The Studies On The Detection Of Five Quarantine Plant Viruses By Nucleic Acid Dipstick Assay

With the acceleration of economic globalization, external quarantine pests is becomingincreasingly serious, it is a direct threat to the safety of our country’s agricultural production.Therefore establishing a rapid, accurate and sensitive detection methods of plant viruses are theimportant means to prevent and control the introduction of the quarantine plant virus effectively,and have important and far-reaching significance for the biosafety of our country. In this study, arapid method for RT-PCR amplicon detection by dipstick assay were developed for the detectionof5quarantine viruses, which were Tomato ringspot virus （ToRSV）、Tobacco ringspot virus（TRSV）、Arabis mosaic virus（ArMV）、Tomato black ring virus （TBRV)、Bean pod mottlevirus（BPMV）repectively, and combined the advantages of the high sensitivity of PCR and thespeediness of GICA together.The detection of5viruses by polymerase chain reaction (PCR):5pairs of virus-specificprimers were designed and synthesized based on the conserved sequence of ToRSV, TRSV,ArMV, TBRV and BPMV gene. Of the two primers for each virus, one primer was labeled withbiotin, another primer with fluorescein (or digoxigenin). The dual-labeled amplicon can begenerated by the routine polymerase chain reaction, and can be detected by dipstick assay.The development of the dipstick assay: The20-30nm of colloidal gold were prepared bytraditional sodium citrate reduction method. The binding ability of avidin, streptavidin and biotinantibody (rabbit polyclonal antibody against biotin) with20~30nm colloidal gold particles wascompared.1μL monoclonal antibody against fluorescein (or digoxigenin) was selected for itshigh bind ability to be spotted on nitrocellulose membrane as T dot (test dot), which is close tothe conjugate pad. And1μL goat anti-rabbit polyclonal antibody was spotted on nitrocellulosemembrane as C dot (control dot), which is close to the blotting paper.The detection of PCR amplicon by dipstick assay: Mixed the colloidal gold labeling withbiotin antibody, the dual-labeled PCR amplicon and PBS buffer solution firstly, then we can testthe virus by inseting the dipstick into the mixture. In15minutes, the result was positive if C andT dot both showed color; the result was negative if only C dot showed color; there is a lack ofresult if C dot did not show color.In this study, the five vireses as above-mentioned are detected by the nucleic acid dipstickassay successfully, this method is simpler and faster than the normal method.The double virus detection is tested based on the nucleic acid dipstick assay which isdeveloped in this study. It can detect two viruses on one dipstick in the mean time. The method had been applicated for the detection of TRSV and ToRSV, of ArMV and TBRV.In this study, the PCR-ELISA detection methods for the five viruses were researched too.The dual-labeled PCR amplicon can be detected by the Enzyme-Linked Immunosorbnent Assay.The PCR amplicon labeled with biotin and digoxigenin which is diluted1000times could bedetected by PCR-ELISA detection methods. But the detection effect for the PCR ampliconlabeled with biotin and fluorescein was not good.