{"title":"建立一种三重 qPCR 检测方法，用于区分中国流行的高致病性基因 I 型重组病毒与低致病性基因 I 型和基因 II 型非洲猪瘟病毒","authors":"Leilei Ding, Tao Ren, Guoxia Bing, Zhigang Wang, Baoyue Wang, Jianqiang Ni, Yuliang Liu, Rui Zhao, Yuanmao Zhu, Fang Li, Renqiang Liu, Qiang Fu, Zhijun Tian, Zhigao Bu, Encheng Sun, Dongming Zhao","doi":"10.1155/2024/6206857","DOIUrl":null,"url":null,"abstract":"<div>\n <p>African swine fever virus (ASFV) poses serious threats to the global swine industry, food safety, and the economy. Since August 2018, different types of ASFVs have successively emerged in China, making ASF diagnostics more challenging. The highly virulent genotype I recombinant virus has gradually become the prevalent dominant strain and is identified by sequencing several of its genes, which is time-consuming and expensive. Here, we developed a triplex real-time quantitative PCR (qPCR) assay based on the ASFV B646L, X64R, and MGF_360-14L genes to differentiate highly virulent genotype I recombinant viruses from low-virulence genotype I and genotype II viruses in China. This method has high sensitivity and a limit of detection of 10 copies/reaction for standard plasmids, as well as good specificity without cross-reactions with the viral nucleic acids of porcine reproductive and respiratory syndrome virus (PRRSV), classical swine fever virus (CSFV), pseudorabies virus (PRV), porcine circovirus 2 (PCV 2), porcine circovirus 3 (PCV 3), porcine epidemic diarrhea virus (PEDV), transmissible gastroenteritis virus (TGEV), or porcine rotavirus (PoRV). Importantly, triplex qPCR can be used to quickly and accurately evaluate clinical samples and cell cultures infected with highly virulent genotype I virus, low-virulence genotype I virus, or genotype II virus. Thus, triplex qPCR provides an alternative tool for ASF surveillance in China.</p>\n </div>","PeriodicalId":234,"journal":{"name":"Transboundary and Emerging Diseases","volume":"2024 1","pages":""},"PeriodicalIF":3.5000,"publicationDate":"2024-09-23","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://onlinelibrary.wiley.com/doi/epdf/10.1155/2024/6206857","citationCount":"0","resultStr":"{\"title\":\"Establishment of a Triplex qPCR Assay for Differentiating Highly Virulent Genotype I Recombinant Virus From Low-Virulence Genotype I and Genotype II African Swine Fever Viruses Circulating in China\",\"authors\":\"Leilei Ding, Tao Ren, Guoxia Bing, Zhigang Wang, Baoyue Wang, Jianqiang Ni, Yuliang Liu, Rui Zhao, Yuanmao Zhu, Fang Li, Renqiang Liu, Qiang Fu, Zhijun Tian, Zhigao Bu, Encheng Sun, Dongming Zhao\",\"doi\":\"10.1155/2024/6206857\",\"DOIUrl\":null,\"url\":null,\"abstract\":\"<div>\\n <p>African swine fever virus (ASFV) poses serious threats to the global swine industry, food safety, and the economy. Since August 2018, different types of ASFVs have successively emerged in China, making ASF diagnostics more challenging. The highly virulent genotype I recombinant virus has gradually become the prevalent dominant strain and is identified by sequencing several of its genes, which is time-consuming and expensive. Here, we developed a triplex real-time quantitative PCR (qPCR) assay based on the ASFV B646L, X64R, and MGF_360-14L genes to differentiate highly virulent genotype I recombinant viruses from low-virulence genotype I and genotype II viruses in China. This method has high sensitivity and a limit of detection of 10 copies/reaction for standard plasmids, as well as good specificity without cross-reactions with the viral nucleic acids of porcine reproductive and respiratory syndrome virus (PRRSV), classical swine fever virus (CSFV), pseudorabies virus (PRV), porcine circovirus 2 (PCV 2), porcine circovirus 3 (PCV 3), porcine epidemic diarrhea virus (PEDV), transmissible gastroenteritis virus (TGEV), or porcine rotavirus (PoRV). Importantly, triplex qPCR can be used to quickly and accurately evaluate clinical samples and cell cultures infected with highly virulent genotype I virus, low-virulence genotype I virus, or genotype II virus. 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引用次数: 0
摘要
非洲猪瘟病毒（ASFV）对全球养猪业、食品安全和经济发展构成严重威胁。2018年8月以来，我国陆续出现了不同类型的ASFV，使得ASF诊断更具挑战性。高致病性的基因Ⅰ型重组病毒已逐渐成为流行的优势毒株，通过对其多个基因进行测序来鉴定，耗时长、成本高。在此，我们开发了一种基于 ASFV B646L、X64R 和 MGF_360-14L 基因的三重实时定量 PCR（qPCR）检测方法，用于区分中国的高致病性基因型 I 重组病毒与低致病性基因型 I 和基因型 II 病毒。该方法灵敏度高，对标准质粒的检测限为 10 拷贝/反应，特异性好，不会与猪繁殖与呼吸综合征病毒（PRRSV）的病毒核酸发生交叉反应、猪繁殖与呼吸综合征病毒 (PRRSV)、猪瘟病毒 (CSFV)、伪狂犬病病毒 (PRV)、猪圆环病毒 2 (PCV 2)、猪圆环病毒 3 (PCV 3)、猪流行性腹泻病毒 (PEDV)、传染性胃肠炎病毒 (TGEV) 或猪轮状病毒 (PoRV)。重要的是，三重 qPCR 可用于快速准确地评估感染了高毒力基因型 I 病毒、低毒力基因型 I 病毒或基因型 II 病毒的临床样本和细胞培养物。因此，三重 qPCR 为中国的 ASF 监测提供了另一种工具。
Establishment of a Triplex qPCR Assay for Differentiating Highly Virulent Genotype I Recombinant Virus From Low-Virulence Genotype I and Genotype II African Swine Fever Viruses Circulating in China
African swine fever virus (ASFV) poses serious threats to the global swine industry, food safety, and the economy. Since August 2018, different types of ASFVs have successively emerged in China, making ASF diagnostics more challenging. The highly virulent genotype I recombinant virus has gradually become the prevalent dominant strain and is identified by sequencing several of its genes, which is time-consuming and expensive. Here, we developed a triplex real-time quantitative PCR (qPCR) assay based on the ASFV B646L, X64R, and MGF_360-14L genes to differentiate highly virulent genotype I recombinant viruses from low-virulence genotype I and genotype II viruses in China. This method has high sensitivity and a limit of detection of 10 copies/reaction for standard plasmids, as well as good specificity without cross-reactions with the viral nucleic acids of porcine reproductive and respiratory syndrome virus (PRRSV), classical swine fever virus (CSFV), pseudorabies virus (PRV), porcine circovirus 2 (PCV 2), porcine circovirus 3 (PCV 3), porcine epidemic diarrhea virus (PEDV), transmissible gastroenteritis virus (TGEV), or porcine rotavirus (PoRV). Importantly, triplex qPCR can be used to quickly and accurately evaluate clinical samples and cell cultures infected with highly virulent genotype I virus, low-virulence genotype I virus, or genotype II virus. Thus, triplex qPCR provides an alternative tool for ASF surveillance in China.
期刊介绍：
Transboundary and Emerging Diseases brings together in one place the latest research on infectious diseases considered to hold the greatest economic threat to animals and humans worldwide. The journal provides a venue for global research on their diagnosis, prevention and management, and for papers on public health, pathogenesis, epidemiology, statistical modeling, diagnostics, biosecurity issues, genomics, vaccine development and rapid communication of new outbreaks. Papers should include timely research approaches using state-of-the-art technologies. The editors encourage papers adopting a science-based approach on socio-economic and environmental factors influencing the management of the bio-security threat posed by these diseases, including risk analysis and disease spread modeling. Preference will be given to communications focusing on novel science-based approaches to controlling transboundary and emerging diseases. The following topics are generally considered out-of-scope, but decisions are made on a case-by-case basis (for example, studies on cryptic wildlife populations, and those on potential species extinctions):
Pathogen discovery: a common pathogen newly recognised in a specific country, or a new pathogen or genetic sequence for which there is little context about — or insights regarding — its emergence or spread.
Prevalence estimation surveys and risk factor studies based on survey (rather than longitudinal) methodology, except when such studies are unique. Surveys of knowledge, attitudes and practices are within scope.
Diagnostic test development if not accompanied by robust sensitivity and specificity estimation from field studies.
Studies focused only on laboratory methods in which relevance to disease emergence and spread is not obvious or can not be inferred (“pure research” type studies).
Narrative literature reviews which do not generate new knowledge. Systematic and scoping reviews, and meta-analyses are within scope.
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