Establishment And Comparision Of High-throughput Assay For Several Plant Quarantine Viruses

Plant virus can cause huge losses to crops and economic crops yield. Nepovirus include Arabis mosaci virus (ArMV),Tobacco ringspot virus(TRSV) and Tomator ingspot virus(ToRSV). ArMV and TRSV are quarantine virus of second level,and ToRSV is quarantine virus of frist level.Tospovirus is the only one of the bunyaviridae infecting plant,including Tomato spotted wilt virus(TSWV),Impatiens necrotic spot virus(INSV),and iris yellow spot virus (IYSV).Plant viruses unevenly distribution,low content, and the sampling limitations will affect the detection sensitivity.In the study,lily bulbs imported adding ArMV were chosen as experimental materials for two differen pre-treatment methods—immunomagnetic beads adsorption and PEG precipitation.The result shows that pretreatment can greatly enriched plant virus improving the detection rate. In this experiment,PEG precipitation effect is better than that of immunomagnetic beads adsorption.This study uses ily bulb infected with ArMV, ToRSV and TRSV purchased from the Agdia company as the experimental material.Several pairs of primers were synthesised according to the CP gene of ArMV, ToRSV and TRSV for single PCR,optimizing reaction conditions for each prime and verifying the specificity and sensitivity. On the basis of single PCR,by properly assembling the three pairs of virus-specific primers and regulating the conditions of the multiple PCR reactions, the conditions of the multiple PCR reactions and SYBR-Green PCR was determined, which shoren testing time, simplifies operation steps. Multiple PCR reactions and SYBR-Green PCR have strong application value suitable for rapid detection in the port and have broad application prospects.In addition, a diplex liquichip assay for TSWV, INSV and IYSV was established in this study.Polyclonal antibodies of virus were conjugated to fluorescent beads produced by Luminex corporation. The fluorescent assay system was comprised of streptavidin R-phycoerythrin and the other monoclonal antibodies biotinylated. The Luminex200was used to measure fluorescent intensity. The results showed that several viruses could be detected in the same tube without cross-reaction.By comparing the sensitivity of the liquichip with those of DAS-ELISA,the results showed that the liquichip was100and10times more sensitive than DAS-ELISA.So the liquichip technique has a wide application prospectin the detection of viruses, and the establishment ofthis method plays a significant role in further research.