Development and validation of a multiplex real‐time <scp>RT</scp>‐<scp>PCR</scp> test for the screening of tomato (<i>Solanum lycopersicum</i>) for the detection of pepino mosaic virus

Abstract Pepino mosaic virus (PepMV) is known to cause a wide range of symptoms in cultivated tomato plants, damaging fruits and crops. Currently, five genotypes of PepMV are known (US1, CH2, EU, LP and PES), which can all infect tomato plants. Tomato seed is a proven pathway for PepMV, and the virus is known to easily spread mechanically during crop handling. A multiplex real‐time reverse transcriptase‐polymerase chain reaction (RT‐PCR) test was developed and validated for the specific identification of PepMV isolates from tomato seeds. This test is designed to be used for diagnostics and seed health tests as a pre‐screen to distinguish negative PepMV samples from suspect samples or as a confirmation test of a positive ELISA or bioassay test result. The multiplex real‐time RT‐PCR test consists of two PepMV‐specific real‐time RT‐PCR tests and a Bacopa chlorosis virus real‐time RT‐PCR test as an internal amplification control. The analytical specificity of the developed test was evaluated by in vitro and in silico analyses. The analytical sensitivity was evaluated by creating three dilution series of naturally PepMV‐infected leaf tissue in healthy tomato seed extracts and the diagnostic specificity and sensitivity were validated on a collection of seed lots.