Objective:To explore a method to solve the issue of interference in fluorescence quantitative PCR non-specific amplification for gene detection.Method:A three-step method was used for amplification,and the quantitativ...Objective:To explore a method to solve the issue of interference in fluorescence quantitative PCR non-specific amplification for gene detection.Method:A three-step method was used for amplification,and the quantitative fluorescence signal collection process was set in the extension stage.Results:Three-step amplification has the advantages of wide application range;improved accuracy;and reduced primer design requirements.Conclusion:The interference of non-specific amplification signals was effectively avoided,the melting curve plotting process was omitted,the reaction time was shortened,and the detection accuracy was improved.展开更多
[ 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
采用实时荧光定量PCR技术,以蜜蜂β-act in基因为内参,利用特异引物对滇南中蜂蜂王的卵黄原蛋白基因进行了转录水平上的定量分析。结果表明:所建立的SYBR Gr een-II实时荧光定量PCR技术扩增效率E=99.9%,R 2=0.99,最低检出量为13个循环...采用实时荧光定量PCR技术,以蜜蜂β-act in基因为内参,利用特异引物对滇南中蜂蜂王的卵黄原蛋白基因进行了转录水平上的定量分析。结果表明:所建立的SYBR Gr een-II实时荧光定量PCR技术扩增效率E=99.9%,R 2=0.99,最低检出量为13个循环。检测灵敏度高,能准确定量滇南中蜂初生蜂王中的Vg基因。展开更多
文摘Objective:To explore a method to solve the issue of interference in fluorescence quantitative PCR non-specific amplification for gene detection.Method:A three-step method was used for amplification,and the quantitative fluorescence signal collection process was set in the extension stage.Results:Three-step amplification has the advantages of wide application range;improved accuracy;and reduced primer design requirements.Conclusion:The interference of non-specific amplification signals was effectively avoided,the melting curve plotting process was omitted,the reaction time was shortened,and the detection accuracy was improved.
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.
文摘采用实时荧光定量PCR技术,以蜜蜂β-act in基因为内参,利用特异引物对滇南中蜂蜂王的卵黄原蛋白基因进行了转录水平上的定量分析。结果表明:所建立的SYBR Gr een-II实时荧光定量PCR技术扩增效率E=99.9%,R 2=0.99,最低检出量为13个循环。检测灵敏度高,能准确定量滇南中蜂初生蜂王中的Vg基因。
