摘要
为建立一种快速测定鼠痘病毒(ECTV)含量的检测方法,针对鼠痘病毒的基因序列,建立了靶向ECTV EVM009基因的荧光定量PCR检测方法。根据ECTV全基因序列,设计出4对qPCR引物并评价其特异性及敏感性。最终通过筛选得到靶向于ECTV EVM009基因的一对特异性高、灵敏度强的荧光定量PCR引物。利用构建的pCAGGS-EVM009真核表达质粒绘制标准曲线获得标准曲线y=-3.5099 x+41.30,线性相关系数R^(2)=0.9992。扩增效率达到92.7%;对重组质粒标准品的最低检测限为4.91×10^(0)拷贝/μL;组内与组间的重复试验变异系数均小于2%;仅能有效检测ECTV,与VACV、GPTV、SPPV、ORFV和LSDV均无交叉反应。结果表明,建立的荧光定量检测方法特异性强、敏感性高及重复性好,可以用于ECTV感染小鼠模型中病理组织中病毒载量的测定。
To establish a rapid assay for the detection of Ectromelia virus(ECTV),a real-time quantitative PCR method targeting the EVM009 gene of ECTV was developed in this study.Based on the whole gene sequence of ECTV,four pairs of qPCR primers were designed and evaluated for their specificity and sensitivity,and a pair of real-time quantitative PCR primers with high specificity and sensitivity targeting the EVM009 gene of ECTV were obtained by screening.A standard curve was constructed based on the pCAGGS-EVM009 eukaryotic expression plasmid.The standard curve was y=-3.5099x+41.30,linear correlation coefficient R^(2)=0.9992.The amplification efficiency reached 92.7%,the lowest detection limit of the recombinant plasmid standard was 4.91×10^(10) copies/μL,and the coefficients of variation of the repeated tests within and between the groups were all less than 2%,it could only effectively detect ECTV,with no cross-reactivity with VACV,GPTV,SPPV,ORFV,or LSDV.This shows that the qPCR method is specific,sensitive,and reproducible and can be used for the determination of viral load in pathological tissues of ECTV-infected mice.
作者
赵立磊
任善会
王会宝
高小龙
姚威
杨雪
张红强
王小龙
张强蓉
喇萍
叶国荣
陈豪泰
孙跃峰
樊江峰
ZHAO Li-lei;REN Shan-hui;WANG Hui-bao;GAO Xiao-long;YAO Wei;YANG Xue;ZHANG Hong-qiang;WANG Xiao-long;ZHANG Qiang-rong;LA Ping;YE Guo-rong;CHEN Hao-tai;SUN Yue-feng;FAN Jiang-feng(College of Veterinary Medicine,Gansu Agricultural University,Lanzhou Gansu,730070,China;Key Laboratory of Veterinary Etiological Biology,Lanzhou Veterinary Research Institute,CAAS,Lanzhou,Gansu,730046,China;Academy of Environment Engineering,Gansu Forestry Voctech University,Tianshui,Gansu,741020,China;College of Agriculture and Animal Husbandry,Qinghai University,Xining,Qinghai,810016,China;Animal Husbandry Industry Development Center,Wanzhou District,Chongqing,400000,China)
出处
《动物医学进展》
北大核心
2026年第1期9-14,共6页
Progress In Veterinary Medicine
基金
甘肃省自然科学基金项目(24JRRE011)
高校教师创新基金项目(2025A-326)
国家自然科学基金项目(32460876,32302850)。
