Development of rapid Immunofilter paper assay for chrysanthemum stem necrosis orthotospovirus using polyclonal antibody generated in Rabbits

Abstract

A simple detection method using a rapid immunofilter paper assay was developed in this study. First, the N protein gene-coding region of a chrysanthemum stem necrosis orthotospovirus (CSNV) isolate collected in Japan was amplified by reverse transcription-polymerase chain reaction and introduced into the multi-cloning site of the pMAL-c5X vector. The vector was introduced into Escherichia coli (Rosetta DE3) competent cells, and ca. 75 kDa fusion protein of CSNV-N and maltose binding protein was obtained by liquid culture. Antiserum was obtained from rabbits immunized with the fusion protein. IgG was purified from the antiserum, and its titer was determined by enzyme-linked immunosorbent assay to be approximately 200-fold. Immunochromatograms were prepared by rapid immunofilter paper assay using the IgG. CSNV was detected in diseased plants using the immunochromatograms and red polystyrene particles at a concentration of 8%.