A Colloidal Gold Immunochromatographic Assay And RT-Lamp Detection Of Soybean Mosaic Virus

Soybean mosaic virus (SMV) is a widely distributed and highly destructive plant virus worldwide. Infection by SMV usually results in severe yield losses and seed quality reduction. SMV infected disease has caused a huge economic losses in many countries. Therefore, methods for rapidly detecting SMV would be useful.The objective of this study is to use two methods:one-step immunochromatographic assay is the use of colloidal gold labeled monoclonal antibody (mAb) immunological detection of SMV, and RT-LAMP (Reverse-transcription loop-mediated isothermal amplification) to achieve rapid, sensitive and accurate detection method for SMV in soybean leaves and seeds.SMV one-step immunochromatographic assay:The research method was coat protein in vitro expression of soybean mosaic virus in prokaryotic cell using intraperitoneal immunization with coat protein immunized mice. The cell fusion technique to obtain hybridoma cell lines producing monoclonal antibodies was successful. Additionally, double antibody sandwich model using two different monoclonal antibody preparation of immunochromatographic strip was achieved. An anti SMV monoclonal antibody (mAb3Ei2C6) fixed to nitrocellulose membrane in the detection area and another kind of anti SMV monoclonal antibody (mAb4B8D9) coupled to colloidal gold particles as chromogenic agent. Subsequently the plant sample solution by capillary action reaction with anti SMV IgG antibody was added to the detection area, the SMV retention in the detection area which appeared red gave positive results. Strip preparation detection was sensitive and specific to the leaves and seeds of SMV. This is a rapid and convenient detection method, without special equipment, reagents and technical personnel.RT-LAMP detection of SMV:Using SMV coat protein gene sequences from GenBank database, the RT-LAMP primers sets were designed and the best primers set was selected by the screening test. Then they were used for the optimization of the RT-LAMP parameters. The sensitivity of the RT-LAMP was 10-100 times higher than the conventional RT-PCR. When crude RNA was integrated in this assay, the developed one-step RT-LAMP can rapidly and sensitively detect SMV in soybean leaves. Addition of fluorescent dye SYBR Green I make visualization the reaction product in-tube. The developed RT-LAMP can successfully detect SMV in soybean seeds.In this work, we first develop a simple and rapid detection of SMV test using a strip and first report of the application of the RT-LAMP assay to detect SMV in soybean leaves and seeds. The methods are highly specific, sensitive and rapid, applicable to the field with great potential.