A sensitive semi-quantitative biotin-streptavidin ELISA for the detection of potyviruses infecting cucurbits

Abstract

The biotin-streptavidin (BA)-ELISA system was optimized for the detection of three potyviruses infecting cucurbits, the watermelon strain of papaya ringspot virus (PRSV-W), zucchini yellow mosaic virus (ZYMV) and watermelon mosaic virus 2. BA-ELISA was four to eight times more sensitive than double antibody sandwich ELISA and detected all three viruses in the nanogram range. BA-ELSA was virus-specific but did not differentiate between different pathotypes of ZYMV. The use of egg-white avidin-enzyme conjugate or the simultaneous incubation of biotinylated antibody and streptavidin-enzyme conjugate decreased the sensitivity of BA-ELISA. The concentrations of PRSV-W and ZYMV in field-infected cucurbit specimens or in resistant breeding lines were estimated from calibration curves derived from a dilution series of purified virus in extracts of uninfected plants on each test plate. Thus it was possible to determine semi-quantitatively the degree and uniformity of resistance in plants from these lines and to differentiate resistant from tolerant selections.