摘要
为快速检测水貂圆环病毒(MiCV),本试验根据MiCV Cap基因序列设计合成特异性引物,建立了水貂圆环病毒SYBR Green II实时荧光定量PCR检测方法。结果显示,该方法在模板浓度为1.0×10^(6)~1.0×10^(1) copies/μL范围内呈良好线性关系,相关系数为0.9983。本试验建立的检测方法对水貂阿留申病毒、猪圆环病毒2型、水貂肠炎病毒、犬瘟热病毒和猪伪狂犬病毒进行检测均无特异性扩增,批内和批间CV均小于2%,对重组阳性质粒的检测下限为1.0×10^(1) copies/μL,比普通PCR的敏感度高100倍,对阳性样品DNA的检测下限为2.38×10^(-2) pg/μL,比普通PCR的敏感度高1000倍。应用该方法对吉林省部分毛皮动物养殖场的水貂、狐狸、貉和环境样品进行检测,结果其阳性率分别为57.23%、48.42%、39.71%和68.48%。上述结果表明,本研究建立的实时荧光定量PCR检测方法具有良好的灵敏性、特异性和重复性,为MiCV的诊断及流行病学调查提供技术支持。
Specific primers were designed according to the Mink circovirus(MiCV)cap gene sequence and synthesized for development of a SYBR Green II real-time fluorescence quantitative PCR.The results showed that the method had a good linear relationship in the template concentration range of 1.0×10^(6)copies/μL to 1.0×10^(1)copies/μL and the correlation coefficient was 0.9983.This detection method also had no amplifications for AMDV,PCV2,MEV,CDV and PRV.The intra-assay and inter-assay CV were less than 2%.The lower limit of recombinant positive plasmid was 1.0×10^(1)copies/μL,which was 100 times more sensitive than the conventional PCR.In addition,the lower limit of detection for DNA of positive samples was 2.38×10^(-2)pg/μL,which was 1000 times more sensitive than the conventional PCR.Samples were collected from minks,foxes,raccoon dogs and environmental resources on some fur animal farms in Jilin province and tested using this method.The results showed that the positive rates were 57.23%for minks,48.42%for foxes,39.71%for racoon dogs and 68.48%for environmental resources,respectively.The above results indicated that the real-time fluorescence quantitative PCR method developed in this study had good sensitivity,specificity and repeatability,and provided technical support for the diagnosis and epidemiological investigation of MiCV.
作者
盛陈艳
麻宝艺
李健明
宫庆龙
刘菲
时坤
孙志博
刘艺
冷雪
杜锐
SHENG Chenyan;MA Baoyi;LI Jianming;GONG Qinglong;LIU Fei;SHI Kun;SUN Zhibo;LIU Yi;LENG Xue;DU Rui(College of Animal Science and Technology,Jilin Agricultural University,Changchun 130118,China;College of Chinese Medicine Materials,Jilin Agricultural University,Changchun 130118,China;Key Laboratory of Animal Production and Product Quality Safety of Ministry of Education,Changchun 130118,China;Jilin Provincial Engineering Research Center for Efficient Breeding and Product Development of Sika Deer,Changchun 130118,China;Jilin Agricultural Science and Technology University,Jilin 132101,China)
出处
《中国动物传染病学报》
CAS
北大核心
2024年第2期131-138,共8页
Chinese Journal of Animal Infectious Diseases
基金
吉林省科技发展计划项目(20190304004YY)。
