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Improved Detection of Citrus psorosis virus and Coat Protein‐Derived Transgenes in Citrus Plants: Comparison Between RT‐qPCR and TAS‐ELISA
Author(s) -
De Francesco Agustina,
Costa Norma,
Plata María I.,
García María L.
Publication year - 2015
Publication title -
journal of phytopathology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.53
H-Index - 60
eISSN - 1439-0434
pISSN - 0931-1785
DOI - 10.1111/jph.12392
Subject(s) - biology , genetically modified crops , plant virus , transgene , gene , coat protein , virology , virus , titer , microbiology and biotechnology , citrus tristeza virus , citrus fruit , horticulture , rna , genetics
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.