检测南方水稻黑条矮缩病毒胶体金免疫试纸条的建立

Development of colloidal gold immunochromatographic strip for detecting Southern rice black-streaked dwarf virus

由白背飞虱Sogatella furcifera(Horváth)传播的南方水稻黑条矮缩病毒(Southern rice blackstreaked dwarf virus,SRBSDV)是目前我国南方水稻上危害最严重的病毒,为开发简便、快速、准确的SRBSDV病毒检测技术和检测试剂,以感染SRBSDV的植物粗提液为免疫原,利用杂交瘤技术制备了2株抗SRBSDV的单抗(14A8和15G6),并利用制备的单抗建立了可快速、特异、灵敏地检测SRBSDV的胶体金免疫试纸条。结果表明,2株制备单抗的抗体类型及亚类均为Ig G1、kappa链,单抗腹水的间接ELISA效价均达到10^(-7);Western blot分析表明,2株单抗均与SRBSDV的外壳蛋白亚基有特异反应,而不与水稻黑条矮缩病毒(Rice black-streaked dwarf virus,RBSDV)反应。以制备14A8和15G6单抗分别为捕获抗体和胶体金标记抗体,开发成能在5 min内准确、特异地检测水稻植物和白背飞虱传毒介体体内SRBSDV的胶体金免疫试纸条;灵敏度分析表明,该检测试纸条的检测水稻病叶的灵敏度达到1∶6 400倍(g/m L),检测单头携毒白背飞虱的灵敏度达到1∶51 200倍(单头/μL)。田间样品检测结果表明,该试纸条的检测结果与RT-PCR的符合率达到100%。建立的SRBSDV胶体金免疫试纸条可对南方水稻黑条矮缩病毒进行快速、特异、灵敏的诊断和检测。

Southern rice black-streaked dwarf virus （SRBSDV） transmitted by white-backed planthopper, Sogatella furcifera Horvath, is the most important rice virus in southern rice production region of China, causing serious losses in rice production. The developments of simple, rapid and accurate virus detection techniques and detection reagents are crucial to forecast and early warning, and scientific prevention and control of this viral disease. For this purpose, two monoclonal antibodies （MAbs, 14A8 and 15G6） against SRBSDV were produced using the SRBSDV-infected rice crude extract as the immunogen, and a colloidal gold immunochromatographic strip based on the MAbs were developed for rapid, specific, sensitive detection of SRBSDV in this study. Both MAbs were isotyped as IgG1, kappa light chain. The titers of ascitic fluids of two MAbs were up to 10^-7 by an indirect-ELISA. Western blot analysis indicated that both MAbs could specifically react with the coat protein of SRBSDV, and not with Rice black-streaked dwarf virus （RBSDV）. Using the prepared 14A8 and 15G6 as the capture antibody and the colloidal gold conjugate antibody, a colloidal gold immunochromatographic strip was successfully developed to specifically and accurately detect SRBSDV in rice plants and white-backed planthop- per vectors in 5 min. The sensitivity analysis revealed that the strip could detect the virus in SRBSDV- infected rice plant crude extracts diluted at 1 ： 6 400 （g/mL）, and in SRBSDV-infected white-backed planthopper homogenate diluted at 1 ： 51 200 （individual/μL）, respectively. The detections of field samples indicated that the coincidence rate of detection results of the strip and RT-PCR reached 100%. The development of the colloidal gold immunochromatographic strip of SRBSDV provides the technology and materiel for diagnosis and detection of this virus disease.