Simple and Quantitative Detection of Apple latent spherical virus Vector by a Spot Hybridization

Apple latent spherical virus (ALSV) vector has been widely used as a vector for virus-induced gene silencing, gene expression, and vaccination for protection from virus diseases in wide variety of plants. Here we describe a simple and quantitative method for ALSV detection in plant materials. ALSV infected plant leaves were homogenized in a buffer, and added with NaOH, which was found to be optimal at the final concentration of 10 mM. After serial dilution with 10 mM NaOH, the samples were spotted onto nylon membrane. After neutralization with acidic solution and fixation of nucleic acid by UV irradiation, the membrane was subjected to a prehybridization for an hour, followed by a hybridization with digoxygenin-labelled RNA probe for overnight. ALSV was quantitatively detected using a chemiluminescent substrate. The entire protocol took two days with hands-on time of a couple of hours.