Development of an EU protocol for the detection and diagnosis of Potato spindle tuber pospiviroid*

Abstract

The work described here formed part of the EU SMT DIAGPRO project, to develop diagnostic protocols for 18 regulated pests. The Potato spindle tuber pospiviroid (PSTVd) protocol was developed primarily for testing in vitro-and glasshouse-grown potato plants for the purposes of post-entry quarantine and the production of pathogen-tested nuclear stock. After a performance audit of methods used by 12 laboratories in Europe and America by ring testing, four methods were chosen for multilaboratory validation. For most laboratories, the detection limits were 10-20 mg of PSTVd-infective tissue for R-PAGE; 0.25-0.5 mg for DIG-probe; 0.062 mg for RT-PCR; and 0.0155 mg for TaqMan (this was the lowest weight of infective tissue tested). Some laboratories were able to extend the detection limit to 0.0155 mg for DIG-probe and RT-PCR. The DIG-probe and R-PAGE are recommended as primary detection methods, with confirmation of viroid presence by any of the four validated detection methods. Specific diagnosis requires the viroid to be sequenced. Other methods may be used for primary detection, providing that they preferably detect all PSTVd isolates and other Pospiviroids that have the potential to infect potato, and detect viroid in at least 1/10 of the tissue weight normally tested per plant.

Interlaboratory evaluation of two Reverse‐transcriptase Polymeric Chain Reaction‐based methods for detection of four fruit tree viruses

“…In conclusion, a careful documentation of the results combined with a preliminary statistical analysis selected the interpretable results for statistical analysis. Specificity and sensitivity values for ASPV, ASGV and ACLSV were similar or higher than those obtained in the literature for specificity (82-100%) and sensitivity (38-96%) (Malorny et al, 2003;Josefsen et al, 2004;Jeffries & James, 2005;Taha et al, 2005;Lò pez et al, 2006;Truyen et al, 2006). Nevertheless, the specificity value of ASPV detection was low (67.9%), reflecting the high percentage of false positives.…”

Inter-laboratory evaluation of a duplex RT-PCR method using crude extracts for the simultaneous detection of Prune dwarf virus and Prunus necrotic ringspot virus

“…The estimated sensitivities of the duplex RT-PCR protocol were 98% and 90% for PDV and PNRSV, respectively. During previous inter-laboratory evaluations of (RT)-PCR protocols for single plant, animal or human pathogen detection, the sensitivity values ranged between 72% to 96% (Jeffries and James 2005;Josefsen et al 2004;Paton et al 2000;Taha et al 2005;Truyen et al 2006) or 38% to 85% (Lopez et al 2006). So, while using crude extracts and detecting two viruses simultaneously, the specificity and the sensitivity values were very high and equal or higher than the average values published in the literature for detection of single pathogens using purified RNA or DNA.…”

“…Compared to most of the previous inter-laboratory assays of (RT-)PCR detection methods (Jeffries and James 2005;Lopez et al 2006;Paton et al 2000), the experiment presented three additional difficulties: (1) the processing of vascular tissues (more complicated to process than leaves), (2) the use of crude extract preparations, and (3) the simultaneous identification of two different RNA targets.…”

“…Other interlaboratory evaluations of (RT)-PCR protocols for single plant or human pathogen detection obtained specificity values ranging from 82% to 100% (Jeffries and James 2005;Josefsen et al 2004;Lopez et al 2006;Malorny et al 2003;Taha et al 2005). The estimated sensitivities of the duplex RT-PCR protocol were 98% and 90% for PDV and PNRSV, respectively.…”

The Challenge for Rapid Detection of High-Structured Circular RNA: Assay of Potato Spindle Tuber Viroid Based on Recombinase Polymerase Amplification and Lateral Flow Tests

“…The European and Mediterranean Plant Protection Organization (EPPO) recommended conducting some of these tests for highly sensitive PSTVd detection [ 26 ]. While the official standards of the EPPO state that the dilution rate is the limit of detection (LOD) [ 27 ], only one LOD was found: 17 pg of PSTVd RNA (8.4·10 7 copies) for infected samples after specific RNA extraction [ 28 ]. RT-qPCR requires the use of expensive equipment, which may be an obstacle for introducing the method into the field as a detection practice.…”