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实时荧光定量PCR技术在植物检疫中应用的研究进展

实时荧光定量PCR技术在植物检疫中应用的研究进展

摘要:实时荧光定量PCR技术是在PCR反应体系中加入荧光染料或荧光探针,利用荧光信号积累实时监测整个PCR反应进程,最后通过标准曲线对未知模板进行定量分析,具有操作简便?快速高效?高通量?高敏感性等特点?近年来,实时荧光定量PCR技术在植物检疫研究上不断深入,大大提高了植物疫情的检测效率和监测防治水平?综合论述了实时荧光定量PCR技术在由真菌?细菌?病毒和线虫引发的植物疫情的检测与应用?

关键词:实时荧光定量PCR技术;植物检疫;植物病害

Research Progress of Application of Real Time Fluorescent Quantitative PCR Technology in Plant Quarantine

Abstract: FQ-PCR involves the process of adding fluorescent dye or fluorescent probes in the PCR reaction system. Based on the accumulation of fluorescent signal, the polymerase chain reaction (PCR) process could be monitored in order to quantitatively analyze unknown template by means of making standard curve. FQ-PCR has good features such as simplicity of operation with high efficiency, high throughput, and high sensitivity etc. In recent years, application of FQ-PCR in plant quarantine was studied and improved the detecting efficiency and monitoring level of plant epidemic. The application of quantitative fluorescence detection in plants epidemic caused by fungi, bacteria, viruses and nematodes was comprehensively described.

Key words: real-time fluorescent quantitative PCR; plant quarantine; plant disease

实时荧光定量PCR(Real-time fluorescent quantitative PCR,FQ-PCR)技术是20世纪90年代中期发展起来的一种新型核酸定量技术?最早是由Higuchi等[1]于1992年提出来的,1996年由美国Applied Biosystems公司推出?与普通PCR相比,它具有快速?灵敏?高通量?特异性强?自动化程度高?重复性好?定量准确等特点?过去实时荧光定量PCR技术主要应用在医学诊断?新型农业?基础研究等领域[2],而在植物检疫中的应用相对较少[3],随着分子生物学的不断发展,特别是在检疫检验系统,实时荧光定量PCR技术已经被广泛用来检测植物病害,有效地遏制了国外有害生物的入侵,保证了生物入境安全?

1 实时荧光定量PCR技术原理

实时荧光定量PCR技术是指在PCR反应体系中加入荧光基团,利用荧光信号累积实时监测整个PCR技术进程,最后通过标准曲线对未知模板进行定量分析的方法?在实时荧光定量PCR技术中有一个很重要的概念,即Ct值?C代表循环(Cycle),t代表阈值(Threshold)?Ct值是指每个反应管内的荧光信号到达设定的阈