番茄环斑病毒半巢式荧光定量RT-PCR检测方法的建立

Establishment of semi-nested real-time fluorescence quantitative RT-PCR method for the detection of tomato ringspot virus

为准确高效地检测出植物样品中的番茄环斑病毒,提高检疫效率,本研究结合了巢式PCR技术和实时荧光定量PCR技术,根据番茄环斑病毒外壳蛋白基因保守区序列设计了3条特异性引物和一条TaqMan探针,成功建立了半巢式荧光定量RT-PCR检测番茄环斑病毒的方法。该方法通过两轮实时荧光PCR扩增发现只有在番茄环斑病毒侵染的植物样品中出现荧光扩增曲线,而感染TRSV、SBMV、TSWV、PDV、BPMV的植物样品和健康叶片的c DNA及对照ddH2O未检测到荧光信号,表明该方法具有良好的特异性。根据梯度稀释法测得荧光扩增信号的最低浓度为498 ag/μL植物总RNA,说明该方法灵敏度较高,且重复性良好。两套引物探针的扩增效率对比试验结果表明第一套引物探针扩增效率小于第二套引物探针扩增效率。本研究建立的半巢式荧光定量RT-PCR检测番茄环斑病毒的方法具有特异性强、灵敏度高等特点,通过两轮实时荧光PCR检测相互验证,大大降低了假阳性结果的出现,是一种准确高效的检测方法。

In order to accurately and efficiently detect tomato ringspot virus in plant samples and improve quarantine efficiency,this study combined nested PCR technology and real-time fluorescence quantitative RT-PCR technology.Three specific primers and one TaqMan probe were designed according to the coat protein gene of tomato ringspot virus,and a semi-nested real-time fluorescence quantitative RT-PCR method for detection tomato ringspot virus was established successfully. This method found that fluorescence amplification curves only appeared in plant samples infected with tomato ringspot virus through two rounds of real-time fluorescence quantitative PCR amplification,while no fluorescence signals were detected in cDNA of plant samples infected with TRSV,SBMV,TSWV,PDV,BPMV,healthy leaves,and control DPEC sterile water,indicating that this method has good specificity. The minimum concentration of fluorescence amplification signal measured by gradient dilution method is 498 ag/μL total plant RNA,indicating that the method has high sensitivity and good repeatability. The comparison of the amplification efficiency of two sets of primer probes showed that the amplification efficiency of the first set of primer probes was lower than that of the second set of primer probes. The semi-nested real-time fluorescence quantitative RT-PCR method developed in this study for the detection of tomato ringspot virus was highly specific and high sensitive,which can greatly reduces the occurrence of false positive results through two rounds of real-time fluorescence PCR detection and mutual verification. And it is an accurate and efficient detection method.