Improved Detection of Citrus psorosis virus and Coat Protein‐Derived Transgenes in Citrus Plants: Comparison Between RT‐qPCR and TAS‐ELISA

Citrus is one of the most economically important fruit crops in the world. Citrus psorosis is a serious disease affecting mainly oranges and mandarins in A rgentina and U ruguay. The causal agent is C itrus psorosis virus ( CP s V ), an ophiovirus with a tripartite ss RNA genome of negative polarity. The coat protein ( CP ), the most abundant viral protein in infected plants, has been used to detect CP s V by TAS ‐ ELISA , but only biological indexing, requiring 1 year, is the current and validated technique for diagnosis of citrus psorosis. In this study, a SYBR G reen RT ‐ qPCR protocol was developed, with primers designed to the most conserved region of the cp gene. We tested their specificity and sensitivity in comparison with TAS ‐ ELISA . This RT ‐ qPCR was applied successfully to field samples from A rgentina, to a variety of isolates from different countries maintained in the greenhouse, to young seedlings and old trees from a psorosis natural transmission plot, and to transgenic citrus expressing the cp gene of CP s V or a fragment thereof. This method allowed accurate quantification of viral titer and cp gene expression in transgenic plants, which could not be detected previously. The sensitivity and reliability of quantitative CP s V detection were improved with greater speed using commercial reagents, and the sensitivity was three orders of magnitude higher than that of TAS ‐ ELISA . All these data encourage its validation.