Sensitive and specific qPCR and nested RT-PCR assays for the detection of Tobacco streak virus in soybean

Abstract

Tobacco streak virus (TSV) is a re-emerging and understudied pathogen of soybean (Glycine max). Management of TSV is challenging due to the multiple modes of transmission, widespread susceptibility of commercial soybean, and lack of reliable diagnostic tests for the virus. Soybean plants with TSV-like, virus-like, or no symptoms were collected from commercial and research fields in seven counties in Wisconsin. Two sensitive assays were developed for the detection of TSV, a fluorescent dye-based quantitative RT-PCR (qPCR) assay and a nested RT-PCR (nRT-PCR). Tobacco streak virus was detected in 47 percent and 91 percent of symptomatic samples using the qPCR assay and the nRT-PCR assay, respectively, suggesting that the nRT-PCR assay has higher sensitivity for detecting TSV. The qPCR assay’s limit of detection was determined at 10 fg and the assay was used to estimate the viral load in TSV-symptomatic samples. The titer of TSV in these samples was determined by absolute quantification and ranged from 15 fg to 0.796 ng. The two assays reported here provide diagnostic tools for the rapid and accurate detection of TSV that can aid in monitoring outbreaks, assessing management strategies, or screening soybean cultivars or accessions for resistance to the virus.