Rapid diagnosis of soybean mosaic virus N soybean by tissue-print immunoassay and DIBA in comparison to other serological methods.
Soybean mosaic virus (SMV) is an important disease in soybean and is widely distributed in northern of Iran. SMV transmitted by soybean seed and detection of it is very important for disease management. In this study, several detection methods including DAS-ELISA, indirect-ELISA, tissue-print immunoassay (TPIA) and Dot immunobinding assay (DIBA) were optimized and compared with each other to identify the virus, using polyclonal antibody. For TPIA, nitrocellulose membrane was used to imprint fresh sections of healthy and infected plant materials, and for DIBA 10 microl of extracts was doted onto nitrocellulose membranes. Both membranes were incubated 1 hour in blocking buffer, and then incubated 2 h in 1:1000 dilution of IgG-conjugate. After incubation the membranes were washed three times with PBS-T buffer for 15 min. Then the membranes were incubated in substrate solution containing NBT/BCIP. After some minutes prints or blots of infected tissues turned dark violet, whereas prints or blots of healthy ones did not show any color changes. In some cases, substrate solution was Fast red, containing 0.2M Tris-HCl buffer and 2mM MgCl2, pH = 7.8, producing red color in infected prints or blots. Both methods are simple and TPIA is rapidly and easily applicable in the field. However, TPIA had some advantages over the others. TPIA is time-saving as there is no need for conventional sap extraction and also nitrocellulose membranes used for printing can be used in the field and stored for a long time or transported to another laboratory for process. These two methods can be used routinely for detection of SMV in many samples.
Ahangaran
A
A
Department of Plant Protection, Faculty of Agriculture, Tehran University, Karaj, Iran. akbarahangaran@yahoo.com
Mohammadi
Gh Mosahebi
GM
Habibi
M Koohi
MK
Shahraeen
N
N
Khezri
S
S
D004797:Enzyme-Linked Immunosorbent Assay; D007118:Immunoassay / Q000379:methods; D009029:Mosaic Viruses / Q000145:classification / Q000302:isolation & purification / Q000472:pathogenicity*; D010935:Plant Diseases / Q000382:microbiology*; D012703:Serotyping; D013025:Glycine max / Q000821:virology*