Ding Leilei, Ren Tao, Bing Guoxia, Wang Zhigang, Wang Baoyue, Ni Jianqiang, Liu Yuliang, Zhao Rui, Zhu Yuanmao, Li Fang, Liu Renqiang, Fu Qiang, Tian Zhijun, Bu Zhigao, Sun Encheng, Zhao Dongming
State Key Laboratory for Animal Disease Control and Prevention National High Containment Facilities for Animal Disease Control and Prevention National African Swine Fever Para-reference Laboratory Harbin Veterinary Research Institute Chinese Academy of Agricultural Sciences, Harbin 150069, China.
College of Veterinary Medicine Xinjiang Agricultural University, Urumqi 830052, China.
Transbound Emerg Dis. 2024 Sep 24;2024:6206857. doi: 10.1155/2024/6206857. eCollection 2024.
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.
非洲猪瘟病毒(ASFV)对全球养猪业、食品安全和经济构成严重威胁。自2018年8月以来,不同类型的ASFV相继在中国出现,这使得非洲猪瘟的诊断更具挑战性。高致病性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,这既耗时又昂贵。在此,我们基于ASFV的B646L、X64R和MGF_360 - 14L基因开发了一种三重实时定量PCR(qPCR)检测方法,以区分中国的高致病性I型重组病毒与低致病性I型和II型病毒。该方法具有高灵敏度,对标准质粒的检测限为10拷贝/反应,且特异性良好,与猪繁殖与呼吸综合征病毒(PRRSV)、经典猪瘟病毒(CSFV)、伪狂犬病病毒(PRV)、猪圆环病毒2型(PCV 2)、猪圆环病毒3型(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)的病毒核酸无交叉反应。重要的是,三重qPCR可用于快速准确地评估感染高致病性I型病毒、低致病性I型病毒或II型病毒的临床样本和细胞培养物。因此,三重qPCR为中国的非洲猪瘟监测提供了一种替代工具。