A multiplex RT-PCR assay combined with co-extraction of DNA and RNA for simultaneous detection of TYLCV and ToCV in whitefly

Abstract

Tomato yellow leaf curl virus (TYLCV) and tomato chlorosis virus (ToCV) were transmitted by the sweet potato whitefly Bemisia tabaci (Gennadius) and cause serious yield losses on tomato around the world. To understand the actual situation of co-infection of TYLCV and ToCV of individual whiteflies, we developed multiplex RT-PCR combined with co-extraction of DNA and RNA from a single whitefly. First, a nucleic acid extraction method previously reported was modified and adopted to obtain the RNA-DNA mixture including TYLCV and ToCV in a simple form without manual homogenization. Second, primers were newly designed in actin gene of B. tabaci for the confirmation of extraction and PCR success, and multiplex RT-PCR method was developed using specific primer sets for TYLCV, ToCV and B. tabaci. This method enables the detection of TYLCV and ToCV from a single insect and efficient use of field samples obtained using sticky traps. The method will be useful to monitor infection status of TYLCV and ToCV in the field while reducing labor and cost.