Apple stem grooving virus(ASGV),apple stem pitting virus(ASPV),and apple necrotic mosaic virus(ApNMV)pose significant hazards to the growth and development of fruit trees,and the mixed infection rate of apple virus is high.To achieve rapid,sensitive,and efficient detection of these three viruses,we designed specific primers based on the conserved sequences of ASPV,ASGV,and ApNMV genomes,and established a highly sensitive real-time fluorescence quantitative PCR detection system(RT-qPCR)for the above three viruses.The results showed that the primers ASGV-qF/qR-1,ASPV-qF/R-2 and ApNMV-qF/R-3 displayed high specificity,and the optimal annealing temperatures were 60 ℃,58 ℃,and 58 ℃,respectively.The RT-qPCR system for ASGV,ASPV and ApNMV was 100 times more sensitive than the conventional RT-PCR detection system with minimum detection limits of 82.6,1.49× 102 and 13.3 copies/μL,respectively.The coefficients of variation of the Ct values of the detection system were all less than 5％ ,and the coefficients of variation between groups were within 5％ .The detection rate of RT-qPCR for 88 apple trees with viruses confirmed by RT-PCR in the orchard of Hebei Agricultural University were 100％ .This suggested that the RT-qPCR method we established were highly reliable and stable and is suitable for the detection of three viruses in field apple trees,providing technical support for accurate diagnosis of apple viruses.
关键词
苹果茎沟病毒/苹果茎痘病毒/苹果坏死花叶病毒/荧光定量RT-PCR检测体系
Key words
apple stem grooving virus/apple stem pitting virus/apple necrotic mosaic virus/fluorescence quantitative RT-PCR detection system
维普
万方数据