Development and validation of a real-time RT-PCR test for screening pepper and tomato seed lots for the presence of pospiviroids.

Potato spindle tuber viroid and other pospiviroids can cause serious diseases in potato and tomato crops. Consequently, pospiviroids are regulated in several countries. Since seed transmission is considered as a pathway for the introduction and spread of pospiviroids, some countries demand for the testing of seed lots of solanaceous crops for the presence of pospiviroids. A real-time RT-PCR test, named PospiSense, was developed for testing pepper (Capsicum annuum) and tomato (Solanum lycopersicum) seeds for seven pospiviroid species known to occur naturally in these crops. The test consists of two multiplex reactions running in parallel, PospiSense 1 and PospiSense 2, that target Citrus exocortis viroid (CEVd), Columnea latent viroid (CLVd), pepper chat fruit viroid (PCFVd), potato spindle tuber viroid (PSTVd), tomato apical stunt viroid (TASVd), tomato chlorotic dwarf viroid (TCDVd) and tomato planta macho viroid (TPMVd, including the former Mexican papita viroid). Dahlia latent viroid (DLVd) is used as an internal isolation control. Validation of the test showed that for both pepper and tomato seeds the current requirements of a routine screening test are fulfilled, i.e. the ability to detect one infested seed in a sample of c.1000 seeds for each of these seven pospiviroids. Additionally, the PospiSense test performed well in an inter-laboratory comparison, which included two routine seed-testing laboratories, and as such provides a relatively easy alternative to the currently used tests.

A semi-automated and highly sensitive streptavidin magnetic capture-hybridization RT-PCR assay: application to genus-wide or species-specific detection of several viruses of ornamental bulb crops.

A semi-automated, rapid and sensitive method that combines magnetic capture-hybridization and reverse-transcription polymerase chain reaction (MCH/RT-PCR) for the detection of plant viruses is described. The assay uses a target specific biotin-labelled oligoprobe for RNA capture and streptavidin-coated magnetic beads for subsequent RNA-oligoprobe hybrid isolation from plant lysate. Detection and specific identification was accomplished by RT-PCR. This approach was investigated for the specific detection of Tobacco rattle virus and for the detection of viruses within the Potexvirus group in leaves, dormant bulbs and corms of flower bulbs of different species. Dilution series of TRV-infected tulip leaf sap showed that MCH/RT-PCR was 70,588 times more sensitive than enzyme-linked immunosorbent assay (ELISA) and was similar to that of RT-PCR. ELISA underestimated the infection levels of TRV in field samples compared to MCH/RT-PCR. The ability of MCH/RT-PCR to be performed in a microtiter plate on an automatic nucleic acid isolation station facilitates high throughput virus diagnostics. RNA isolation and purification was rapid, specific, sensitive, contamination-free and reproducible making this method amenable for routine indexing of stock plants as part of a management plan to reduce the propagation of virus-infected plants.