荧光定量RT-PCR方法在EV71感染猕猴血液病毒含量监测中的应用

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• 英文论文题名：Application of Real time RT-PCR for monitoring viral content in blood of macaque infected with EV71
• 论文作者：徐婧 ; 邹瑶 ; 张露 ; 陈正礼 ; 黄超 ; 刘文涛 ; 罗启慧 ; 景波
• 年：2016
• 作者机构：四川农业大学动物医学院实验动物疾病模型研究室;四川农业大学动物医学院/动物疫病与人类健康中心四川省重点实验室;
• 论文关键词：肠道病毒71型(EV71) ; 实时荧光定量RT-PCR ; 病毒载量
• 英文论文关键词：Enterovirus 71(EV71) ; Real-time RT-PCR ; viral load
• 会议召开时间：2016-11-04
• 会议录名称：中国畜牧兽医学会动物微生态学分会第五届第十二次全国学术研讨会论文集
• 语种：中文
• 分类号：S855.3
• 学会代码：ZGXJ
• 会议名称：中国畜牧兽医学会动物微生态学分会第五届第十二次全国学术研讨会
• 会议地点：中国山东青岛
• 主办单位：中国畜牧兽医学会动物微生态学分会
• 学会名称：中国畜牧兽医学会
• 页数：1
• 文件大小：86k
• 原文格式：O
• 会议级别：全国

摘要

通过建立肠道病毒71型(EV71)的荧光定量RT-PCR检测方法,为构建EV71猕猴模型提供检测技术。以克隆EV71病毒VP1基因构建重组质粒为标准品,计算其拷贝数,经倍比稀释构建标准曲线,并进行特异性、灵敏性和重复性检测。同时经气管注射EV71病毒建造手足口猕猴模型,采用注射剂量分别为1×10~(6.5)CCID_(50)、4.29×10~(6.5)CCID_(50)、1.86×10~(7.5)CCID_(50)感染新生猕猴和断奶期猕猴,并用建立的实时荧光定量RT-PCR方法监测感染后0~14天血液样品中病毒含量的动态变化。结果表明,建立的方法最低可检测到10~2 copies的病毒RNA,比普通RT-PCR方法灵敏度高100倍,标准曲线的线性范围在10`3~10~8 copies/μL之间,PCR扩增率达到99.37%,对其他肠道病毒检测结果均为阴性,具有特异性强、灵敏性高、稳定性好的特点;血液样品测定分析结果显示,病毒的增殖拷贝数与感染剂量和猕猴的年龄具有相关性。表明该荧光定量PCR技术可用于EV71猕猴模型病毒载量的定量检测。
To develop real-time RT-PCR assays for detection of EV71.Cloning EV71 VP1 gene to construct recombinant plasmid for standard,after calculating the copy numbers,the recombinant plasmid was diluted by times to establish standard curve,and tested the sensitivity,specificity and reproducibility.Newborn macaques and weaning macaques were infected 1×10~(6.5) CCID_(50)、4.29×10~(6.5) CCID_(50)、1.86×<10~(7.5) CCID_(50) EV71 virus through trachea respectively to establish Hand foot and mouth macaque models.Then,the newly developed real-time RT-PCR assay was applied to determine the viral loads in blood at various times following infecting.All assays had a linear dynamic range of 10~3 to 10~8 copies/μL,and could detect the least 10~2 copies of viral RNA,which is 100 times as much as regular RT-PCR,with 99.37%amplification rate,and did not cross-react with other enterovirus.It was high specificity,high sensitivity and good stability.The results of blood samples detection analysis showed that the infective dose and age of macaques were correlated with the copy number of virus.The experimental results show that the detection method of EV71 can be used in quantitative detection of EV71 virus load.

引文

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