Comparison of Techniques for Detection of Barley Yellow Dwarf Virus-PAV-IL

Abstract

Detection of barley yellow dwarf virus (BYDV)-PAV-IL by an improved nucleic acid hybridization technique, using a nonradioactive probe with chromogenic and chemiluminescent substrates, was compared with detection by polymerase chain reaction (PCR), double antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA) with polyclonal antibodies, and triple antibody sandwich ELISA with polyclonal and monoclonal antibodies. Each method was used to detect purified virus and virus in sap extracts from infected oat leaves. The detection limits for both ELISA procedures were 1 ng of purified BYDV-PAV-IL and the equivalent of 78 ng of infected tissue. Nucleic acid hybridization with either chemiluminescent or chromogenic substrates also detected as little as 1 ng of purified BYDV-PAV-IL, but it was slightly more sensitive at detecting virus in tissue extracts (25 ng of infected tissue). The most sensitive detection technique was PCR amplification, which could detect as little as 0.1 pg of RNA extracted from purified virus and detected viral RNA in the equivalent of 0.5 pg of infected leaf tissue.