Purification of Banana Bunchy-top Virus

Banana bunchy-top virus (BBTV) has been purified from diseased banana tissues in Fujian province,China.The banana tissues are pulverized with liquid nitrogen in a coffee grinder.The extracts are clarified with chloroform-butanol,and concentrated by one cycle of differential centrifugation.The suspension of the pellets is further centrifuged on the top of two layer sucrose cushions with 10% and 30% concentrations.The particles in the cushions are collected, concentrated, resuspended and centrifuged on the top of 0 - 40 % sucrose density gradient.The virus particles in a UV-absorbing band on the uppper tube of the sucrose density gradient are further purified in a discontinuous density gradient of cesium sulfate and form a narrow visible band containing a large number of highly purified isometric virus with the diameter about 18 nm.The virus is able to be detected in the process of purification by ELISA with the monoclonal antibody a-gainst BBTV which provided from Taiwan University, and be trapped by ISEM from the clarified extract.The monoclonal antibody has been used to diagnose the field-growing banana samples and tissue culture seedlings.