Abstract

ALMEYDA-LEON, Isidro H. et al. A simple method for the multiple detection of citrus viroids. Agrociencia [online]. 2007, vol.41, n.1, pp.87-93. ISSN 2521-9766.

RT-PCR for viroid detection is commonly used by certified laboratories in México to determine sanitary quality of citrus plants, when donor trees are to be used to propagate plants in nursery. This paper describes a simple methodology for multiple detection of the viroids Citrus exocortis viroid (CEVd), CVd II and Citrus viroid III (CVdIII) in a single RT-PCR reaction. Nucleic acids were extracted with minimum quantities of tissue (250 mg) and 1% polyvinylpyrrodolidone was added to a saline extraction solution of glycine-phosphate. Detection of the three viroids was successful when the RT reactions were performed with 2.0 μg of RNA, 1.0-1.5 mM of dNTPs, 0.90 μM of the complementary primer (C) for CEVd, and 0.49 μM for the corresponding C primers for CVd II and CVd III, respectively, followed by PCR reactions with 2-4 μL of cDNA from RT and 10, 5, 5 pmols of the set of homologous (H) and C set of primers for CEVd, CVd II and CVd III. The use of less than 1.0 μM of dNTPs and equal quantities of the C primers in the RT reactions and equal quantities of both C and H primers for CEVd and CVd II and CVd III in the PCR reactions provided reliable simultaneous detection for CVd II and CVd III, but erratic results with CEVd.

Keywords : Citrus cachexia; citrus exocortis; nucleic acid extraction; polyvinylpyrrolidon.

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