土拨鼠肝炎病毒检测方法的建立
发布时间：2018-12-13 01:36
【摘要】：目的建立土拨鼠肝炎病毒(Woodchuck hepatitis virus, WHV)核酸的荧光定量PCR (Real-time PCR)检测方法及土拨鼠肝炎病毒表面抗原(WHsAg)的改良双抗加心ELISA检测方法,应用于土拨鼠肝炎病毒模型的创制研究。 方法核酸检测：分别根据土拨鼠肝炎病毒核心抗原(WHcAg)和表面抗原(WHsAg)的DNA序列设计13对扩增引物,分别以混合土拨鼠肝脏DNA的WHV核酸DNA为模板进行PCR,从中筛选无非特异性扩增及引物二聚体,且扩增灵敏度高的引物,用于建立土拨鼠血清中WHV DNA的Real-time PCR检测方法,并利用该方法对土拨鼠血清进行了筛查。表面抗原检测：原核表达WHsAg的N端区域,His·Tag镊柱亲和层析纯化目的蛋白；用该蛋白分别免疫兔和蛋鸡制备多克隆抗体并纯化。建立用于检测感染土拨鼠肝炎病毒的土拨鼠血清中WHsAg的双抗夹心ELISA方法。 结果核酸检测：根据WHsAg基因的5’端设计的一对引物WHVSF1与WHVSR1,检测灵敏度可达1×10’拷贝／u L,病毒拷贝数与Real-time PCR Ct值的标准曲线的R2值为0.997,且电泳未见明显非特异性条带及引物二聚体。表面抗原检测：纯化后的WHsAg蛋白纯度达到95%,免疫兔和蛋鸡后分别制备相应的抗WHsAg血清,纯化得到相应抗体,ELISA结果显示兔抗WHsAg抗体的效价达1：1,024,000,鸡抗WHsAg抗体的效价为1：64,000。本实验建立的双抗加心ELISA法检测WHsAg的检测限为10ng/mL。 结论初步建立了土拨鼠血清中WHV DNA的Real-time PCR检测方法及感染土拨鼠肝炎病毒的土拨鼠血清中WHsAg的改良双抗加心ELISA检测方法,上述方法为进一步研究土拨鼠肝炎病毒模型奠定了基础。
[Abstract]:Objective to establish a fluorescence quantitative PCR (Real-time PCR) method for the detection of groundhog hepatitis virus (Woodchuck hepatitis virus, WHV) nucleic acid and an improved double-antibody plus heart ELISA method for detection of groundhog hepatitis virus surface antigen (WHsAg). It was applied to the creation of groundhog hepatitis virus model. Methods nucleic acid detection: according to the DNA sequence of groundhog hepatitis virus core antigen (WHcAg) and surface antigen (WHsAg) (WHsAg), 13 pairs of amplified primers were designed. The WHV nucleic acid DNA of mixed groundhog liver DNA was used as template for PCR,. The nonspecific amplification and primer dimer were screened from the samples and the primers with high sensitivity were used to establish a method for the detection of WHV DNA in groundhog serum. The method was used to screen the groundhog serum. Surface antigen detection: the target protein was purified by affinity chromatography of N-terminal, His Tag tweezers which expressed WHsAg in prokaryotic cells, and the polyclonal antibody was prepared and purified by immunizing rabbits and laying hens respectively. A double antibody sandwich ELISA method was developed for the detection of WHsAg in the serum of groundhog infected with groundhog hepatitis virus. Results nucleic acid detection: the sensitivity of a pair of primers WHVSF1 and WHVSR1, designed according to the 5 'end of WHsAg gene could reach 1 脳 10' copy / u L, and the R2 value of the standard curve of virus copy number and Real-time PCR Ct value was 0.997. Moreover, there were no obvious nonspecific bands and primer dimer on electrophoresis. Surface antigen detection: the purity of purified WHsAg protein reached 95%. After immunizing rabbits and laying hens, the corresponding anti-WHsAg serum was prepared, and the corresponding antibody was purified. The ELISA results showed that the titer of rabbit anti-WHsAg antibody was 1: 1 024000. The titer of chicken anti WHsAg antibody was 1: 64000. The detection limit of double antibody plus center ELISA method for detection of WHsAg is 10 ng / mL. Conclusion the Real-time PCR detection method for WHV DNA in groundhog serum and the modified double antibody plus heart ELISA method for detection of WHsAg in groundhog serum infected with groundhog hepatitis virus were established. These methods lay a foundation for further study of groundhog hepatitis virus model.
【学位授予单位】：北京协和医学院
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R-332;R373
[Abstract]:Objective to establish a fluorescence quantitative PCR (Real-time PCR) method for the detection of groundhog hepatitis virus (Woodchuck hepatitis virus, WHV) nucleic acid and an improved double-antibody plus heart ELISA method for detection of groundhog hepatitis virus surface antigen (WHsAg). It was applied to the creation of groundhog hepatitis virus model. Methods nucleic acid detection: according to the DNA sequence of groundhog hepatitis virus core antigen (WHcAg) and surface antigen (WHsAg) (WHsAg), 13 pairs of amplified primers were designed. The WHV nucleic acid DNA of mixed groundhog liver DNA was used as template for PCR,. The nonspecific amplification and primer dimer were screened from the samples and the primers with high sensitivity were used to establish a method for the detection of WHV DNA in groundhog serum. The method was used to screen the groundhog serum. Surface antigen detection: the target protein was purified by affinity chromatography of N-terminal, His Tag tweezers which expressed WHsAg in prokaryotic cells, and the polyclonal antibody was prepared and purified by immunizing rabbits and laying hens respectively. A double antibody sandwich ELISA method was developed for the detection of WHsAg in the serum of groundhog infected with groundhog hepatitis virus. Results nucleic acid detection: the sensitivity of a pair of primers WHVSF1 and WHVSR1, designed according to the 5 'end of WHsAg gene could reach 1 脳 10' copy / u L, and the R2 value of the standard curve of virus copy number and Real-time PCR Ct value was 0.997. Moreover, there were no obvious nonspecific bands and primer dimer on electrophoresis. Surface antigen detection: the purity of purified WHsAg protein reached 95%. After immunizing rabbits and laying hens, the corresponding anti-WHsAg serum was prepared, and the corresponding antibody was purified. The ELISA results showed that the titer of rabbit anti-WHsAg antibody was 1: 1 024000. The titer of chicken anti WHsAg antibody was 1: 64000. The detection limit of double antibody plus center ELISA method for detection of WHsAg is 10 ng / mL. Conclusion the Real-time PCR detection method for WHV DNA in groundhog serum and the modified double antibody plus heart ELISA method for detection of WHsAg in groundhog serum infected with groundhog hepatitis virus were established. These methods lay a foundation for further study of groundhog hepatitis virus model.
【学位授予单位】：北京协和医学院
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R-332;R373
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