Development of a multiplex RT-PCR method to detect six orthotospovirus species reported in Japan

Abstract

We improved an established one-step multiplex RT-PCR method to detect six orthotospoviruses that infect Solanaceae plants (tomato spotted wilt virus [TSWV], newly reported tomato zonate spot virus [TZSV], impatiens necrotic spot virus [INSV], chrysanthemum stem necrosis virus [CSNV], watermelon silver mottle virus [WSMoV], capsicum chlorosis virus [CaCV]). Specific primer sets for one-step multiplex RT-PCR were designed for TSWV, TZSV, INSV, CSNV, WSMoV, and CaCV, and this optimized RT-PCR amplified DNA fragments corresponding to the target virus species: TSWV (831 bp), TZSV (734 bp), INSV (592 bp), CSNV (502 bp), WSMoV (395 bp), CaCV (324 bp). All combinations of orthotospoviruses were detected in mixed samples. The method was applied to Solanaceae plants infected with orthotospoviruses under field conditions.