru...

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Autores principales: Ruan, Shengnan, Ren, Wenhui, Yu, Bin, Yu, Xuexiang, Wu, Hao, Li, Wentao, Jiang, Yunbo, He, Qigai
Formato: Online Artículo Texto
Lenguaje:English
Publicado: MDPI 2023
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10536281/
https://www.ncbi.nlm.nih.gov/pubmed/37766352
http://dx.doi.org/10.3390/v15091946
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author Ruan, Shengnan
Ren, Wenhui
Yu, Bin
Yu, Xuexiang
Wu, Hao
Li, Wentao
Jiang, Yunbo
He, Qigai
author_facet Ruan, Shengnan
Ren, Wenhui
Yu, Bin
Yu, Xuexiang
Wu, Hao
Li, Wentao
Jiang, Yunbo
He, Qigai
author_sort Ruan, Shengnan
collection PubMed
description Background: Porcine reproductive and respiratory syndrome virus (PRRSV) causes porcine reproductive and respiratory syndrome (PRRS), leading to abortion in sows and respiratory distress in breeding pigs. In China, PRRSV1 and PRRSV2 are the two circulating genotypes in swine herds, with distinct virulence. PRRSV2 further consists of classical (C-PRRSV2), highly pathogenic (HP-PRRSV2), and NADC30-Like (N-PRRSV2) subtypes. The diversity of PRRSV poses challenges for control and eradication, necessitating reliable detection assays for differentiating PRRSV genotypes. Methods: A new TaqMan-based RT-qPCR assay with four sets of primers and probes targeting conserved regions of the ORF7 and NSP2 genes of PRRSV was developed, optimized, and evaluated by us. Reaction conditions such as annealing temperature, primer concentration, and probe concentration were optimized for the assay. Specificity, sensitivity, repeatability, stability, limit of detection (LOD), concordance with the reference method were evaluated for the assay. Results: The assay could detect and type PRRSV1, C-PRRSV2, HP-PRRSV2, and N-PRRSV2 simultaneously with 97.33% specificity, 96.00% sensitivity, 12 copies/μL LOD, 97.00% concordance with reference assays. We applied the assay to 321 clinical samples from swine farms in China. The assay successfully detected and typed 230 PRRSV-positive samples, with 24.78% (57/230) of them further confirmed by ORF5 gene sequencing. The prevalence of PRRSV subtypes among the positive samples was as follows: C-PRRSV2 (15.22%), HP-PRRSV2 (23.48%), and N-PRRSV2 (61.30%). Two samples showed coinfection with different PRRSV subtypes. Conclusion: The quadruple RT-qPCR assay is a powerful tool for detecting and typing the currently circulating PRRSV strains in Chinese swine populations. It can assist in the surveillance of PRRSV prevalence and the implementation of prevention and control strategies.
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spelling pubmed-105362812023-09-29 Development and Implementation of a Quadruple RT-qPCR Method for the Identification of Porcine Reproductive and Respiratory Syndrome Virus Strains Ruan, Shengnan Ren, Wenhui Yu, Bin Yu, Xuexiang Wu, Hao Li, Wentao Jiang, Yunbo He, Qigai Viruses Article Background: Porcine reproductive and respiratory syndrome virus (PRRSV) causes porcine reproductive and respiratory syndrome (PRRS), leading to abortion in sows and respiratory distress in breeding pigs. In China, PRRSV1 and PRRSV2 are the two circulating genotypes in swine herds, with distinct virulence. PRRSV2 further consists of classical (C-PRRSV2), highly pathogenic (HP-PRRSV2), and NADC30-Like (N-PRRSV2) subtypes. The diversity of PRRSV poses challenges for control and eradication, necessitating reliable detection assays for differentiating PRRSV genotypes. Methods: A new TaqMan-based RT-qPCR assay with four sets of primers and probes targeting conserved regions of the ORF7 and NSP2 genes of PRRSV was developed, optimized, and evaluated by us. Reaction conditions such as annealing temperature, primer concentration, and probe concentration were optimized for the assay. Specificity, sensitivity, repeatability, stability, limit of detection (LOD), concordance with the reference method were evaluated for the assay. Results: The assay could detect and type PRRSV1, C-PRRSV2, HP-PRRSV2, and N-PRRSV2 simultaneously with 97.33% specificity, 96.00% sensitivity, 12 copies/μL LOD, 97.00% concordance with reference assays. We applied the assay to 321 clinical samples from swine farms in China. The assay successfully detected and typed 230 PRRSV-positive samples, with 24.78% (57/230) of them further confirmed by ORF5 gene sequencing. The prevalence of PRRSV subtypes among the positive samples was as follows: C-PRRSV2 (15.22%), HP-PRRSV2 (23.48%), and N-PRRSV2 (61.30%). Two samples showed coinfection with different PRRSV subtypes. Conclusion: The quadruple RT-qPCR assay is a powerful tool for detecting and typing the currently circulating PRRSV strains in Chinese swine populations. It can assist in the surveillance of PRRSV prevalence and the implementation of prevention and control strategies. MDPI 2023-09-18 /pmc/articles/PMC10536281/ /pubmed/37766352 http://dx.doi.org/10.3390/v15091946 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
Ruan, Shengnan
Ren, Wenhui
Yu, Bin
Yu, Xuexiang
Wu, Hao
Li, Wentao
Jiang, Yunbo
He, Qigai
Development and Implementation of a Quadruple RT-qPCR Method for the Identification of Porcine Reproductive and Respiratory Syndrome Virus Strains
title Development and Implementation of a Quadruple RT-qPCR Method for the Identification of Porcine Reproductive and Respiratory Syndrome Virus Strains
title_full Development and Implementation of a Quadruple RT-qPCR Method for the Identification of Porcine Reproductive and Respiratory Syndrome Virus Strains
title_fullStr Development and Implementation of a Quadruple RT-qPCR Method for the Identification of Porcine Reproductive and Respiratory Syndrome Virus Strains
title_full_unstemmed Development and Implementation of a Quadruple RT-qPCR Method for the Identification of Porcine Reproductive and Respiratory Syndrome Virus Strains
title_short Development and Implementation of a Quadruple RT-qPCR Method for the Identification of Porcine Reproductive and Respiratory Syndrome Virus Strains
title_sort development and implementation of a quadruple rt-qpcr method for the identification of porcine reproductive and respiratory syndrome virus strains
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10536281/
https://www.ncbi.nlm.nih.gov/pubmed/37766352
http://dx.doi.org/10.3390/v15091946
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