[ 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
核酸扩增反应能够在短时间内将特定DNA模板的数量增加106~109倍,显著提高微量核酸检测的灵敏度,但同时扩增产物的遗漏极易造成交叉污染,影响后续检测。扩增反应通过添加荧光染料实现的实时闭管检测,不仅可以避免反应结束后开管操作所导...核酸扩增反应能够在短时间内将特定DNA模板的数量增加106~109倍,显著提高微量核酸检测的灵敏度,但同时扩增产物的遗漏极易造成交叉污染,影响后续检测。扩增反应通过添加荧光染料实现的实时闭管检测,不仅可以避免反应结束后开管操作所导致的交叉污染,还可以通过反应动力学对样品中模板的起始拷贝数定量,以及通过熔解曲线分析鉴别多种靶标来源、进行基因突变扫描和基因分型检测。目前常用的商业化实时检测荧光染料,如SYBR Green I、Eva Green等,其精确结构信息尚未披露,因此只能通过实验“试错法”选取合适染料,无法从根本上获知染料结构和分析性能之间的关系。本文将通过对目前常用的核酸实时检测荧光染料性质和应用进行总结,为其优化选择、设计和开发提供参考依据。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.
文摘该研究针对单增李斯特氏菌的特异性基因hlyA设计引物,优化反应条件,建立实时荧光环介导等温扩增(realtime loop-mediated isothermal amplification,real-time LAMP)的检测方法,在此基础上,建立检测单增李斯特氏菌的基于淬灭基团释放环介导等温扩增检测方法(detection of amplification by release of quenching-LAMP,DARQ-LAMP),分析其特异性和灵敏度。结果表明,该方法的适宜反应条件为反应温度63℃、Bst DNA 3.0聚合酶0.32 U/μL、淬灭探针双链(quenching probe double chain,QPD)探针浓度10%、Mg2+浓度6 mmol/L;对单增李斯特菌的检测限为7.3×101copies/mL,灵敏度是普通聚合酶链式反应(polymerase chain reaction,PCR)的100倍。该方法效率高、特异性好、灵敏度高,为环介导等温扩增技术检测食源性致病菌的研究提供参考。
文摘核酸扩增反应能够在短时间内将特定DNA模板的数量增加106~109倍,显著提高微量核酸检测的灵敏度,但同时扩增产物的遗漏极易造成交叉污染,影响后续检测。扩增反应通过添加荧光染料实现的实时闭管检测,不仅可以避免反应结束后开管操作所导致的交叉污染,还可以通过反应动力学对样品中模板的起始拷贝数定量,以及通过熔解曲线分析鉴别多种靶标来源、进行基因突变扫描和基因分型检测。目前常用的商业化实时检测荧光染料,如SYBR Green I、Eva Green等,其精确结构信息尚未披露,因此只能通过实验“试错法”选取合适染料,无法从根本上获知染料结构和分析性能之间的关系。本文将通过对目前常用的核酸实时检测荧光染料性质和应用进行总结,为其优化选择、设计和开发提供参考依据。
