建立一种快速定量检测混合肉制品中牛肉成分含量的方法。以牛线粒体细胞色素b基因为目的基因,真核生物核糖体18S rRNA为内参基因,设计引物;采用基于SYBR green I的实时荧光PCR技术对已知牛肉添加比例的混合肉样标准品建立标准曲线模型...建立一种快速定量检测混合肉制品中牛肉成分含量的方法。以牛线粒体细胞色素b基因为目的基因,真核生物核糖体18S rRNA为内参基因,设计引物;采用基于SYBR green I的实时荧光PCR技术对已知牛肉添加比例的混合肉样标准品建立标准曲线模型。结果显示,该方法在0.05%-100%区间具有良好的线性和扩增效率;通过熔解曲线验证了具有良好的特异性;通过对模拟样品的验证试验,证实该方法适用于对牛肉含量的检测,对市售加工食品具有较好的应用性。此方法可作为测定混合肉制品中牛肉含量的检测方法。展开更多
Burkholderia glumae causing seedling rot and grain rot of rice was listed as a plant quarantine disease of China in 2007. It＇s quite necessary to set up effective detection methods for the pathogen to manage further ...Burkholderia glumae causing seedling rot and grain rot of rice was listed as a plant quarantine disease of China in 2007. It＇s quite necessary to set up effective detection methods for the pathogen to manage further dispersal of this disease. The present study combined the real-time PCR method with classical PCR to increase the detecting efficiency, and to develop an accurate, rapid and sensitive method to detect the pathogen in the seed quarantine for effective management of the disease. The results showed that all the tested strains of B. glumae produced about 139 bp specific fragments by the real-time PCR and the general PCR methods, while others showed negative PCR result. The bacteria could be detected at the concentrations of 1×10^4 CFU/mL by general PCR method and at the concentrations below 100 CFU/mL by real-time fluorescence PCR method. B. glumae could be detected when the inoculated and healthy seeds were mixed with a proportion of 1：100.展开更多
目的:建立一种用于单核细胞增生李斯特菌的实时荧光赖解旋酶恒温核酸扩增(helicase-dependent isothermal DNA amplification,HDA)快速检测方法。方法:针对单核细胞增生李斯特菌hly基因序列设计引物对,基于荧光定量聚合酶链式反应(polym...目的:建立一种用于单核细胞增生李斯特菌的实时荧光赖解旋酶恒温核酸扩增(helicase-dependent isothermal DNA amplification,HDA)快速检测方法。方法:针对单核细胞增生李斯特菌hly基因序列设计引物对,基于荧光定量聚合酶链式反应(polymerase chain reaction,PCR)仪的平台,提取单核细胞增生李斯特菌基因组DNA,以此作为模板,优化反应温度、反应时间及引物浓度。利用单核细胞增生李斯特菌及10株对照菌株,并与实时荧光PCR对比,来验证实时荧光HDA方法的特异性和灵敏度,并初步用于样品检测。结果:实时荧光HDA体系的最适引物浓度为0.075μmol/L,反应温度及反应时间为65℃、80 min(40个循环),具有良好的特异性和灵敏度。结论:建立了一种特异性强、灵敏度高的实时荧光HDA检测单核细胞增生李斯特菌的方法。展开更多
文摘建立一种快速定量检测混合肉制品中牛肉成分含量的方法。以牛线粒体细胞色素b基因为目的基因,真核生物核糖体18S rRNA为内参基因,设计引物;采用基于SYBR green I的实时荧光PCR技术对已知牛肉添加比例的混合肉样标准品建立标准曲线模型。结果显示,该方法在0.05%-100%区间具有良好的线性和扩增效率;通过熔解曲线验证了具有良好的特异性;通过对模拟样品的验证试验,证实该方法适用于对牛肉含量的检测,对市售加工食品具有较好的应用性。此方法可作为测定混合肉制品中牛肉含量的检测方法。
基金supported by National Natural Science Foundation of China(Grant No.30671397 and No.30871655)the Public Beneficial Research Project of Agricultural Ministry,China(Grant No.nyhyzx07-056)
文摘Burkholderia glumae causing seedling rot and grain rot of rice was listed as a plant quarantine disease of China in 2007. It＇s quite necessary to set up effective detection methods for the pathogen to manage further dispersal of this disease. The present study combined the real-time PCR method with classical PCR to increase the detecting efficiency, and to develop an accurate, rapid and sensitive method to detect the pathogen in the seed quarantine for effective management of the disease. The results showed that all the tested strains of B. glumae produced about 139 bp specific fragments by the real-time PCR and the general PCR methods, while others showed negative PCR result. The bacteria could be detected at the concentrations of 1×10^4 CFU/mL by general PCR method and at the concentrations below 100 CFU/mL by real-time fluorescence PCR method. B. glumae could be detected when the inoculated and healthy seeds were mixed with a proportion of 1：100.
