摘要
为建立一种绝对定量的赤羽病病毒(Akabane virus,AKAV)痕量检测方法,以AKAV的S基因为靶基因,选择引物和探针,优化反应体系和反应条件,建立了一种检测AKAV的芯片式数字RT-PCR(chip-based digital RT-PCR,cdRT-PCR)方法,并对其敏感性、特异性和重复性进行了评估,使用行标推荐的实时荧光定量PCR(qPCR)方法对64份临床样品进行了符合性验证。结果显示:建立的cdRT-PCR方法检测限可达到1.36 copies/μL,组内和组间重复性试验变异系数分别为2.09%和3.44%,与常见的牛羊病毒无交叉反应。临床样品检测显示,本研究建立的cdRT-PCR方法与行业标准推荐的qPCR方法检测结果符合率达到100%。结果表明,建立的cdRT-PCR方法灵敏度高,特异性及重复性好,可为AKAV核酸标准物质制备、标准样品定值、痕量病毒检测提供方法支撑。
In order to establish an absolutely quantitative trace detection method for the Akabane virus(AKAV),a chip-based digital RT-PCR(cdRT-PCR)assay for Akabane virus(AKAV)was developed through optimizing reaction system and conditions of primers and probes that were designed taking S gene of AKAV as a target gene,and assessed for its sensitivity,specifi city and repeatability,followed by conformance verifi cation targeting 64 clinical samples using the qPCR assay recommended by the industry standard.The results showed that the limit of detection(LOD)of the established cdRT-PCR could reach 1.36 copies/μL,the coefficients of variation(CVs)for intraand inter-assay repeatability tests were 2.09%and 3.44%,respectively,and no cross-reactivity was observed with other common bovine or ovine viruses.Based on the testing of clinical samples,it was revealed that the results by the established cdRT-PCR were 100%conformant with those by the recommended qPCR.In conclusion,value assignment for AKAV nucleic acid reference materials or samples and trace viral detection could be supported by the established cdRT-PCR that was highly sensitive,specific and repeatable.
作者
刘硕
李超
李祥雨
王素春
李小慧
范春艳
王楷宬
Liu Shuo;Li Chao;Li Xiangyu;Wang Suchun;Li Xiaohui;Fan Chunyan;Wang Kaicheng(College of Life Science and Food Engineering,Hebei University of Engineering,Handan 056038,Hebei,China;China Animal Health and Epidemiology Center,Key Laboratory of Animal Biosafety Risk Prevention and Control(South China),Ministry of Agriculture and Rural Aff airs,Key Laboratory of Animal Biosafety,Qingdao 266032,Shan dong,China)
出处
《中国动物检疫》
2025年第12期120-125,共6页
China Animal Health Inspection
基金
国家重点研发计划项目(2024YFD1800101)。
