实时荧光定量PCR技术的概述及应用     

梁子英  刘芳 《现代农业科技》2020,(6)

实时荧光定量PCR技术是在传统PCR技术上发展而来的,不仅能判断某一基因的有无,而且还能对其进行定量分析。由于该技术与常规PCR相比,能够进行实时监测、且自动化程度高而被广大研究者青睐。本文对实时荧光定量PCR的原理、数据分析、定量方法、分类以及应用进行了系统而详细地综述。  相似文献   

文冠果实时荧光定量PCR内参基因的筛选     

王旭  敖妍  刘阳  张宁  郑雅琪  刘金凤  张行杰  张永明 《分子植物育种》2020,(9):2977-2986

为筛选适用于文冠果(Xanthoceras sorbifolium Bunge)性别分化过程研究和不同器官的内参基因,本研究以文冠果性别分化关键期的顶芽和侧芽、根、茎、叶、种仁、雌能花和雄能花为材料,通过实时荧光定量PCR(qRT-PCR)鉴定7个候选内参基因(Actin,UBQ,EF-1α,TUB,eIF-4α,18S rRNA和GAPDH)在各样品中的表达量,并评价7个基因的表达稳定性。结果表明,7个候选内参基因在不同样品中的表达量存在明显差异,各基因在雌能花中的表达均显著高于其他材料,在雄能花中的表达处于较低水平;适用于文冠果性别分化过程的最优内参基因为UBQ和eIF-4α,适用于不同器官的最优内参基因为EF-1α和GAPDH。研究结果将对今后进行文冠果性别分化过程研究和不同器官中的基因表达分析提供参考。  相似文献   

EMS诱导水稻‘一目惚’表型变异和部分基因表达变异的比较     

张安星  孟卫隆  李喆  吴楠  刘威  马建 《分子植物育种》2020,(2):488-494

本研究利用甲基磺酸乙酯(Ethy methan sulfonate)试剂胁迫诱导粳稻品种‘一目惚’(Hitomebore),获得性状稳定遗传的水稻矮杆突变体(E58),对株高、分蘖、千粒重等表现与相同环境生长下的野生型‘一目惚’对照(E63)进行对比分析,结果表明,该突变体田间表现为植株矮化,分蘖增多,千粒重减少,且株高与对照相比呈极显著差异,分蘖呈显著差异,千粒重呈极显著差异。EMS诱导的序列变异可能对基因表达产生影响,进而引起水稻矮化的表型变异。根据前人试验结果,应用Real-time PCR对与胁迫及矮化相关的39对引物中筛选出24对引物进行Real-Time PCR扩增,结果表明:引物OsNR1、OsP5CS、OsGS1;1、OsGDH1、OsHKT1;5、OsNRT1;1、OsGDH3、OsAKT1、OsAMT2;3、OsGS1;2、OsSOS1、OsNHX2、OsNRT2;1、OHAK16、OsGS2、OsHAK1、OsGDH2、OsAMT3;3在E63、E58表达差异极显著;OsHAK10、OsNHX1、OsAMT1;1表达差异显著;OsNADHGOGAT、OsFd-GOGAT、OsGS1;3表达差异不显著。EMS诱导对部分水稻基因表达产生影响,从而影响水稻表现型,为进一步工作提供科学依据。  相似文献   

纳米磁珠联合PCR检测甘蔗杆状病毒方法的建立和初步应用     

李书巧  王坤  陈开闯  王凤林  陈保善  温荣辉 《分子植物育种》2020,(7):2268-2272

本研究通过对DNA提取裂解液的成分和用量以及裂解时间等因素进行系统优化,建立了一种高通量的磁珠法提取甘蔗总DNA,再进行PCR检测甘蔗杆状病毒(SCBV)的方法。采用核酸自动提取仪,应用纳米磁珠提取甘蔗总DNA,经PCR检测甘蔗杆状病毒,并与以试剂盒提取DNA为模板的PCR检测结果进行比较。结果显示,经优化的裂解液成分为EDTA·Na20.08 mol/L、NaCl 0.8 mol/L、SDS 2%和Tris-HCl0.10 mol/L,裂解时间为20 min,可用于纳米磁珠提取甘蔗总DNA进行SCBV检测。所建立的方法通量高、成本低、耗时短,可用于SCBV的批量检测。  相似文献   

非洲猪瘟病毒锁核酸探针荧光定量PCR检测方法的建立     

李艳  郭怡德  勾红潮  卞志标  孙铭飞  蔡汝健  宋帅  蒋智勇  楚品品  徐民生  杨东霞  李春玲 《中国兽医学报》2021,(2):208-212,230

根据非洲猪瘟病毒(African swine fever virus,ASFV)P72基因核苷酸序列设计特异性引物和锁核酸(locked nucleic acid,LNA)-TaqMan探针,建立了基于P72基因的LNA-TaqMan探针的ASFV荧光定量PCR方法.结果显示,所建立的LNA-TaqMan探针荧光定量P...  相似文献   

牛星状病毒EvaGreen实时荧光定量PCR检测方法的建立及应用     

师志海  王文佳  张彬  孟红丽  王亚州  金磊  兰亚莉  徐照学 《中国兽医学报》2021,(1):50-55

牛星状病毒(BAstV)是我国新发的犊牛腹泻病原,本试验的目的是建立检测BAstV的Real-time PCR方法.根据BAstV流行株的ORFla基因序列设计引物,通过优化反应条件和体系,成功建立基于EvaGreen检测BAstV的Real-time PCR方法.结果表明,该检测方法的Ct值与标准品模板在1.36×1...  相似文献   

High vegetative compatibility diversity of Cryphonectria parasitica infecting sweet chestnut (Castanea sativa) in Britain indicates multiple pathogen introductions     

A. Pérez-Sierra  P. Romón-Ochoa  C. Gorton  A. Lewis  H. Rees  S. Van Der Linde  J. Webber 《Plant pathology》2019,68(4):727-737

Chestnut blight, caused by Cryphonectria parasitica, was identified in Devon, UK, in December 2016. Intensive surveys detected the disease at further sites in Devon (seven), Berkshire (one), Dorset (one), Derbyshire (four) and a cluster of eight sites in southeast London. Over 570 survey samples were tested, and 227 were positive for C. parasitica by isolation and real-time PCR. A total of 227 isolates were tested for mating type, and 197 screened for vegetative compatibility group (VCG) and compared with VCGs known from mainland Europe. The same isolates were also screened for the presence of Cryphonectria hypovirus 1 (CHV-1). Eleven VCGs were identified within the UK population. Five corresponded to already known European VCGs but six were unique. The European VCGs mainly came from the Devon, Dorset, Berkshire and Derbyshire disease outbreaks, whilst unique VCGs were almost exclusively from the southeast London cluster. Both mating types were detected, but only one mating type was present at each site, with the exception of a single Devon site. Perithecia of C. parasitica were never observed at any site. CHV-1 was found in seven isolates from three different locations and was always subtype-I, which has limited hypovirulence. Therefore, although CHV-1 is associated with C. parasitica at some outbreaks, it probably has limited impact on virulence. The diversity of VCGs and their distribution at outbreak sites, together with findings of CHV-1, suggests C. parasitica has been introduced to the UK multiple times over at least two decades through international plant trade.  相似文献   

魔芋软腐病病原菌TaqMan荧光探针PCR技术的建立及应用     

卢美欢  李利军  马英辉  王晓兵  郭邦利  王晓娥 《植物保护学报》2019,46(5):1100-1109

为实现对田间土壤软腐病病原菌的定量检测,基于魔芋软腐病优势病原菌胡萝卜软腐果胶杆菌胡萝卜亚种Pectobacterium carotovorum subsp. carotovorum的FyuA基因序列,设计特异性引物PCC1/PCC2/PCC3,建立TaqMan荧光探针实时荧光定量PCR技术,并对魔芋根系土壤中软腐病病原菌进行动态监测。结果显示:基于FyuA基因序列设计的引物特异性好,仅能特异性检出胡萝卜软腐果胶杆菌胡萝卜亚种;当模拟带菌土壤中病原菌浓度低至1.88 CFU/g时也能检出,灵敏度高;发病魔芋根际土壤中软腐病病原菌检出率为100.00%,病原菌DNA浓度最高达到了7.52×10~7ng/μL,健康魔芋根际土壤中也存在病原菌,检出率为40.00%;不同种植模式中,林下魔芋土壤中软腐病病原菌数量较少;连作时间与病原菌数量、病情指数存在正相关关系,连作时间越长,病原菌积累越多,魔芋病情指数也越高,魔芋连作4年土壤中病原菌DNA浓度最高达到4.03×10~4ng/μL;对魔芋土壤软腐病病原菌进行全年监测,病原菌数量随着月份增长逐渐上升,在8—10月达到峰值543.20 ng/μL后下降,病原菌数量与魔芋病情指数变化规律一致,但田间魔芋软腐病的发生相对滞后。表明建立的TaqMan荧光探针实时荧光定量PCR技术可用于田间魔芋软腐病的监测。  相似文献   

Comparison of dot blot and PCR diagnostic techniques for detection of white spot syndrome virus in different tissues of Penaeus monodon     

M.S. Shekhar  I.S. Azad  P. Ravichandran 《Aquaculture (Amsterdam, Netherlands)》2006,261(4):1122-1127

Comparison of PCR and dot blot diagnostic techniques for detection of white spot syndrome virus (WSSV) was made on different tissues of infected Penaeus monodon including eye stalk, eye stalk with eye, gills, cuticle, pleopod, periopods, uropods and telson. Dot blots of crude DNA extracted from infected tissue samples showed positive reactions with all the samples; however, the sensitivity of the dot blot was reduced with the purification of DNA samples extracted from pleopod, telson and uropod. PCR was found to be more sensitive when compared to dot blot. Both crude DNA and purified DNA samples extracted from all the tissues except for eye stalk with eye showed single step nested PCR positive reaction. The amplification of all or either of the three bands of 941 bp, 525 bp and 204 bp size varied with the tissues analysed. The severity of infection assessed by PCR amplification was found to be maximum in cuticle and telson followed by gill. Other tissues such as eye stalk, pleopod, periopods and uropod were observed to have mild infection. The maximum intensity of the PCR product was for the smallest amplified product of 204 bp followed by 525 bp and the weakest intensity was observed for the 941 bp size. The limitation of PCR due to inhibiting factors present in tissues could be overcome with the use of dot blot which gave positive reaction from the DNA extracted from eye stalk including the eye but yielded no amplification by PCR.  相似文献   

显微注射生长激素基因导入中国对虾（Penaeus chinensis）受精卵的研究   总被引：4，自引：0，他引：4  

刘萍  孔杰  王清印  李健  杨丛海  梁利群  孙孝文 《中国水产科学》1996,3(4):35-38

本实验采用显微微量注射方法，交羊生长激素基因导入中轻地虾受精卵，受精卵发育到蚤状幼体第三期后采样检测。ＰＣＲ检测结果表明：７个样品共９３尾幼体，有３个样品呈出阳性信号，基因转移比率至少在３％以上。同时斑点杂交结果也证明有两个明显阳性斑点。  相似文献