Inter-Laboratory Comparison of RT-PCR-Based Methods for the Detection of Tomato Brown Rugose Fruit Virus on Tomato

Marta Luigi,Sabrina Bertin,Ariana Manglli,Antonio Tiberini,Laura Tomassoli,Luca Ferretti,Francesco Faggioli,Anna Taglienti

doi:10.3390/pathogens11020207

Abstract

In 2020, a test performance study (TPS) for the specific detection of tomato brown rugose fruit virus (ToBRFV) was organized in the frame of the H2020 Valitest project. Since no validated tests were available, all the protocols reported in the literature were at first screened, performing preliminary studies in accordance with the EPPO standard PM 7/98 (4). Five molecular tests, two conventional RT-PCR and three real-time RT-PCR were found to be suitable and were included in the TPS. Thirty-four laboratories from 18 countries worldwide took part in TPS, receiving a panel of 22 blind samples. The panel consisted of sap belonging to symptomatic or asymptomatic leaves of Solanum lycopersicum and Capsicum annuum. The results returned by each laboratory were analyzed and diagnostic parameters were assessed for each test: reproducibility, repeatability, analytical sensitivity, diagnostic sensitivity and diagnostic specificity. All the evaluated tests resulted in being reliable in detecting ToBRFV and were included in an EPPO Standard PM 7/146—Diagnostics.

Highlights

Tomato brown rugose fruit virus (ToBRFV) has a single-stranded positive sense RNA genome located in rigid elongated particles
2019, ToBRFV was reported in the Asian continent, in Turkey [5] and China [6]
Due to the lack of some validation data and the difficulty to compare those already available, preliminary trials were performed in-house to select the most suitable tests to be included in the test performance study (TPS)

Summary

Introduction

Tomato brown rugose fruit virus (ToBRFV) (genus Tobamovirus, family Virgaviridae) has a single-stranded positive sense RNA genome located in rigid elongated particles It first emerged in Israel and Jordan in 2014 and 2015, respectively [1,2], and later on was reported in several European countries as well as in Central and North America, where it was first reported in Mexico in Autumn 2018 [3], and it spread in California [4]. ToBRFV virions can survive for a long time in infected plant residues, contaminated soil, on tools and worker clothes, irrigation systems and greenhouse structures (such as poles, nets, pallets, etc.) contaminated with raw sap from infected plants [10] For these reasons, the probability of further entry and the establishment of ToBRFV in the EPPO region is reported to be high, with a low uncertainty [11]

Methods

Results

Conclusion