Selection,optimization and characterization of molecular tests for the detection of Tobacco ringspot virus (TRSV)

The aim of the study was to select, optimize and characterize RT‐PCR tests for the detection of Tobacco ringspot virus (TRSV) in post‐entry quarantine. Among five different tests, the Poojari et al. (Journal of Virological Methods 235, 112–118) and Jossey & Babadoost (Plant Disease 90, 1361) conventionnal RT‐PCR tests were chosen and optimized for this purpose. Ten TRSV isolates, 17 healthy plants from the genera Vitis, Prunus and Malus, four other Nepoviruses or Secoviridae isolates and serial dilutions of three TRSV isolates were used for the complete characterization of the optimized tests. In the tested conditions, the Poojari and Jossey & Babadoost tests respectively showed 100% and 95% inclusivity, 100% and 100% exclusivity, 100% and 96.4% analytical specificity, 100% and 100% diagnostic specificity, 10^?7; 10^?1] and 10^?8; 10^?2] limit of detection dilution factors (analytical sensitivity), 91.1% and 90.0% repeatability, 90.3% and 91.1% reproducibility and 100% and 100% selectivity. Due to its higher inclusivity, the optimized Poojari test is recommended for the detection of TRSV isolates for post‐entry quarantine purposes, but can also be used for global surveys. The optimized Jossey & Babadoost test showed the best analytical sensitivity, suggesting that combining both tests can further enhance detection.