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

Development and Application of Taq Man MGB Probe real-time Fluorescence Quantitative PCR for Rapid Detection of Ectromelia virus

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。

Objective To develop a Taq Manminor groove binder（ MGB） probe-based,sensitive and specific realtime fluorescence quantitative polymerase chain reaction（ q PCR） assay for rapid detection of Ectromelia virus（ ECTV）. Method Primers and probes specific to HA（ hemagglutinin） gene of Ectromelia virus were designed.Plasmid containing the sequence of HA gene was constructed as ECTV-DNA standard for quantitative analysis. A Taq Man MGB probeq PCR assay was developed. The sensitivity,specificity and stability of the assay were assessed.Then,the developed Taq Man MGB probe q PCR assay was applied to detect Ectromelia virus inclinical specimens.Result The developed Taq Man MGB probe q PCR assay was shown to be 100% specific to test a panel of fourteen organisms consisting of ECTV,other orthopoxvirus species,non-orthopoxvirus species,bacteria,fungi,parasites and cells. The technology was demonstrated to be highly sensitive,allowing a precise ECTV DNA quantitation over a range of ten orders of magnitude（ from 100 to 109copies of standard DNA）,and the limit of detection（ LOD） of the assay was determined to be 4 copies of target DNA. The reproducibility of this method was excellent as the Coefficient of Variation（ CV） for each point was less than 3%. Measurements of linearity such as Correlation coefficient（ R2）,slope（ S） and efficiency（ Eff%） did not vary significantly among the test suggesting good accuracy and precision. The Taq Man MGB probe q PCR assay was successfully applied to quantifiable detection of viral genomic load in clinical specimens,and confirmed by using conventional PCR and sequence analysis. The assay described in this report generates complete result in 2 h and can be used as a fast diagnostic method.Conclusion This newly developed Taq Man MGB probe real-time fluorescence quantitative PCR method,has the characteristics of quick and easy,strong specificity and high sensitivity,suitable for Ectromelia virus detection in animal origin products,food and drug safety inspection,environmental monitoring and epidemiology investigation.It provides a specific and effective evaluation tool for rapid and quantitative detection of Ectromelia virus in clinical specimens,is worthy of popularization and application.