[Objective]To investigate the expression of zebrafish vascular endothelial growth factor-2（VEGFR-2） at different developmental stages.[Method]Total RNAs were extracted from 12,24,48,72 and 96 hpf stage zebrafish emb...[Objective]To investigate the expression of zebrafish vascular endothelial growth factor-2（VEGFR-2） at different developmental stages.[Method]Total RNAs were extracted from 12,24,48,72 and 96 hpf stage zebrafish embryos and larvae.Real-time quantitative RT-PCR was performed to examine the expression of VEGFR-2.The data were analyzed by 2^-△△Ct method.[Result]The expression level of VEGFR-2 gene increased gradually from 12 to 72 hpf,and subsequently decreased at 96 hpf.The expression level was lowest at 12 hpf,highest at 72 hpf,and had significant differences when compared with that of other developmental stages.[Conclusion]The expression level of VEGFR-2 increases gradually before blood vessel maturation and decreases as blood vessels mature.展开更多
Real-time fluorescent quantitative PCR （RQ-PCR） is a detection method by adding fluorescent dye or fluorescent probe into the PCR reaction system, using fluorescent signal accumulation to monitor amplification react...Real-time fluorescent quantitative PCR （RQ-PCR） is a detection method by adding fluorescent dye or fluorescent probe into the PCR reaction system, using fluorescent signal accumulation to monitor amplification reactions of PCR reaction process, and finally the unknown template can be quantitatively analyzed through the standard curve. So the detection level of PCR has improved from the qualitative to the quantitative. In order to provide a theoretical reference for further application, the principle, classification, advantages and disadvantages of RQ-PCR were intro- duced, and its application and progress in plants in recent years were reviewed.展开更多
仙人掌X病毒(cactuSviruSX,CVX)严重威胁世界火龙果产业的发展。为实现对该病毒的定量检测,本研究根据CVX外壳蛋白(CP)的保守序列设计特异性引物CVX-F/-R,建立了引物浓度150 nmol/L,退火温度62℃的SYBR Green I实时荧光定量PCR检测方法...仙人掌X病毒(cactuSviruSX,CVX)严重威胁世界火龙果产业的发展。为实现对该病毒的定量检测,本研究根据CVX外壳蛋白(CP)的保守序列设计特异性引物CVX-F/-R,建立了引物浓度150 nmol/L,退火温度62℃的SYBR Green I实时荧光定量PCR检测方法。该引物扩增效率为97.38%,R^(2)为0.9965,对标准品的检测极限浓度为8×10^(2)拷贝/μL,其灵敏度是普通PCR的100倍。对火龙果不同组织中CVX的相对表达量进行检测发现,病毒在花丝中的含量最高,之后依次为花萼、花瓣、嫩枝、老枝和根。对采自贵州黔南州的40份火龙果样品进行检测,共检测出32份阳性样品。上述结果表明,本研究建立的实时荧光定量PCR特异性强、灵敏度高,为今后CVX的检测提供了重要的技术补充。展开更多
目的建立炭疽芽孢杆菌的微滴式数字聚合酶链式反应(Droplet digital PCR,ddPCR)方法对炭疽芽孢杆菌实验室活动污染的定量评估提供技术支持。方法以炭疽芽孢杆菌pXO1质粒编码保护性抗原pagA基因为靶序列,优化微滴数字PCR方法的反应条件,...目的建立炭疽芽孢杆菌的微滴式数字聚合酶链式反应(Droplet digital PCR,ddPCR)方法对炭疽芽孢杆菌实验室活动污染的定量评估提供技术支持。方法以炭疽芽孢杆菌pXO1质粒编码保护性抗原pagA基因为靶序列,优化微滴数字PCR方法的反应条件,建立实验室微环境中炭疽芽孢杆菌核酸定量方法;对比微滴式数字PCR方法和平板计数法的定量评估效果,分析ddPCR的灵敏性、特异性和重复性。结果建立的ddPCR方法最佳引物和探针终浓度分别为900nmol·L^(-1)和250nmol·L^(-1),最佳退火温度为60℃,最佳升降温速度为1℃/s,本方法的最低检测下限为1.12copies·μL^(-1),未发现与常见疫病存在交叉反应,重复性试验的变异系数小于5%。结论本研究中建立的炭疽芽孢杆菌的微滴数字PCR方法敏感性高、特异性强、重复性好,为疫情监测、流行病学调查和实验室污染微环境检测提供重要技术。展开更多
[Objective] This study aimed to develop a quantitative competitive assay for detecting Mycoplasma hyopneumoniae in culture. [Method] One pair of Mhp-specific primers was designed for detecting Mhp in culture. Another ...[Objective] This study aimed to develop a quantitative competitive assay for detecting Mycoplasma hyopneumoniae in culture. [Method] One pair of Mhp-specific primers was designed for detecting Mhp in culture. Another pair of primers was designed based on the conserved gene sequences of Mycoplasma. The competitive template, which carried the same primer binding site with the target fragment, was constructed using enzyme digestion method. [Result] The logarithm of concentration of competitive template was treated as the abscissa（X-axis）, and the logarithm of corrected optical density ratio between amplification products by competitive template and target template was treated as the ordinate（Y-axis）. Thus the standard curve was drawn, and the regression equation was also obtained. When Y was assigned as 0, the concentration of the competitive plate was calculated, and then the concentration of Mhp was deduced. The logarithms of Color change unit（CCU） were treated as the abscissa（X-axis）, and the copy numbers of Mhp were treated as the ordinate（Y-axis）, so the standard curve was generated. It was found that the copy number of Mhp was highly correlated to CCU. [Conclusion] A quantitative competitive PCR assay was successfully established for the rapid detection of Mhp in culture.展开更多
[Objective] To explore the feasibility of using SYBR Green real-time quantitative PCR technique to estimate the copy numbers of exogenous gene in a transgenic plant.[Methods] Using SYBR Green real-time quantitative PC...[Objective] To explore the feasibility of using SYBR Green real-time quantitative PCR technique to estimate the copy numbers of exogenous gene in a transgenic plant.[Methods] Using SYBR Green real-time quantitative PCR technique,we have determined the copy numbers of the exogenous CYCD3;1 in transgenic Arabidopsis by comparing an endogenous single copy reference gene with CYCD3;1 copy numbers in transgenic plant,meanwhile comparing CYCD3;1 copy numbers between wild plant and transgenic plant.[Results]The exogenous CYCD3;1 copy numbers calculated by this method is identical with results of traditional Southern blot analysis which is highly accurate.[Conclusion]This method is simple,effective and safe for estimating transgene copy numbers.展开更多
基金Supported by National Natural Science Foundation of Shandong Province (No. SY2008C179)~~
文摘[Objective]To investigate the expression of zebrafish vascular endothelial growth factor-2（VEGFR-2） at different developmental stages.[Method]Total RNAs were extracted from 12,24,48,72 and 96 hpf stage zebrafish embryos and larvae.Real-time quantitative RT-PCR was performed to examine the expression of VEGFR-2.The data were analyzed by 2^-△△Ct method.[Result]The expression level of VEGFR-2 gene increased gradually from 12 to 72 hpf,and subsequently decreased at 96 hpf.The expression level was lowest at 12 hpf,highest at 72 hpf,and had significant differences when compared with that of other developmental stages.[Conclusion]The expression level of VEGFR-2 increases gradually before blood vessel maturation and decreases as blood vessels mature.
基金Supported by National Natural Science Foundation of China(31260406)Natural Science Fund Project of Inner Mongolia(2012MS0502)~~
文摘Real-time fluorescent quantitative PCR （RQ-PCR） is a detection method by adding fluorescent dye or fluorescent probe into the PCR reaction system, using fluorescent signal accumulation to monitor amplification reactions of PCR reaction process, and finally the unknown template can be quantitatively analyzed through the standard curve. So the detection level of PCR has improved from the qualitative to the quantitative. In order to provide a theoretical reference for further application, the principle, classification, advantages and disadvantages of RQ-PCR were intro- duced, and its application and progress in plants in recent years were reviewed.
文摘仙人掌X病毒(cactuSviruSX,CVX)严重威胁世界火龙果产业的发展。为实现对该病毒的定量检测,本研究根据CVX外壳蛋白(CP)的保守序列设计特异性引物CVX-F/-R,建立了引物浓度150 nmol/L,退火温度62℃的SYBR Green I实时荧光定量PCR检测方法。该引物扩增效率为97.38%,R^(2)为0.9965,对标准品的检测极限浓度为8×10^(2)拷贝/μL,其灵敏度是普通PCR的100倍。对火龙果不同组织中CVX的相对表达量进行检测发现,病毒在花丝中的含量最高,之后依次为花萼、花瓣、嫩枝、老枝和根。对采自贵州黔南州的40份火龙果样品进行检测,共检测出32份阳性样品。上述结果表明,本研究建立的实时荧光定量PCR特异性强、灵敏度高,为今后CVX的检测提供了重要的技术补充。
文摘目的建立炭疽芽孢杆菌的微滴式数字聚合酶链式反应(Droplet digital PCR,ddPCR)方法对炭疽芽孢杆菌实验室活动污染的定量评估提供技术支持。方法以炭疽芽孢杆菌pXO1质粒编码保护性抗原pagA基因为靶序列,优化微滴数字PCR方法的反应条件,建立实验室微环境中炭疽芽孢杆菌核酸定量方法;对比微滴式数字PCR方法和平板计数法的定量评估效果,分析ddPCR的灵敏性、特异性和重复性。结果建立的ddPCR方法最佳引物和探针终浓度分别为900nmol·L^(-1)和250nmol·L^(-1),最佳退火温度为60℃,最佳升降温速度为1℃/s,本方法的最低检测下限为1.12copies·μL^(-1),未发现与常见疫病存在交叉反应,重复性试验的变异系数小于5%。结论本研究中建立的炭疽芽孢杆菌的微滴数字PCR方法敏感性高、特异性强、重复性好,为疫情监测、流行病学调查和实验室污染微环境检测提供重要技术。
基金Supported by Jiangsu Agricultural Science and Technology Innovation Fund[CX(133066]~~
文摘[Objective] This study aimed to develop a quantitative competitive assay for detecting Mycoplasma hyopneumoniae in culture. [Method] One pair of Mhp-specific primers was designed for detecting Mhp in culture. Another pair of primers was designed based on the conserved gene sequences of Mycoplasma. The competitive template, which carried the same primer binding site with the target fragment, was constructed using enzyme digestion method. [Result] The logarithm of concentration of competitive template was treated as the abscissa（X-axis）, and the logarithm of corrected optical density ratio between amplification products by competitive template and target template was treated as the ordinate（Y-axis）. Thus the standard curve was drawn, and the regression equation was also obtained. When Y was assigned as 0, the concentration of the competitive plate was calculated, and then the concentration of Mhp was deduced. The logarithms of Color change unit（CCU） were treated as the abscissa（X-axis）, and the copy numbers of Mhp were treated as the ordinate（Y-axis）, so the standard curve was generated. It was found that the copy number of Mhp was highly correlated to CCU. [Conclusion] A quantitative competitive PCR assay was successfully established for the rapid detection of Mhp in culture.
基金Supported by National Natural Science Foundation Project(30270086)~~
文摘[Objective] To explore the feasibility of using SYBR Green real-time quantitative PCR technique to estimate the copy numbers of exogenous gene in a transgenic plant.[Methods] Using SYBR Green real-time quantitative PCR technique,we have determined the copy numbers of the exogenous CYCD3;1 in transgenic Arabidopsis by comparing an endogenous single copy reference gene with CYCD3;1 copy numbers in transgenic plant,meanwhile comparing CYCD3;1 copy numbers between wild plant and transgenic plant.[Results]The exogenous CYCD3;1 copy numbers calculated by this method is identical with results of traditional Southern blot analysis which is highly accurate.[Conclusion]This method is simple,effective and safe for estimating transgene copy numbers.
