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Detection of Double‐Stranded RNA (DSRNA) in Crude Extracts of Virus‐Infected Plants by Indirect ELISA
Author(s) -
Aramburu J.,
Moreno P.
Publication year - 1994
Publication title -
journal of phytopathology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.53
H-Index - 60
eISSN - 1439-0434
pISSN - 0931-1785
DOI - 10.1111/j.1439-0434.1994.tb04512.x
Subject(s) - antiserum , biology , nicotiana benthamiana , virology , rna silencing , virus , rna , inoculation , polyacrylamide gel electrophoresis , gel electrophoresis , microbiology and biotechnology , antibody , horticulture , biochemistry , enzyme , rna interference , gene , immunology
An antiserum against polyinosinic‐polycytidylic acid (In‐Cn) was used to detect double‐stranded RNA (dsRNA) by indirect ELISA (ELISA‐I). DsRNA from cucumber mosaic virus (CMV) and plum pox virus (PPV)‐infected plants was detected using different types of extracts. The pH of the extraction buffer was very important in dsRNA detection, the highest optical density values being obtained at pH 6 or in aqueous extracts. Extracts heated at 80°C for 2 min showed increased optical density values compared with unheated extracts. DsRNA from Nicotiana benthamiana plants infected with each of six PPV isolates was readily detected by ELISA‐I 50 days after inoculation. ELISA values then obtained with the In‐Cn antiserum were generally higher than those obtained by double antibody sandwich ELISA using an antiserum to virus coat protein. Purified dsRNA from the same infected plants showed no visible band, but it produced a fluorescent background when analysed by polyacrylamide gel electrophoresis.