Journal of Food, Agriculture and Environment

Development of a specific serological kit for detection of Polymyxa betae, transmitting agent of sugar beet rhizomania disease

Author(s):

Hossein Safarpour ^{1, 2}, Mohammad Reza Safarnejad ^{1, 3} *, Marzieh Basirat ¹, Fatemeh Hasanzadeh ⁴, Fereshteh Kamyab ⁴

Recieved Date: 2012-07-02, Accepted Date: 2012-10-04

Abstract:

Sugar beet is one of the most important industrial crops in Iran and during the last two decades has been mainly affected by a destructive virus, beet necrotic yellow vein virus (BNYVV). The Polymyxa betae (Keskin) is the only natural transmitting agent of the disease between the plants. Developing accurate diagnostic methods has a major impact on epidemiological studies and for screening sugar beet in resistance breeding programs. The fungus is an obligate parasite and could not be cultured in the media, then selection of specific antigen presented in all growth statue is a main concern. The glutathione-S-transferase (GST) as a specific immunogenic protein is a critical enzyme expressed in zoospores, sporangia and resting spores and could be a good candidate to develop a serological test for P. betae.  For this aim, the DNA region encoding fungal GST gene was isolated and cloned into pET28a bacterial expression vector. Large-scale expression of the recombinant protein was performed in Escherichia coli (Migula). Purification was carried out under native conditions. The purified recombinant GST protein was used for immunization of rabbit. Purification of immunoglobulin was performed by affinity chromatography using protein A column. The purified immunoglobulin was conjugated with alkaline phosphatase enzyme. The purified antibodies and conjugates were applied for efficient detection of infected plant in serological assays including double antibody sandwich-enzyme linked immunosorbent assay (DAS-ELISA) and dot immune binding assay. The results obtained in immunoassay analysis proved high efficiency of the produced kit for detection of infected plants. 

Keywords:

Glutathione-S-transferase (GST), Polymyxa betae, rhizomania, serological kit, sugar beet, polyclonal antibody

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Journal: Journal of Food, Agriculture and Environment
Year: 2012
Volume: 10
Issue: 3&4
Category: Agriculture
Pages: 729-732

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