Application of real‐time RT‐PCR for large‐scale testing of potato for Potato spindle tuber pospiviroid*

Abstract

The real-time RT-PCR protocol of Boonham and coworkers performed extremely well in a recent ring test, comparing different methods for detection of Potato spindle tuber pospiviroid (PSTVd) in several laboratories. Since, in addition, real-time PCR technology has proved suitable for highthroughput testing, this method was chosen as the starting point for the development of a protocol for the large-scale testing of potato. The initial experiments focused on the specificity of the primers and probes with regard to different isolates of PSTVd and other (pospi-) viroids. Further experiments were performed with leaf material from both primarily and secondarily infected plants. The parameters studied were sampling position, growing-on temperature and bulking rate. In addition, different grinding, nucleic-acid extraction and disinfection methods were compared. To monitor false negatives and positives, different controls were included and tested in duplex and triplex formats. The final protocol was tested using a hundred samples from the Dutch potato-monitoring programme. The results of this pilot experiment were promising. Future plans include the development of a protocol for direct tuber testing and inter-laboratory ring testing of the protocols.

Epidemiological evidence that vegetatively propagated, solanaceous plant species act as sources ofPotato spindle tuber viroidinoculum for tomato

“…Real‐time RT‐PCR was performed according to Boonham et al . (2004), and modified by Roenhorst et al . (2005).…”

Diagnostic Values and Utility of Immunological, Morphological, and Molecular Methods for In Planta Detection of Phytophthora ramorum

“…The workflow was organized in a way that contamination risk was minimized. Chemical decontamination of surfaces and equipment was performed with 1% sodium hypochlorite (29,31).…”

Management of plant health risks associated with processing of plant-based wastes: A review

“…Phytophthora kernoviae sporangia oospores similar to P. ramorum no data Schena et al (2006) Polymyxa spp cystosori in roots or soil 100-150 cystosori detection of spores from soil using Real Time PCR 1.39 × 10 3 cystosori g -1 soil Ward et al (2004) bait tests followed by glutathione-S-transferase ELISA or microscopy no data √ Kingsnorth et al (2003) MPN method bait test method 50-100 cystosori √ Tuitert (1990) 58 Roenhorst et al (2005) …”