双重荧光定量RT-PCR检测猪O型口蹄疫病毒GX和XJ株方法的建立及应用
发布时间：2018-08-18 13:29
【摘要】：为了建立检测O型口蹄疫病毒不同基因型的双重荧光定量RT-PCR方法,本试验根据口蹄疫病毒O/GX/09-7株和O/XJ/10-11株VP1基因序列设计合成1对通用引物和2个Taqman探针。通过制备GX与XJ株标准品RNA,优化反应条件,建立双重荧光定量RT-PCR,并评价其灵敏性,特异性、重复性。利用建立的双重荧光定量RT-PCR检测方法对猪O型FMD疫苗中的GX和XJ株进行鉴别检测,以及对疫苗中病毒RNA含量进行定量测定。结果显示,检测GX与XJ株双重定量RT-PCR标准曲线的相关系数分别为R2=0.997和R2=0.995；建立的双重荧光定量RT-PCR方法能够同时鉴别检测灭活病毒中口蹄疫病毒不同基因型,具有良好的特异性与灵敏性；RNA含量最低检测限为10拷贝/μL。应用此方法能够满足对企业生产车间的不同批次口蹄疫灭活病毒和种毒的RNA含量进行有效的检测,还可用于种毒的纯化检验,并且为疫苗的生产质量控制提供了一种新的检测方法。
[Abstract]:In order to establish a double fluorescent quantitative RT-PCR method for the detection of different genotypes of foot-and-mouth disease virus type O (FMDV), a pair of universal primers and two Taqman probes were designed and synthesized according to the VP1 gene sequences of FMDV O/GX/09-7 and O/XJ/10-11 strains. By preparing GX and XJ strain standard RNAs, optimizing the reaction conditions, establishing double fluorescence quantitative RT-PCRs, and evaluating its sensitivity, specificity and repeatability. The GX and XJ strains of porcine O type FMD vaccine were identified and detected by double fluorescence quantitative RT-PCR assay, and the content of virus RNA in the vaccine was determined quantitatively. The results showed that the correlation coefficient of double quantitative RT-PCR curve between GX and XJ strain was R2O0.997 and R2O0.995.The dual fluorescence quantitative RT-PCR method could identify and detect different genotypes of foot-and-mouth disease virus in inactivated virus simultaneously. The minimum detection limit of RNA content with good specificity and sensitivity was 10 copies / 渭 L. The method can be used to detect the RNA content of inactivated virus and seed virus in different batches of foot-and-mouth disease virus, and can also be used to purify the virus. It also provides a new detection method for the quality control of vaccine production.
【学位授予单位】：内蒙古农业大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S852.65
[Abstract]:In order to establish a double fluorescent quantitative RT-PCR method for the detection of different genotypes of foot-and-mouth disease virus type O (FMDV), a pair of universal primers and two Taqman probes were designed and synthesized according to the VP1 gene sequences of FMDV O/GX/09-7 and O/XJ/10-11 strains. By preparing GX and XJ strain standard RNAs, optimizing the reaction conditions, establishing double fluorescence quantitative RT-PCRs, and evaluating its sensitivity, specificity and repeatability. The GX and XJ strains of porcine O type FMD vaccine were identified and detected by double fluorescence quantitative RT-PCR assay, and the content of virus RNA in the vaccine was determined quantitatively. The results showed that the correlation coefficient of double quantitative RT-PCR curve between GX and XJ strain was R2O0.997 and R2O0.995.The dual fluorescence quantitative RT-PCR method could identify and detect different genotypes of foot-and-mouth disease virus in inactivated virus simultaneously. The minimum detection limit of RNA content with good specificity and sensitivity was 10 copies / 渭 L. The method can be used to detect the RNA content of inactivated virus and seed virus in different batches of foot-and-mouth disease virus, and can also be used to purify the virus. It also provides a new detection method for the quality control of vaccine production.
【学位授予单位】：内蒙古农业大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S852.65
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