In silico re-assessment of RT-qPCR assays for detection of tomato brown rugose fruit virus
DOI:
https://doi.org/10.61308/s931g684Keywords:
ToBRFV, RT-qPCR, EPPO PM 7/146 (2), sequence tracer, entropy calculatorAbstract
Tomato brown rugose fruit virus (ToBRFV) is an emerging and rapidly spreading RNA virus that infects tomato and pepper, with tomato as the primary host. In this study, we aimed to evaluate the variability in primer/probe binding regions of the Real-time RT-PCR (RT-qPCR) assays recommended by the European and Mediterranean Plant Protection Organization (EPPO) Standard on Diagnostics PM 7/146 (2). By analyzing ToBRFV sequences from the NCBI Virus database, variations in primer/probe binding sites were identified, which may impact detection accuracy. The exact match rates for the assays were as follows: ISF-ISHI-Veg CaTa 28: 76.58%, CSP1325: 93.2%, Menzel and Winter: 93.6%, and Bernabé-Orts: 95.63%. This study systematically assesses the inclusivity of these assays, highlighting specific mismatches that could lead to false-negative results. These findings emphasize the importance of continuously validating RT-qPCR methods to ensure accurate detection across diverse viral strains. Given the observed genetic variability, region-specific sequencing and in silico analysis are crucial to improving the robustness and reliability of RT-qPCR assays for ToBRFV.
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