[ Objective] This study aimed to establish a simultaneous detection method of shrimp viruses by real-time fluorescence quantitative RT-PCR, to improve the efficiency of inspection and quarantine. [ Method] A novel rea...[ Objective] This study aimed to establish a simultaneous detection method of shrimp viruses by real-time fluorescence quantitative RT-PCR, to improve the efficiency of inspection and quarantine. [ Method] A novel real-time fluorescence quantitative RT-PCR assay was established and optimized for simultaneously detecting DNA/RNA of four shrimp viruses （WSSV, IHHNV, TSV and YHV ）. [ Result] The optimized real-time fluorescence quantitative RT-PCR system gener- ated typical amplification curves with high amplification efficiencies （E = 1.06, 1.07, 0.92 and 0.92, respectively）, good hnear relationship （ r = 1 ）, uniform repeatability （ standard deviation = 0.05 - 0.46 ; variation coefficient = 0.26% - 1.62% ） and high sensitivity, exhibiting no significant differences compared with re- al-time fluorescence quantitative PCR （average error of Ct value = 0.04 -0.40; T = 0.53 -2.50; P 〉 0.05 ）. The total detection time was about 1 h. [ Conclusion] The optimized real-time fluorescence quantitative RT-PCR system can be used for rapid detection of WSSV, IHHNV, TSV and YHV.展开更多
AtERF4 （ethylene response factor） is a negative regulator in jasmonic acid mediated signal transduction pathway and ethylene mediated signal transduction pathway of Arabidopsis. It could respond to abscisic acid （...AtERF4 （ethylene response factor） is a negative regulator in jasmonic acid mediated signal transduction pathway and ethylene mediated signal transduction pathway of Arabidopsis. It could respond to abscisic acid （ABA） and ethylene stimulus ATSYR1 gene encodes a syntaxin localizing at the plasma membrane in Arabidopsis, which can be induced by abiotic stress. To identify mutation lines for gene functional analysis, real-time PCR was employed to detect the expression level of AtERF4 and ATSYR1 in homozygous T-DNA insertion mutant line, respectively. Real-time PCR is a powerful tool which can be used to detect steady-state mRNA levels specifically, sensitively and reproducibly. Comparing to other forms of quantitative RT-PCR, the amount of amplified products can be detected by real-time PCR instantly and thus is a preferable alternative. In this study, RNA with T-DNA inserting into exon could be detected in AtERF4 knock-out mutation line. The results indicated that AtERF4 had been trucked in transcription level. On the other hand, T-DNA inserting into the promoter of gene ATSYR1 had no effect on reducing the expression level ofATSYR1 gene. Further molecular and phenotype studies now are ongoing to clarify the potential consequences of AtERF4 and ATSYR1 deficiency in Arabidopsis展开更多
利用磁珠法对甘蓝、白菜、芥菜、洋葱、黄瓜、番茄、辣椒等7种形状、大小不同的蔬菜单粒种子和幼苗进行DNA提取,采用内参基因Actin进行实时荧光定量PCR评价DNA质量,利用KASP(kompetitive allele specific PCR)标记检验DNA质量是否达到K...利用磁珠法对甘蓝、白菜、芥菜、洋葱、黄瓜、番茄、辣椒等7种形状、大小不同的蔬菜单粒种子和幼苗进行DNA提取,采用内参基因Actin进行实时荧光定量PCR评价DNA质量,利用KASP(kompetitive allele specific PCR)标记检验DNA质量是否达到KASP检测要求。结果显示,单粒种子磁珠法提取的各蔬菜DNA平均Ct值均处于19~24范围内。KASP检测进一步证实种子提取DNA的质量达到KASP标记分型的要求。综上所述,本研究建立了一种简单、经济、实用性强的单粒种子DNA提取方法,该方法对不同大小的蔬菜种子具有较好的稳定性,且大幅缩短了获得基因型数据的时间。展开更多
文摘[ Objective] This study aimed to establish a simultaneous detection method of shrimp viruses by real-time fluorescence quantitative RT-PCR, to improve the efficiency of inspection and quarantine. [ Method] A novel real-time fluorescence quantitative RT-PCR assay was established and optimized for simultaneously detecting DNA/RNA of four shrimp viruses （WSSV, IHHNV, TSV and YHV ）. [ Result] The optimized real-time fluorescence quantitative RT-PCR system gener- ated typical amplification curves with high amplification efficiencies （E = 1.06, 1.07, 0.92 and 0.92, respectively）, good hnear relationship （ r = 1 ）, uniform repeatability （ standard deviation = 0.05 - 0.46 ; variation coefficient = 0.26% - 1.62% ） and high sensitivity, exhibiting no significant differences compared with re- al-time fluorescence quantitative PCR （average error of Ct value = 0.04 -0.40; T = 0.53 -2.50; P 〉 0.05 ）. The total detection time was about 1 h. [ Conclusion] The optimized real-time fluorescence quantitative RT-PCR system can be used for rapid detection of WSSV, IHHNV, TSV and YHV.
基金Supported by National High Technology Program (2008ZX08004-002, 2009ZX08009-032B)Key Research Plan of Heilongjiang Province (GA06B103)Education Department Plan of Heilongjiang Province(11521021, 1152024)
文摘AtERF4 （ethylene response factor） is a negative regulator in jasmonic acid mediated signal transduction pathway and ethylene mediated signal transduction pathway of Arabidopsis. It could respond to abscisic acid （ABA） and ethylene stimulus ATSYR1 gene encodes a syntaxin localizing at the plasma membrane in Arabidopsis, which can be induced by abiotic stress. To identify mutation lines for gene functional analysis, real-time PCR was employed to detect the expression level of AtERF4 and ATSYR1 in homozygous T-DNA insertion mutant line, respectively. Real-time PCR is a powerful tool which can be used to detect steady-state mRNA levels specifically, sensitively and reproducibly. Comparing to other forms of quantitative RT-PCR, the amount of amplified products can be detected by real-time PCR instantly and thus is a preferable alternative. In this study, RNA with T-DNA inserting into exon could be detected in AtERF4 knock-out mutation line. The results indicated that AtERF4 had been trucked in transcription level. On the other hand, T-DNA inserting into the promoter of gene ATSYR1 had no effect on reducing the expression level ofATSYR1 gene. Further molecular and phenotype studies now are ongoing to clarify the potential consequences of AtERF4 and ATSYR1 deficiency in Arabidopsis
文摘利用磁珠法对甘蓝、白菜、芥菜、洋葱、黄瓜、番茄、辣椒等7种形状、大小不同的蔬菜单粒种子和幼苗进行DNA提取,采用内参基因Actin进行实时荧光定量PCR评价DNA质量,利用KASP(kompetitive allele specific PCR)标记检验DNA质量是否达到KASP检测要求。结果显示,单粒种子磁珠法提取的各蔬菜DNA平均Ct值均处于19~24范围内。KASP检测进一步证实种子提取DNA的质量达到KASP标记分型的要求。综上所述,本研究建立了一种简单、经济、实用性强的单粒种子DNA提取方法,该方法对不同大小的蔬菜种子具有较好的稳定性,且大幅缩短了获得基因型数据的时间。
