Interlaboratory test performance study (TPS) for the evaluation of molecular methods to detect Xylella fastidiosa in the vector Philaenus spumarius
Ente finanziatore EU EUPHRESCO (17-XFAST-EU)
Unità di Ricerca DAFNE UNITUS
Responsabile scientifico Prof. BALESTRA G.M.
Partecipanti: Dr. A. Mazzaglia, PhyDia srl
Durata Maggio 2017 – Febbraio 2018
Abstract
The test performance study (TPS) is a way in which the performances of one or more methods are assessed. For this test, standardized samples were prepared with known status regarding the presence of the target pathogen. In addition naturally infected samples were also used.
These were sent out to participating laboratories that analyze them using the provided protocols. The organizer analyzed the results and provided a report detailing all participants’ results in confidential manner together with actual sample status.
This test performance study aims to assess the efficiency and accurateness of different molecular methods used for the detection of the bacterium Xylella fastidiosa into the insect vector Philaenus spumarius. The study is part of the research activities aiming at implementing and validating reliable and sensitive detection procedures for X. fastidiosa in the framework of the following ongoing European projects:
- EUPHRESCO project (2015-F-146) “Harmonized protocol for monitoring and detection of Xylella fastidiosa in its host plants and its vectors”
- H2020 “POnTE – Pest Organisms Threatening Europe (635646)” – Work package 4: “Implementation and validation of diagnostic kits for early and rapid detection of target pathogens in host plants and vectors”
This test performance study has been organized in accordance with the EPPO 7/122 guideline and the following performance criteria have been considered in the data analyses:
- Diagnostic sensitivity: proportion of positive samples giving a positive result.
- Diagnostic specificity: proportion of negative samples giving a negative result.
- Accuracy: the accuracy is the closeness of agreement between a test result and the accepted reference value.
- Repeatability (or accordance): level of agreement between replicates of a sample tested under the same conditions.
- Reproducibility: ability of a test to provide consistent results when applied to aliquots of the same sample tested under different conditions (time, persons, equipment, location, etc.).
- Limit of detection (analytical sensitivity): Smallest amount of target that can be detected reliably in matrix.
The diagnostic methods evaluated during this TPS included several molecular approaches described in the EPPO diagnostic protocol 7/24 (2). The detailed laboratory procedures tested in the different laboratories are reported in the technical sheet in Annex I. The samples were processed either manually or, according to the kit or protocol, using automatized platforms.
IMPORTANT NOTES: In order to provide to all laboratories the same materials and to avoid any risk related to the movement of infective materials, samples were prepared by spiking inactivated bacterial suspensions of Xylella fastidiosa subsp. fastidiosa (ATCC35879) into crushed insects prepared according to each extraction method. This condition may have affected the yield of bacterial target DNA using the different extraction methods. Thus, the results obtained may not reflect the performances of these methods achieved when using fresh infected samples.
1.2 Organizers
This study has been conceived and promoted by the French Agency for food, environmental and occupational health and safety (Anses), Plant health laboratory, Bacteriology Virology and GMO
unit, Angers (France), and implemented in collaboration with Anses - Plant health laboratory,
RAPT, Saint Pierre de La Réunion, France.
1.3 Participating Laboratories
Participant laboratories are listed below. During the test they have been identified with an anonymous identification code, ensuring results confidentiality. Laboratories AGES, Department for Molecular
Diagnostics of Plant Diseases, Biology and Ecology of Soil Microbiota, Institute for Sustainable Agriculture ( CSIC), Central laboratory for plant quarantine, Centro di difesa e certificazione, CREA, CIHEAM-IAMB, Danish Veterinary and Food Agency, Ringsted Laboratory, DEFRA (FERA Science LTD), Departement for Agriculture and Forest Sciences (DAFNE), University of Tuscia, French Agency for food, environmental and occupational health and safety (Anses), LSV, UBVO, ILVO Plant Health Belgium Instituto por la protezione sostenibile, CNR UNIBA, Dipartimento di Scienze del Suolo, della Planta e delli Alimenti, Institute Valenciano de Investigaciones agrarias (IVIA), Bacteriology, Julius Kuehn-Institute - (JKI), Institute for national and international plant health, Braunschweig.