Establishment of semi-nested real-time fluorescence quantitative RT-PCR method for the detection of tomato ringspot virus
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 am-plification 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 cD-NA 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 theoccurrence 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.
万方数据
维普
