为建立牛副流感病毒5型(BPIV5)的SYBR Green Ⅰ荧光定量PCR(qPCR)检测方法,本研究经PCR扩增BPIV5 L基因的保守片段,构建重组质粒p MD18-T-BPIV5,并经PCR、双酶切与测序鉴定正确后作为标准品,经各反应条件优化后初步建立检测BPIV5的qPCR...为建立牛副流感病毒5型(BPIV5)的SYBR Green Ⅰ荧光定量PCR(qPCR)检测方法,本研究经PCR扩增BPIV5 L基因的保守片段,构建重组质粒p MD18-T-BPIV5,并经PCR、双酶切与测序鉴定正确后作为标准品,经各反应条件优化后初步建立检测BPIV5的qPCR方法。利用10倍倍比稀释的质粒标准品作为模板,利用优化的qPCR扩增,建立该方法的标准曲线,结果显示,重组质粒标准品的拷贝数与其Ct值呈良好的线性关系,相关系数为0.998,扩增效率为1.09。以BPIV5、牛肠道病毒(BEV)、牛病毒性腹泻病毒(BVDV)、牛诺如病毒(BNOV)、牛副流感病毒3型(BPIV3)和牛冠状病毒(BCOV)的cDNA作为模板,采用该qPCR扩增,评估该方法的特异性;以10倍倍比稀释的重组质粒标准品pMD18-T-BPIV5作为模板,利用本研究建立的qPCR方法扩增,评估该方法的敏感性;以3种不同浓度的质粒标准品作为模板,利用本研究建立的qPCR方法在同一时间和不同时间分别进行批内和批间重复性试验。结果显示,该方法仅能特异性扩增BPIV5,其他相关病毒均为阴性结果;对质粒标准品的检测限为6.43拷贝/μL;批内和批间重复性试验的变异系数均小于2%。利用该方法和已发表的qPCR方法分别检测采自内蒙古东部部分牛场的80份鼻拭子样品,结果显示,两种方法对样品的阳性检测率均为10%(8/80),阴性样品的检测率均为90%(72/80),二者的阳性符合率和总符合率均达100%。本研究建立的检测BPIV5的qPCR方法特异性较强、敏感性较高、重复性较好,可以用于临床样品的检测,为BPIV5的检测和流行病学调查提供了一种新的检测手段。展开更多
The gut microbiota is a complex ecosystem composed of many bacteria and their metabolites.It plays an irreplaceable role in human digestion,nutrient absorption,energy supply,fat metabolism,immune regulation,and many o...The gut microbiota is a complex ecosystem composed of many bacteria and their metabolites.It plays an irreplaceable role in human digestion,nutrient absorption,energy supply,fat metabolism,immune regulation,and many other aspects.Exploring the structure and function of the gut microbiota,as well as their key genes and metabolites,will enable the early diagnosis and auxiliary diagnosis of diseases,new treatment methods,better effects of drug treatments,and better guidance in the use of antibiotics.The identification of gut microbiota plays an important role in clinical diagnosis and treatment,as well as in drug research and development.Therefore,it is necessary to conduct a comprehensive review of this rapidly evolving topic.Traditional identification methods cannot comprehensively capture the diversity of gut microbiota.Currently,with the rapid development of molecular biology,the classification and identification methods for gut microbiota have evolved from the initial phenotypic and chemical identification to identification at the molecular level.This review integrates the main methods of gut microbiota identification and evaluates their application.We pay special attention to the research progress on molecular biological methods and focus on the application of high-throughput sequencing technology in the identification of gut microbiota.This revolutionary method for intestinal flora identification heralds a new chapter in our understanding of the microbial world.展开更多
文摘为建立牛副流感病毒5型(BPIV5)的SYBR Green Ⅰ荧光定量PCR(qPCR)检测方法,本研究经PCR扩增BPIV5 L基因的保守片段,构建重组质粒p MD18-T-BPIV5,并经PCR、双酶切与测序鉴定正确后作为标准品,经各反应条件优化后初步建立检测BPIV5的qPCR方法。利用10倍倍比稀释的质粒标准品作为模板,利用优化的qPCR扩增,建立该方法的标准曲线,结果显示,重组质粒标准品的拷贝数与其Ct值呈良好的线性关系,相关系数为0.998,扩增效率为1.09。以BPIV5、牛肠道病毒(BEV)、牛病毒性腹泻病毒(BVDV)、牛诺如病毒(BNOV)、牛副流感病毒3型(BPIV3)和牛冠状病毒(BCOV)的cDNA作为模板,采用该qPCR扩增,评估该方法的特异性;以10倍倍比稀释的重组质粒标准品pMD18-T-BPIV5作为模板,利用本研究建立的qPCR方法扩增,评估该方法的敏感性;以3种不同浓度的质粒标准品作为模板,利用本研究建立的qPCR方法在同一时间和不同时间分别进行批内和批间重复性试验。结果显示,该方法仅能特异性扩增BPIV5,其他相关病毒均为阴性结果;对质粒标准品的检测限为6.43拷贝/μL;批内和批间重复性试验的变异系数均小于2%。利用该方法和已发表的qPCR方法分别检测采自内蒙古东部部分牛场的80份鼻拭子样品,结果显示,两种方法对样品的阳性检测率均为10%(8/80),阴性样品的检测率均为90%(72/80),二者的阳性符合率和总符合率均达100%。本研究建立的检测BPIV5的qPCR方法特异性较强、敏感性较高、重复性较好,可以用于临床样品的检测,为BPIV5的检测和流行病学调查提供了一种新的检测手段。
文摘The gut microbiota is a complex ecosystem composed of many bacteria and their metabolites.It plays an irreplaceable role in human digestion,nutrient absorption,energy supply,fat metabolism,immune regulation,and many other aspects.Exploring the structure and function of the gut microbiota,as well as their key genes and metabolites,will enable the early diagnosis and auxiliary diagnosis of diseases,new treatment methods,better effects of drug treatments,and better guidance in the use of antibiotics.The identification of gut microbiota plays an important role in clinical diagnosis and treatment,as well as in drug research and development.Therefore,it is necessary to conduct a comprehensive review of this rapidly evolving topic.Traditional identification methods cannot comprehensively capture the diversity of gut microbiota.Currently,with the rapid development of molecular biology,the classification and identification methods for gut microbiota have evolved from the initial phenotypic and chemical identification to identification at the molecular level.This review integrates the main methods of gut microbiota identification and evaluates their application.We pay special attention to the research progress on molecular biological methods and focus on the application of high-throughput sequencing technology in the identification of gut microbiota.This revolutionary method for intestinal flora identification heralds a new chapter in our understanding of the microbial world.
