Enzyme-Linked Immunosorbent Assay Testing of Shoots Grown In Vitro and the Use of Immunocapture-Reverse Transcription-Polymerase Chain Reaction Improve the Detection of Prunus necrotic ringspot virus in Rose

Abstract

We developed and evaluated two different methods to improve the detection of the most prevalent virus of rose in Europe, Prunus necrotic ring-spot virus (PNRSV). Immunocapture-reverse transcription-polymerase chain reaction was estimated to be about 100 times more sensitive than double-antibody sandwich-enzyme-linked immunosorbent assay (DAS-ELISA) and showed an equivalent specificity. Based on the observation that PNRSV multiplies actively in young growing tissues (axillary shoots and cuttings), an in vitro culture method allowing rapid (about 15 days) and homogeneous development of dormant axillary buds with high virus titers was standardized. ELISA tests of these young shoots showed, in some cases, a 10(4) to 10(5) increase in sensitivity in comparison to adjacent leaf tissues from the rose mother plants. Between 21 and 98% (depending on the season) more samples were identified as positive by using ELISA on samples from shoot tips grown in vitro rather than on leaves collected directly from the PNRSV-infected mother plants. This simple method of growing shoot tips in vitro improved the confidence in the detection of PNRSV and eliminated problems in sampling appropriate tissues.

Enhanced Detection of Prune Dwarf Virus in Peach Leaves by Immunocapture‐Reverse Transcription‐Polymerase Chain Reaction with Nested Polymerase Chain Reaction (IC‐RT‐PCR Nested PCR)

“…The IC‐RT‐PCR nested PCR should have circumvented the occurrence of potential interference in amplifications (Olmos et al., 1999). Inconsistencies in IC‐RT‐PCR have been mentioned by other authors (Spiegel et al., 1996; Moury et al., 2000), that improved the assay performances by adding extra steps such as further hybridization with nucleic acid probes. In the present study problems were also observed in detecting IC‐RT‐PCR products (data not shown) that might have been due to lower stability of viral RNA and the addition of RNase inhibitors may have been beneficial.…”

Development of a non-radioactive molecular hybridization probe for detecting Strawberry mottle virus in strawberry

“…Even though this technique is highly sensitive and specific, it turns out to be impractical because of the effort involved in making a large number of simultaneous analyses, and also because PCR testing is rather costly. Analyzing many samples at the same time is possible through serological techniques such as the enzyme-linked immunosorbent assay (ELISA); it has been shown, however, that they are not as sensitive as molecular testing (Stevens et al, 1997;Sanchez-Navarro et al, 1998;Moury et al, 2000;Lunello et al, 2005). Molecular hybridization probes are an interesting alternative for this purpose.…”

Biological and molecular characterization of Prunus necrotic ringspot virus isolates from three rose cultivars

“…Apart from almond, apricot, peach, plum, sour-and sweet-cherry trees, roses are also attacked by this virus (Németh 1986). PNRSV is the most commonly found rose virus in the United Kingdom, France and Poland (Thomas 1981(Thomas , 1984aMoury et al 2000Moury et al , 2001Paduch-Cichal 2003). Some field-grown rose cultivars show no symptoms of PNRSV infection, whereas others develop line patterns, ringspots or yellow nets on leaves (Thomas 1981(Thomas , 1984aCurtis and Moran 1986;Wong and Horst 1988;Paduch-Cichal 2003).…”