牛结节性皮肤病病毒与山羊痘病毒双重TaqMan实时荧光定量PCR方法的建立
Establishment and application of a dual TaqMan real-time quantitative PCR method for lumpy skin disease virus and goatpox virus
余翰维 1冯秋媛 2苏洋 3张慧 3邵晓龙 2杨云凤 2何小兵 3陈国华 3房永祥 3景志忠 1陈轶霞2
作者信息
- 1. 西北民族大学生命科学与工程学院,甘肃兰州 730030;中国农业科学院兰州兽医研究所动物疫病防控全国重点实验室农业部兽医公共卫生重点实验室,甘肃兰州 730046
- 2. 西北民族大学生命科学与工程学院,甘肃兰州 730030
- 3. 中国农业科学院兰州兽医研究所动物疫病防控全国重点实验室农业部兽医公共卫生重点实验室,甘肃兰州 730046
- 折叠
摘要
为建立可快速鉴别牛结节性皮肤病病毒(LSDV)与山羊痘病毒(GTPV)的方法,通过对GenBank中公布的LSDV和GTPV的全基因序列进行比对分析,靶向LSDV基因组的保守区域设计了1对特异性的引物和2条TaqMan探针,经反应条件优化后,建立了一种LSDV和GTPV的双重TaqMan实时荧光定量PCR检测方法,并评估其敏感性、重复性、特异性.结果表明,本方法能从LSDV、GTPV、绵羊疽病毒、羊口疮病毒、牛病毒性腹泻病毒、口蹄疫病毒6种常见的牛源性疾病病原中成功鉴别LSDV和GTPV,具有良好的特异性.LSDV和GTPV基因组的最低检测下限都为3.37×101 copies/μL.组内、组间变异系数均小于1％.运用本方法对10份临床样品进行检测,符合率达到100％.综上所述,本方法具有良好的特异性、敏感性和重复性,可快速鉴别出样品中的LSDV和GTPV,解决了目前国内容易造成的牛结节性皮肤病漏诊的问题,为全面控制国内牛结节性皮肤病疫情提供了新的技术手段.
Abstract
In order to establish a method to rapidly identify lumpy skin disease virus(LSDV)and goatpox virus(GTPV),the whole gene sequences of LSDV and GTPV published in GenBank were compared and analyzed,and one pair of specific primers and two TaqMan probes were designed based on the conserved sequences of LSDV.After optimization of the reaction conditions,a dual TaqMan real-time quantitative PCR assay for LSDV and GTPV was established and evaluated for sensitivity,reproducibility,and specificity.The results showed that this method can successfully identify LSDV and GTPV from LSDV,GTPV,sheeppox virus,orf virus,bovine viral diarrhea virus and foot-and-mouth disease virus,which indicates good specificity.The lowest limit of detection for both LSDV and GTPV was 3.37 × 101 copies/μL.The coefficient of variation was less than 1％for both intra-and inter-group.The method was applied to 10 clinical samples,and the compliance rate was 100％.In conclusion,this method has good specificity,sensitivity and repro-ducibility,and can rapidly identify LSDV and GTPV in sample.It solves the current problem of overdiag-nosis of bovine lumpy skin disease,which is easily caused in China,and provides a new technical means for the comprehensive control of lumpy skin disease in China.
关键词
牛结节性皮肤病病毒/山羊疽病毒/双重TaqMan实时定量PCRKey words
lumpy skin disease virus/goatpox virus/dual TaqMan real-time quantitative PCR引用本文复制引用
基金项目
国家自然科学基金项目(32260874)
国家动物疫病监测与流行病学调查计划项目(125161031)
家畜疫病病原生物学国家重点实验室(应用)基础重大成果培育项目(SKLVEB2021CGQD03)
甘肃省科技计划项目(22JR5RA780)
出版年
2024
国家科技期刊平台
NSTL
万方数据