3种苹果病毒实时荧光定量RT-PCR检测体系的建立

Establishment of real-time fluorescence PCR detection system for three apple viruses

苹果茎沟病毒(Apple stem grooving virus,ASGV)、苹果茎痘病毒(Apple stem pitting virus,ASPV)和苹果坏死花叶病毒(Apple necrotic mosaic virus,ApNMV)对果树的生长发育危害严重,而且复合侵染几率较高,为快速、灵敏和高效的检测3种病毒,本研究根据ASPV、ASGV和ApNMV基因组保守序列设计特异引物建立了3种病毒高灵敏度的实时荧光定量PCR检测体系(Real-time fluorescence quantitative reverse transcription PCR,RT-qPCR)。结果表明:引物ASGV-qF/qR-1、ASPV-qF/R-2和ApNMV-qF/R-3有较高的特异性,最适宜的退火温度分别为60℃、58℃和58℃,ASGV、ASPV和ApNMV 3种病毒的RT-qPCR体系比常规RT-2 PCR检测体系灵敏度高100倍,最低检出限分别为82.6、1.49×10^(2)和13.3 copies/μL。检测体系的Ct值的变异系数均小于5%,组间的变异系数在5%以内。河北农业大学果园中经RT-PCR确定带毒的88棵苹果树RT-qPCR检出率为100%,表明建立的RT-qPCR方法的可靠性和稳定性高,适用于田间果树3种病毒的检测,为苹果病毒的准确诊断提供了技术支撑。

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°C,58°C,and 58°C,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 82.6,21.49×10^(2)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.