Real-time RT-PCR fluorescent detection of tomato spotted wilt virus

Abstract

A real-time reverse transcription-polymerase chain reaction assay based on TaqMan™ chemistry was developed for the detection and quantification of tomato spotted wilt virus (TSWV). This method enabled sensitive, reproducible and specific detection of TSWV in ‘leaf soak’ and total RNA extracts from infected plants. TaqMan reliably detected TSWV in as little as 500 fg total RNA. The assay was 10-fold more sensitive than visualisation of ethidium bromide-stained bands following agarose gel electrophoresis. TSWV isolates from various crops and locations were detected with a cycle threshold of 20–26 in 1 ng total RNA extracted from fresh or freeze-dried leaves. A dilution series of in vitro transcripts from a cloned 628 base pair fragment of TSWV S RNA served as standard for quantification of viral template in infected leaf samples. The TaqMan assay detected reproducibly 1000 molecules of the target transcript.