水貂圆环病毒实时荧光定量PCR检测方法的建立与应用
Development and Application of a Real-time Fluorescence Quantitative PCR Detection Method for Mink Circovirus
盛陈艳 1麻宝艺 1李健明 2宫庆龙 1刘菲 1时坤 2孙志博 3刘艺 1冷雪 2杜锐4
作者信息
- 1. 吉林农业大学动物科学技术学院,长春 130118
- 2. 吉林农业大学中药材学院,长春 130118
- 3. 吉林农业科技学院,吉林 132101
- 4. 吉林农业大学中药材学院,长春 130118;动物生产及产品质量安全教育部重点实验室,长春 130118;吉林省梅花鹿高效养殖和产品开发技术工程研究中心,长春 130118
- 折叠
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 × 106～1.0× 101 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 × 101 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.
Abstract
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 1.0×106 copies/μL to 1.0×101 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 1.0×101 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.
关键词
水貂圆环病毒/Cap基因/SYBR/Green/Ⅱ实时荧光定量PCR方法Key words
Mink circovirus/Cap gene/SYBR green Ⅱ real-time fluorescence quantitative PCR引用本文复制引用
基金项目
吉林省科技发展计划(20190304004YY)
出版年
2024
国家科技期刊平台
NSTL
维普
万方数据