鼠痘病毒TaqMan MGB探针实时荧光定量PCR快速检测方法的建立与应用研究

发布时间：2018-03-11 14:47

本文选题：鼠痘病毒　切入点：HA基因　出处：《实验动物科学》2016年02期 　论文类型：期刊论文

【摘要】：目的建立快速、敏感、特异检测鼠痘病毒的Taq Man MGB探针实时荧光定量PCR方法。方法针对鼠痘病毒血凝素HA基因设计特异性引物和探针,构建含有HA基因的标准质粒进行定量分析,建立Taq Man MGB探针实时荧光定量PCR检测方法,评价其敏感性、特异性和稳定性。对临床标本中的鼠痘病毒进行检测。结果研究结果显示,建立的鼠痘病毒Taq Man MGB探针实时荧光定量PCR检测方法特异性为100%,与其他正痘病毒属病毒、非正痘病毒属病毒、细菌、真菌、寄生虫和细胞均无交叉反应。该技术灵敏度高,能精确定量检测鼠痘病毒DAN线性范围达10个数量级(100—109拷贝),最低检测限度为4拷贝。该方法重复性非常好,组内变异系数和组间变异系数均小于3%。测试中相关系数、斜率和效率测量线性没有显著变化,表明该方法准确度高、精密度好。将其成功应用于临床标本中鼠痘病毒载量的定量检测,用普通PCR和测序进行确证。整个检测过程可在2 h内完成,可以用作快速诊断方法。结论本研究新建立的鼠痘病毒Taq Man MGB探针实时荧光定量PCR方法,具有快速简便、特异性强、灵敏度高的特性,适用于动物源性产品中鼠痘病毒检测、食品和药品安全检查、环境监测、流行病毒调查,为临床标本中鼠痘病毒的快速定量检测提供了一种特异有效的评价工具,值得推广应用。
[Abstract]:Objective to establish a rapid, sensitive and specific Taq Man MGB probe real-time quantitative PCR method for the detection of mouse pox virus. Methods specific primers and probes were designed for hemagglutinin HA gene of mouse pox virus. The standard plasmid containing HA gene was constructed for quantitative analysis, and the real-time fluorescent quantitative PCR detection method for Taq Man MGB probe was established to evaluate its sensitivity. Specificity and stability. Mouse pox virus was detected in clinical specimens. The results showed that the specificity of the established real-time fluorescent quantitative PCR detection method of mouse pox virus Taq Man MGB probe was 100%, which was similar to that of other positive poxvirus viruses. There is no cross reaction between viruses, bacteria, fungi, parasites and cells. The linear range of accurate quantitative detection of mouse pox virus DAN is 10 orders of magnitude 100-109 copies and the minimum detection limit is 4 copies. This method has a very good reproducibility, the coefficient of variation within group and the coefficient of variation between groups are less than 3 parts. The linear measurement of slope and efficiency showed that the method had high accuracy and good precision. It was successfully applied to the quantitative detection of rodent pox virus load in clinical samples. The whole detection process can be completed within 2 hours and can be used as a rapid diagnostic method. Conclusion the newly established real-time fluorescent quantitative PCR method for Taq Man MGB probe of mouse pox virus is rapid, simple and specific. High sensitivity, suitable for the detection of mouse pox virus in animal products, food and drug safety inspection, environmental monitoring, epidemic virus investigation, It provides a specific and effective tool for rapid quantitative detection of mouse pox virus in clinical specimens and is worth popularizing and applying.
【作者单位】：中国食品药品检定研究院;
【基金】：国家科技支撑计划资助项目(No.2013BAK11B03)
【分类号】：R511;R440

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