Study On Detection Of CGMMV By Antibody Macroarray

Cucumber green mottle mosaic virus (CGMMV) is the one of the most important virusin cucurbit crops. As China’s entry plant quarantine pest, it leads to mottle, mosaic, deformity,fruit rot, dwarf and other symptoms, and causes serious yield loss. Timely detection ofpathogens is the key to early detection and prevention of cucurbit viruses. Using theconventional immunoassay ELISA technique as a benchmark and a chromogenic dye, theantibody macroarray assay is developed, the optimized procedure was used to detectCGMMV as well. The50lots of cucurbit seeds produced in Hexi Corridor of Gansu provincewere selected as the samples to verify the method sensitive and easy to use that detection ofCGMMV by antibody macroarray. This work provided a rapid and meaningful detection ofCGMMV.(1) The cross-reactions of12common cucurbit virus antibodies were done withDAS-ELISA for expelling the unsutible antibodies. Then the suitable virus antibodies selectedby reacting with NCM carrier antibody macroarray. The result suggested that the12cucurbitvirus antibodies have no cross-reaction and they differ greatly in antibodies macrobodies. Thebetter antibodies are:TRSV, PRSV, CGMMV, WMV, PVY.(2) An antibody macroarray procedure was developed and its usefulness for the detectionof CGMMV was demonstrated.The optimized procedure was used to detect CGMMV,including:coating antibody dilution factor,the condition of sample incubation andenzyme-linked antibody incubation, and the condition of substrate chromogenic reaction. Theresult of good reaction signals based on coating antibody dilution factor1:5,sampleincubated1h(20℃),enzyme-linked antibody incubated3h(20℃),substrate chromogenicreacted15min(20℃).(3) The pilot study on the sensitivity of antibody macroarray and comparison withDAS-ELISA were carried on with the sample of serial diluted CGMMV suspension. Theresults showed that with the increase of CGMMV positive virus dilution, the testsignal intensity decreased.The dot was recogonized on the dilution of28on antibodymacroarray, while the DAS-ELISA’s sensitivity is26(by standard OD405=2.0).The antibodymacroarray has higher sensitivity.(4)This method was further validated by testing50lots of cucurbit seeds and the resultwas recheched by DAS-ELISA and PCR,which demonstrated that the coincidence rate was98.0%between the results of antibody macroarray and that of ELISA,consistency ofthe results was tested by PCR.These results indicate that the developed antibody macroarrayhas larger dot, low integrated degree,convinience,suitable for visual detection and other merits, which would deliver cost effective and have potential for diagnosis use.